Functional characterization of partial recombinant goat conglutinin: Its role as innate immunity marker and use as antigen in sandwich ELISA

Journal Pre-proof Functional characterization of partial recombinant goat conglutinin: its role as innate immunity marker and use as antigen in sandwich ELISA Sasmita Barik, Mohini Saini, Chandra Mohan S, Ramesh D, Praveen K. Gupta

PII:

S0165-2427(19)30153-9

DOI:

https://doi.org/10.1016/j.vetimm.2019.109987

Reference:

VETIMM 109987

To appear in:

Veterinary Immunology and Immunopathology

Received Date:

30 April 2019

Revised Date:

25 October 2019

Accepted Date:

22 November 2019

Please cite this article as: Barik S, Saini M, Chandra Mohan S, Ramesh D, Gupta PK, Functional characterization of partial recombinant goat conglutinin: its role as innate immunity marker and use as antigen in sandwich ELISA, Veterinary Immunology and Immunopathology (2019), doi: https://doi.org/10.1016/j.vetimm.2019.109987

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Original article Functional characterization of partial recombinant goat conglutinin: its role as innate immunity marker and use as antigen in sandwich ELISA Sasmita Barik1* [email protected], Mohini Saini1, Chandra Mohan S1, Ramesh D2, Praveen K Gupta3 Indian Veterinary Research Institute, Izatnagar 243 122 Uttar Pradesh India

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Division of Biochemistry, Indian Veterinary Research Institute, Izatnagar, India Assistant professor, Department if physiology and biochemistry, Hassan veterinary college, KVAFSU-Bidar 3 Division of Veterinary Biotechnology , Indian Veterinary Research Institute , Izatnagar , India

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*Corresponding author

PhD scholar, IVRI, Izatnagar, Bareilly, UP 243122, India.

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E-mail: Telephone No. +91-8859240683

Text word count- 3570

Abstract word count- 250

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Highlights 

Conglutinin is a soluble pattern recognition receptor of the innate immune system.



Goat makes an immense contribution to world livestock industry.



Improvement of goat health may attribute towards world food security.



Estimation of serum conglutinin level can help to assess immunity status of the animal.



As an immunomodulator, conglutinin can stimulate the phagocytic activity of neutrophils through production of reactive oxygen species.

Abstract Conglutinin, a liver synthesized versatile innate immune marker consisting C-type lectin domain belongs to collectin superfamily of proteins. The protein, first detected in bovine serum as soluble pattern recognition receptor (PRR) has wide range of antimicrobial activities. In the present study, open reading frame (ORF) encoding neck and carbohydrate recognition domain (NCRD) of goat conglutinin gene ligated to the vector pRSET-A was expressed in E. coli BL-21(pLys) cells.

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The 27 kDa recombinant protein (rGCGN) purified by single step Ni+2 -NTA affinity

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chromatography was found to cross-react with recombinant anti-buffalo conglutinin antibody raised in poultry. Further, it displayed calcium-dependant sugar binding activity towards yeast mannan and

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calcium-independent binding activity towards LPS. The mannan binding activity of rGCGN was

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inhibited in the presence of N-acetyl-glucosamine because of higher affinity towards this sugar. The recombinant protein was found to stimulate production of superoxide ions and hydrogen peroxide

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in goat neutrophils, which are instrumental in stimulating phagocytic activity of cells. When used as antigen in Sandwich ELISA, straight line (Y= 0.299x + 0.067, R2= 0.997) was observed within the

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concentration range of 200 to 1000 ng/100μl of rGCGN. Using this equation, the native conglutinin concentration in goat sera was estimated to be 0.5 to 7.5 μg/ml. The results indicated that prokaryotically expressed functionally active rGCGN can be used as antigen to assess native serum conglutinin levels in Sandwich ELISA and as immunomodulator in therapeutic applications to

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sequester unwanted immune complexes from the circulation. Keywords: Conglutinin; Capra hircus; Collectin; N-acetyl-glucosamine; neutrophils, C-type lectin; PRR

1. Introduction

The versatile immune mechanism to protect from infection is basically a congregation of complex responses produced by the body to avoid pathogens invasion and to nullify their virulence properties. The innate immune system is the most ancient mechanism to serve as first line of defense, involving various cells expressing germ line-encoded pattern recognition receptors (PRRs) [1, 2, 3]. The soluble PRRs are meant to recognize a set of evolutionarily conserved stereotypic molecular

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features of pathogens i.e. pathogen associated molecular patterns (PAMPs) [4]. Conglutinin, a liver synthesized [5] high molecular weight serum component belonging to collectin superfamily of

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proteins [6], has an important role in innate immune system as a soluble PRR [7]. It is the first animal lectin to be discovered in animals [8] displaying characteristic C-type lectin domains (CTLD).

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Initially, it was reported to be confined to bovines [9] but, recent reports revealed cross reactivity of

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serum conglutinin in other species towards antiserum raised against native bovine conglutinin [10]. Conglutinin is a dodecameric hepatic lectin with individual polypeptides consisting of a

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globular carbohydrate recognition domain (CRD) attached to collagenous domain via an alphacoiled neck region [11]. Further oligomerization into a triple collagen helix through N-terminal

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cysteine residue mediated disulphide bridges form functionally active protein [12]. It binds to microbial surface saccharides either directly or via complement iC3b through CRD [13] causing agglutination [14]. The affinity of conglutinin towards sugar residues has functional significance in early recognition of pathogens, as the binding strength is comparable to that of antibody-antigen

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complex [15]. The putative role of native conglutinin as opsonin has approved its wide range of antimicrobial activities [16]. It activates the C1q receptors on phagocytic cells by the collagenous domain stimulating the microbicidal activity mediated through production of reactive oxygen species [17].

Although liver was reported to be the prime site to synthesize conglutinin [18], its activity has been detected in the serum, neutrophils, follicular dentritic cells, and macrophages [19]. It reflects that conglutinin has specific role in innate immunity mediated phagocytosis and antigen presentation [20]. Recombinant partial conglutinin consisting of the neck and carbohydrate recognition domain (NCRD) of cattle (Bos Taurus) [21], sheep (Ovis aris) [22], buffalo (Bubalus

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bubalis) [23] and nilgai (Boselaphus tragocamelus) [23] has been expressed in prokaryotic system and is found to display in vitro functional activities similar to that of the native form. The native

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bovine conglutinin has been used in standardizing sandwich ELISA to establish the normal serum level in bovines [21]. However, the recombinant protein encoding NCRD of conglutinin in any of

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the ruminant species has not been explored to be used as antigen in ELISA. Goat (Capra hircus) is

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an economically important small ruminant as 90% of 921 million world population of goats is found in the developing countries including India with a major contribution towards world livestock

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industry [23]. Extensive reports are available on disease aspects and related acquired immunity but research on exploration of innate immune markers especially conglutinin in goats is scanty. The

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gene encoding NCRD of goat conglutinin has recently been characterized in silico in our laboratory [25]. The present study demonstrates that prokaryotically expressed recombinant partial goat conglutinin (rGCGN) retains its structural and functional activities and could be used to investigate

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innate immune status of this species.

2. Materials and methods 2.1. Construction of pRSET expression plasmid encoding partial goat conglutinin ORF

The cloned ORF of conglutinin gene [25] encoding NCR domain in goat was sub-cloned directionally into expression vector pRSET-A (Invitrogen, USA) at Eco RI and Xho I restriction sites and transformed into E. coli (BL21 (DE3) pLyS) competent cells using TSS protocol [26] and then

plated on LB agar containing ampicillin (100μg/ml) and chloramphenicol (100μg/ml).

Plasmids were isolated from overnight grown cultures using QIAGEN Plasmid isolation kit. The plasmids

were

confirmed

by

GGCTCGAGGGGGAGAGTGGGCTTGCAGA-3’)

PCR and

using

forward

primers

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recombinant

reverse

primer

(5’(5'-

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GGGAATTCTCAAAACTCGCAGATCACAA-3’) as established for bovine conglutinin [21, 25]. Confirmed plasmids were characterized by restriction digestion using Pst I, Pvu I and Nhe I enzymes

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(MBI Fermentas, Maryland).

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2.2. Prokaryotic expression and affinity purification of recombinant goat conglutinin (rGCGN) The recombinant colonies were bulk cultured and incubated till attainment of mid-log phase

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with OD600 of nearly 0.6-0.8 followed by induction with 1mM isopropyl β-D-thiogalactoside (IPTG) (Sigma, USA.). Aliquots were collected before and after induction at 1h interval up to 4h to check

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the expression kinetics of recombinant protein in 12 % SDS-PAGE [27]. The recombinant rGCGN protein was purified by urea lysis method using nickel-nitrilo-triacetic acid (NTA) agarose column (Invitrogen ProBondTM Purification System). Briefly, the cultures were centrifuged at 3500 rpm for 25 min and the supernatant was discarded. The pellet was resuspended in 4 volumes of ice cold

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lysis buffer (50mM sodium phosphate, 300mM NaCl, 10mM imidazole and 1 mg/ml Lysozyme) followed by sonication at 40 Hz for 6 cycles with 1 min interval. The sonicated mixture was centrifuged at 4000 rpm for 25 min at 4°C to separate soluble proteins. To the pellet, 4 volumes of urea lysis buffer (8M urea, 0.1M sodium phosphate, 0.01M Tris-Cl, pH-8.0) was added in order to release the proteins from inclusion bodies. Contents were mixed until pellet dissolved totally,

centrifuged at 12000 rpm and supernatants were passed through the columns containing Ni-NTA resin. The columns were washed with excess of urea wash buffer (8M urea, 0.1M sodium phosphate, 0.01M Tris-Cl, pH-6.3). The bound protein was eluted by using elution buffer (8M urea, 0.1M sodium phosphate, 0.01M Tris-Cl, pH-4.5) and collected in fractions of 1ml each. Eluted fractions containing purified recombinant protein were analyzed in SDS-PAGE and pooled together for

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dialysis against decreasing concentration of urea as per protocol [28]. Dialysis membrane (Sigma with molecular weight cut off of 18kDa) was cut with sterile blade and was processed by boiling in

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2% sodium bicarbonate and 0.05% EDTA for 10 min followed by boiling in distilled water and stored in 20% Ethanol and 0.1% sodium azide at 4°C. The membrane was tied at one end, filled with

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appropriate volume of eluted protein fractions and then tied from the other end too. Slow dialysis

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allowed at 4°C with constant stirring at different molarities of urea in PBS viz 6M, 4M, 3M, 2M, 1 M, 0.5M and finally kept in PBS overnight to equilibrate. Finally the protein was aliquoted and

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stored at -80°C. The purity of the protein was checked by 12% SDS-PAGE as described earlier and the proteins of was quantified using Quanti-IT protein assay kit in fluorometer (Invitrogen) .

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2.3. Immuno-reactivity of rGCGN in ELISA and Western blotting Reactivity of dialyzed rGCGN towards recombinant buffalo conglutinin (rBuCGN) antiserum raised in our earlier study [23] was analyzed in indirect ELISA. 4μg of rGCGN in coating buffer (15mM-Na2CO3, 35 mM-NaHCO3, pH 9.6) was added onto 96 well ELISA plate (Nunc

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MaxiSorp, USA) and incubated overnight at 4°C along with PBS as control. Followed by washing thrice with PBS-Tween (PBS, pH 7.2; Tween-20, 0.05%) the unbound sites were blocked with 3% casein in PBS at 37°C for 1h. The plate was then incubated with chicken-anti-recombinant-buffaloconglutinin (rBuCGN) serum as primary antibody at 1:5000 dilution at 37°C for 1h followed by incubation with secondary antibody (rabbit-anti chicken-IgG HRPO conjugate, BioRad, USA) at

1:3000 dilution for 1h at 37°C. Colour was developed using TMB as substrate, the reaction was stopped by 5% H2SO4 and the plate was read at 450 nm in ELISA reader (Awareness Technology, USA). The antigenic specificity of recombinant protein (rGCGN) was checked in Western blotting according to Towbin et al. (1979) [29]. Samples prepared from bacterial lysate, eluted fraction and

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dialysate of recombinant protein along with prestained marker (MBI Fermentas, Germany) were electrophoresed in 12% SDS-PAGE under reducing conditions. Bands were electroblotted (Bio-Rad,

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at 0.8 mA for 2h) onto PVDF membrane (Thermo Scientific) and blocked with 5% skimmed milk powder (SMP). The blot was first incubated with primary antibody (chicken-anti-rBu-CGN serum,

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1:500 dilutions in PBS) and then with rabbit-anti-chicken IgG HRPO conjugates (1:600). 4-

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Chloronapthol (Sigma USA) was used as substrate to visualize the immunoreactions. 2.4. Functional characterization of rGCGN in vitro

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2.4.1. Solid phase saccharide binding assay

Saccharide binding assay was performed as per the protocol described by Lu et al., (1993)

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[5] with minor modifications. Briefly, 96 well microtitre plate (Nunc, U.S.A.) coated with yeast mannan (Sigma, USA) (10μg/well) in coating buffer was incubated overnight at 4°C. Each washing step was done with TBS/NTC or TBS/NT solution (20 mM Tris-HCI, 140mM sodium chloride, 0.05% sodium azide, 0.05% Tween 20, pH 7.4, with or without 5mM calcium chloride) as per

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requirements whereas blocking of non-specific sites was done by 3% BSA in TBS/NTC or TBS/NT solution. Followed by washing, coated wells were incubated for 3h at 37°C with purified goat recombinant conglutinin (rGCGN) in dilutions ranging from 0 to 1000 ng/ml in TBS/NTC or TBS/NT containing either 20mM N-acetyl-D-glucosamine (NAGA) or 10mM EDTA. Chicken-antirBuCGN serum (1: 5,000 dilution) was used as primary antibody whereas rabbit anti-chicken-IgG

HRPO conjugate (1: 6,000 dilution) as secondary antibody. TMB was used as substrate for colorigenic reaction which was stopped by 5% H2SO4 and the absorbance was read at 450nm. 2.4.2. Solid phase LPS binding assay LPS (E. coli LPS: Sigma USA) binding activity of rGCGN was assessed by modified ELISA method of Lu et al., (1993) [5]. Overnight incubated (4°C) LPS (10μg/ml in coating buffer) coated

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microtiter plates were washed with TBS/NTC or TBS/NT solution followed by blocking with 3% BSA in TBS/NTC or TBS/NT solution. Purified goat recombinant conglutinin (rGCGN) at

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concentrations of 0 to 1000 ng/ml in TBS/NTC or TBS/NT containing either 20mM N-acetyl-Dglucosamine (NAGA) or 10mM EDTA was added to the coated wells and plate was incubated for

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3h at 37°C. PBS (pH-7.2) was used as negative control. Primary antibody (chicken-anti-rBuCGN

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serum at 1: 5,000 dilution) followed by secondary antibody (rabbit anti-chicken-IgG HRPO conjugate at 1: 6,000 dilution) was added subsequently and incubated, TMB substrate was added for

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colorigenic reaction, which was stopped by 5% H2SO4 and the absorbance was read at 450nm. 2.4.3. Superoxide ion and hydrogen peroxide production assay

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2.4.3.1. Isolation of Neutrophils

Neutrophills were isolated using hypotonic lysis method [30] from goat blood procured from a local abattoir with (2.7%) EDTA as anticoagulant. To the blood, 0.2% NaCl solution was added to lyse RBC followed by addition of chilled 1.6% NaCl solution immediately to restore the cellular

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activity. The contents were centrifuged at 400 × g for 10 min at 4°C to separate the cells. After the centrifugation contents were washed repeatedly in PBS to remove the lysed RBC. Finally cells were resuspended in Hanks balanced salt solution without phenol red and the cell number was counted using Neubauer’s chamber. The viable cell density was then adjusted to 5× 106 cells/ml. 2.4.3.2. Superoxide anion assay

The assay was performed as per modified Pick et al., (1981, 1986) [31, 32] method reported by Dec et al., (2012) [17]. Briefly, wells were coated with yeast mannan (10μg/100μl) in coating buffer incubated at 4°C overnight. Followed by washing with TBS/NTC the unbound sites were blocked with 3% BSA (Sigma-Aldrich, USA) in TBS/NTC at room temperature for 1h. Individual dilutions (0, 5, 10, 25, 50, 100 and 300μg/ml) of goat recombinant conglutinin (rGCGN) dissolved in TBS/NTC were added to the coated plate and incubated for 3 h at 37°C. Neutrophils were added

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at the rate of 50μl/well (5 × 106 cells/ml) to the both coated and non-coated plates (used as control).

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Followed by incubation at room temperature for 3h, 5μL/well of LPS (2mg/ml), PMA (Sigma) in PBS and opsonized zymosan (Sigma) [33] were added to the non-coated plate as positive controls

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whereas 5μL/well HBSS was added as negative control. Upon addition of Nitroblue tetrazolium

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(NBT; Sigma-Aldrich, USA) solution (2 mg/ml) in phenol red free HBSS, plates were incubated at 37°C in 5% CO2 for 60 min. The optical density of the formazan complex produced in each well

2.4.3.3. Hydrogen peroxide assay

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was measured at 550 nm.

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H2O2 production from neutrophils was measured by phenol red assay as per method of Pick et al., (1981 and 1986) [31, 32]. Overnight incubated (4°C) yeast mannan (10μg/100μl) coated plate was washed with TBS/NTC followed by blocking with 3% BSA (Sigma-Aldrich, USA) in TBS/NTC at room temperature for 1h Purified rGCGN at concentrations of 0, 5, 10, 25, 50, 100 and 300μg/ml

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were added and incubvated for 3 h at 37°C. Followed by the addition of neutrophils (5 × 106 cells/ml) as per the protocol mentioned above with LPS, opsonized zymosan [33] and PMA as positive control and HBSS as negative control. Freshly prepared Phenol red (PR) solution (50μL/well) (phenol red 0.2 g/ml and HRPO 20 U/ml final concentration in HBSS) was added and incubated. The reaction was stopped by 10μl/well 1N NaOH and absorbance was recorded at 620 nm.

2.4.4. Sandwich ELISA Sandwich ELISA was standardized by using the purified recombinant goat conglutinin (rGCGN) as standard antigen to screen various goat serum samples. The ELISA plate was coated with 100μl of capturing antibody (anti-buffalo conglutinin rabbit polyclonal antiserum) diluted (1:600) in coating buffer (pH 9.6) and incubated overnight at 4°C. The plates were then washed 3 times with wash buffer (PBS-T, pH 7.4) in automated ELISA plate washer. Blocking was done with

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300μl of blocking buffer (3% Casein in PBS) followed by incubation at 37°C for 3h. Antigen

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(rGCGN protein) was added at different dilutions in PBS (0,100, 200, 400, 600, 800, 1000, 1200, 1400 and 1600ng final concentration of rGCGN) at the rate of 100μl per well and incubated at 37°C Followed by washing, 100μl of detecting antibody (anti-buffalo conglutinin poultry

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for 1h.

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polyclonal serum) diluted in PBS with 2%BSA (1:1600) was added and incubated at 37°C for 1h. Then anti-poultry-rabbit IgG HRPO conjugate (Bio-Rad) diluted in PBS 2% BSA with (1:5000) was

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added at the rate of 100μl in each well and incubated for 1h at 37°C. TMB was used as substrate for the colorigenic reaction and the reaction was stopped by 50μl of 5% H2SO4 per well. Positive control

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(with Ag and detecting Ab), negative control (without Ag, detecting Ab) and Antigen blank (without Ag) were placed in the plate simultaneously. The absorbance was recorded at 450nm in an ELISA reader. The OD values obtained for the standards were used to plot standard curve. In a similar way, collected goat test sera were diluted at the rate of 1:10 in PBS and added as antigen (100 μl/well) in

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place of rGCGN to the ELISA plate. The OD values obtained from the goat test sera were compared with the standard curve generated with rGCGN as standard antigen to find out serum conglutinin concentration.

2.5.Statistical Analysis

The data were analysed with GraphPad Prism 6 for Windows (GraphPad Software, Inc.) using a one-way ANOVA test with P-values <0.0001 as significant. 3. Results 3.1. Expression and purification of GCGN Restriction digestion of the recombinant plasmid encoding NCRD ORF by Pvu I, Nhe I or

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Pst I generated two fragments each (Fig. 1). Digestion with Pvu I, Nhe I and Pst I yielded fragments of size 1.039 kb and 2.3 kb (Lane1), 289 bp and 3.072 kb (Lane2) and 233 bp and 3.1 kb (Lane3)

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respectively. The results obtained were in accordance with those expected in sequence analysis. An evaluation of the expression kinetics shown by uninduced and IPTG-induced bacterial

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cell pellets during 1-4 h interval in 12% SDS-PAGE suggested the maximum expression of 27 kDa

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recombinant protein (rGCGN) at 4 h of induction (Fig. 2A). The pellets obtained from bulk culture were lysed by urea lysis method and the expressed recombinant conglutinin (rGCGN) was purified

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by Ni-NTA affinity column chromatography using urea elution buffer. The eluted fractions were were resolved in 12% SDSPAGE that finally depicted the purified 27kDa recombinant goat

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conglutinin in coomassie blue staining (Fig: 2B). This 27 kDa protein was purified from inclusion bodies under denaturating conditions and then renatured by extensive slow dialysis against

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decreasing molarity of urea and finally PBS. The refolded rGCGN was quantified to be 530µg/ml.

3.2. Characterization of purified rGCGN Cross reactivity of renatured rGCGN towards chicken anti-rBuCGN-serum as primary

antibody was confirmed in ELISA (Fig. 3A) as the absorbance of test antigen (OD450-0.63) was 82%

higher than that of PBS control (OD450-0.07). Likewise, rGCGN in crude (bacterial lysate), purified as well as renatured form revealed cross reactivity to anti-rBuCGN-serum in western blot (Fig. 3B). 3.3. Assessment of biological activities of renatured rGCGN Prokaryotically expressed rGCGN was found to display affinity towards mannan in the presence of Ca+2 in concentration dependent manner and maximum binding activity was observed at

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a concentration of 1000ng/ml (Fig. 4A). However, in the absence of Ca+2, 90% inhibition in this binding activity was observed. Similarly, 80% inhibition was observed in presence of 10mM EDTA

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whereas the extent of inhibition in the presence of 20mM NAGA was up to 72%.

Further characterization in solid phase LPS binding assay revealed that binding of the

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rGCGN (1000ng/ml) to E. coli derived LPS was calcium-independent (Fig. 4B). Absence of Ca2+

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was found to increase the binding activity to 59% as compared to that in the presence of Ca2+. Inclusion of 10mM EDTA in the assay was sufficient enough to chelate Ca2+ thus favouring LPS

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interaction. No apparent activity was observed in wells with NAGA in the presence of calcium. Incubation of goat neutrophils with mannan bound rGCGN at various concentrations

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displayed significant stimulatory effect in generating superoxide radicals only at a concentration as high as 300μg/ml as compared to unstimulated control (Fig. 5A) which was comparable to that produced by LPS, Zymosan and PMA. Similarly the mannan bound rGCGN at 300μg/ml concentration (Fig. 5B) was found to be an effective in producing peroxide radicals in goat

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neutrophils in vitro.

3.4. Standardization of sandwich ELISA Development and standardization of sandwich ELISA using the purified rGCGN as standard

antigen, rabbit anti-rBuCGN serum as capture antibody and poultry anti-rBuCGN serum as detecting antibody led to generation of standard curve (Fig. 6A). Linear regression equation

(Y=0.299x+0.0.067, R2=0.997) was derived within the concentration range of 200 to 1000ng/100μl (Fig. 6B). Out of 131 goat sera, the concentration of conglutinin in the positive sera assessed using the straight line regression equation was found to be in 0.5 to 5.5μg/ml range. 4. Discussion Infectious diseases are the leading cause of morbidity and mortality worldwide in livestock

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sector and are a major challenge for the biomedical sciences. Exploration and functional characterization of innate immune sentinels could direct towards early recognition of invading

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pathogens, thus stimulating the adaptive arm of defense system.

In the present study, partial ORF encoding goat conglutinin NCRD amplified from liver was

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successfully cloned in prokaryotic expression vector. The restriction digestion patterns of

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recombinant plasmid pRSET-GCGN with Pst I, Pvu I and Nhe I was identical with those of cattle [21] and sheep [34] indicating conservation of sequences. Whereas, the pattern differ from those

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obtained in case of buffalo and nilgai [23], indicating possible divergence from wild ruminants. The expressed 27 kDa recombinant goat conglutinin (rGCGN) was higher than 17 kDa predicted from

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ORF due to N-terminal histidine tag and in frame enterokinase amino acids [35]. In spite of lacking the N-terminal cysteine rich and collagenous domain, the purified and extensively renatured rGCGN was found to display in vitro antimicrobial behavior mediated through affinity binding to microbial cell surface glycan residues [14] identical to the native bovine

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conglutinin [16]. Binding affinity for mannan in presence of calcium and inhibition in presence of the calcium chelating agent (EDTA) approved its lectin like properties [36, 37]. The observation is in accordance with the earlier reports [21, 22, and 23] for bovine, sheep, buffalo and nilgai conglutinin respectively. The shift in binding of rGCGN from mannan towards NAGA, corroborated with the existing report that the saccharide binding activity of the native bovine conglutinin is in the

order N-acetyl-Dglucosamine >>>> D-mannose> L-fucose > glucose > maltose D-glucosamine > galactosamine > D-galactose > D-fucose [38]. Observed affinity of rGCGN towards LPS in calcium independent manner adds to the fidelity of in vitro folding, orientation and proper exposure of ligand and calcium binding domain as well. Previously, Ca+2 independent binding activity of surfactant protein A (SP-A) to lipid-A of bacterial LPS has been reported [39]. However, a recent finding suggested Ca+2 dependent binding of

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surfactant protein-D (SP-D) to Helicobacter pylori LPS [40] that need to be explored further. Β-

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linked NAGA and β-linked D-Glucose of core polysaccharide in SP-D is found to be involved in the binding with LPS [41]. Further, presence of EDTA in the solution chelating inhibitory calcium ions,

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favouring protein and LPS interaction was in corroboration with the observation of Wakamiya

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(2005) [21], Chandra Mohan (2014) [22] and Ramesh [23].

Conglutinin along with other collectins act as secretory PRR in opsonising and destroying of

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microorganisms by stimulating phagocytic cells [42]. The antimicrobial activity of conglutinin is primarily based on its binding to microbial surface glycan residues and to the complement, the

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degradation product iC3b deposited on microbes [43]. The complement dependant in vitro and in vivo anti-bacterial activity was observed to be mediated by respiratory burst mechanism of phagocytes [44, 45]. LPS, zymosan, PMA were established potent stimulator of ROI production [46] activating membrane-bound enzyme NAD (P) H-oxidase producing superoxide (O2-) and hydrogen

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peroxide (H2O2) reactive products that are toxic to pathogens [47]. It was reported that mannanbound native bovine conglutinin stimulated the production of superoxide and peroxide radicals by PMNs [17]. Among other lectins, human MBP had displayed superoxide production from human polymorphonuclear leukocytes when coated with mannan [48]. Similarly, binding of pulmonary surfactant proteins A and D to Aspergillus fumigatus conidia enhanced phagocytosis and killing by

human neutrophils and alveolar macrophages [49]. The present study revealed the biological role of mannan bound purified rGCGN in production of ROS in vitro for the first time. However, its role in eliminating infection with various pathogens needs to be experimentally proved. Positive cross reactivity towards anti-rBuCGN-serum in indirect ELISA, sandwich ELISA and western blotting confirms its identity and the exposure of epitopic domain giving space towards

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antigen-antibody reaction. Similar findings also exists reflecting presence of conglutinin reactivity in various other bovid and non-bovid species in western blotting using anti-bovine conglutinin serum

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[51, 10]. Reactivity of sheep serum to anti-native bovine conglutinin serum and anti-rBuCGN-serum has also been observed earlier [52, 22]. However, the potency of binding in this cross species

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antigen-antibody interaction is still unknown. Standardization of sandwich ELISA for the

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assessment of serum level of native conglutinin is first attempt in case of goat (Capra hircus). Observed narrow range of concentration (0.5- 7.5 μg/ml) with mean value of 2.6 μg/ml in case of

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positive goats as compared to cattle may be attributed towards the use of cross species antisera as primary and secondary antibody affecting the sensitivity of the reaction. Previously, native bovine

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conglutinin was employed for the detection of cattle serum level of native conglutinin which was found in the range 1.25-35 µg/ml [53, 54]. Lower plasma conglutinin level in cattle is reported to be positively correlated with occurrence of respiratory deseases [7]. Also, existence of positive correlation between native serum

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conglutinin level and disease resistance status of animal [54] has been used for selection process employed for various cattle breeding programmes. Truncated recombinant bovine conglutinin (rfBC) was reported to be potent in two way bacteriostatic effect against M. bovis BCG in culture [55]. Collectins including conglutinin in goats was found to be involved in the molecular immune response to abomassal helminthes [56]. Use of various plant lectins as diagnostic, prophylactic and

diseases treatment purpose has been reported [57]. Other lectin group of proteins including SP-A, SP-D, MBL has been employed as biomarker in assessing the infection status of the animal. Use of MBL as an immune modulatory molecule has been established in poultry sector [58]. In this context, diminishing the significant gaps in understanding many aspects of the area of research could establish goat conglutinin as a prominent immune marker and also as an

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immunomodulator in assessment of the health status for selective breeding of the species for further use.

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5. Conclusions

In the present study recombinant goat conglutinin (rGCGN) encoding the NCR domain was

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successfully expressed prokaryotically and the expressed rGCGN was found functionally active

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through various in vitro functional assays. Protein was also able to stimulate microbicidal activity of neutrophils through generation of various reactive oxygen radicals. The protein was also found

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suitable as antigen to generate standard curve in sandwich ELISA to quantitate native serum conglutinin concentration. The standard curve generated must be characterized further by screening

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many serum samples to validate the data. Since the serum conglutinin concentration is hereditary, ELISA can be used further to select parents with good innate immunity as breeding stock for better herd.

Conflict of interest statement

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The authors have declared no conflict of interest.

Acknowledgements

The authors are grateful to the Director, Joint Director (Academics) and Joint Director

(Research), Indian Veterinary Research Institute, Izatnagar for providing necessary facilities to

conduct the present study. Financial support from ICAR to the first author as ICAR-JRF is also

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acknowledged.

Reference 1. Beutler B, Jiang Z, Georgel P, et al. (2006) Genetic analysis of host resistance: Toll-like receptor signaling and immunity at large. Annual Review of Immunology 24:353–358. 2. Medzhitov R. (2007) Recognition of microorganisms and activation of the immune response. Nature 449, 819–826. 3. Kawai T, Akira S. (2008) Toll-like receptor and RIG-I-like receptor signaling. Annals of the New York Academy of Sciences 1143, 1–20. 4. Janeway CA. (1989) Approaching the asymptote? Evolution and revolution in immunology.

of

Cold Spring Harb. Symp. Quantitative Biology 54, 1–13.

5. Lu J, Laursen SB, Thiel S, Jensenius JC, Reid KB. (1993) The cDNA cloning of conglutinin and

ro

identification of liver as a primary site of synthesis of conglutinin in members of the Bovidae. Biochem. J. 292, 157-162.

-p

6. van de Wetering JK, van Golde LM, Batenburg JJ. (2004) Collectins, Players of the innate immune system. European Journal of Biochemistry 271, 1229–1249.

re

7. Holmskov, U., Jensenius, J.C., Tornoe, I., Lovendahl, P. 1998. The plasma levels of conglutinin are heritable in cattle and low levels predispose to infection. Immunology 93, 431-436.

lP

8. Ehrlich, P., Sachs, H. 1902. Ueber den Mechanismus der Amboceptorenwirkung. Berliner Klinische Wochenschrift 39, 492-496

9. Bordet, J., Gay, F.P. 1906. Sur les relations des sensibili¬satrices avec l’alexine. Annales de

ur na

L’Institut Pasteur 20, 467–473.

10. Dec, M., Wernicki, A., Puchalski, A., Urban-Chmiel, R. 2011. Conglutinin is not specific to cattle. Veterinarni Medicina 56, 510–519. 11. Strang, J., Slayter, S., Lachmann, J., Davis, E., 1986. Ultrastructure and composition of bovine conglutinin. Biochemical Journal 234, 381-389.

Jo

12. Suzuki, Y., Eda, S., Kawai, T., Ohtani, K., Kase, T., Sakamoto, T., & Wakamiya, N. (1997)

Characterization of recom-binant bovine conglutinin expressed in a mammalian cell. Biochem. Biophys. Res. Commun. 238, 856-860.

13. Hirani, S., Lambris, J.D., Mu¨ller-Eberhard, H.J. 1985. Localization of the conglutinin binding site on the third component of human complement. Journal of Immunology 134, 1105-1109. 14. Lachmann, P.J. 1967. Conglutinin and immunoconglutinins. Advances in Immunology 6, 480527.

15. Holmskov, U., Laursen, S.B., Malhotra, R., Wiedemann, H., Timpl, R., Stuart, G.R., Tornoe, I., Madsen, P.S., Reid, K.B., Jensenius, J.C., 1995. Comparative study of the structural and functional properties of bovine plasma C-type lectin, collectin-43, with other collectins. Biochemical Journal 305, 889–896. 16. Lim, B.L., Lu, J., Reid, K.B.M., 1993. Structural similarity between bovine conglutinin and bovine lung surfactant protein D and demonstration of liver as a site of synthesis of conglutinin. Immunology 78, 159-165.

of

17. Dec, M., Wernicki, A., Puchalski, A., Urban-Chmiel, R., Waśko, A., 2012. The effect of conglutinin on production of reactive oxygen species in bovine granulocytes. Journal of Veterinary Science 13, 33-38.

ro

18. Jinhua, L.U., Steen, B., Theil, S., Jensenius, J.C., Reid, K.B.M., 1993. The cDNA cloning of conglutinin and identification of liver as a primary site of synthesis of conglutinin in members

-p

of the Bovidae. Biochemical Journal 292, 157-162.

19. Hansen, S., Holm, D., Moeller, V., Vitved, L., Bendixen, C., Reid, K.B.M., Skjoedt, K.

Journal of Immunology 169, 5726–5734.

re

Holmskov, U., 2002. CL-46, a novel collectin highly expressed in bovine thymus and liver.

lP

20. Holmskov, U., Teisner, B., Pedersen, N. T., Laursen, S. B., Rasmussen , H. B. and Jensenius, J. C. 1992. Tissue localization of conglutinin, a bovine C-type lectin. Immunology 76,169±17. 21. Wakamiya, N., 2005. Recombinant conglutinin and producing method thereof (US patent

ur na

6979727 B2).

22. Chandra Mohan, S., Saini, M., Ramesh, D., Shynu, M., Barik, S., Das, A., Sharma, A.K., Chaturvedi, V.K., Gupta, P.K. 2014. Prokaryotic expression of Ovis aries conglutinin encoding neck and carbohydrate recognition domain and its functional characterization. Animal Biotechnology 26(1), 29-36.

Jo

23. Ramesh, D., Chandra Mohan, S., Saini, M., Barik, S., Shynu, M., Das, A., Sharma, A. K.,. Chaturvedi, V.K. and Gupta, P.K. 2018. Recombinant partial conglutinin of Buffalo and Nilgai in vitro can mimic the functions of native conglutinin in vivo. Proc. Natl. Acad. Sci., India, Sect. B Biol. https://doi.org/10.1007/s40011-018-0979-8. 24. FAOSTAT (2012): http://faostat.fao.org/default.aspx 25. Barik, S., Chandra Mohan, S., Saini, M., Ramesh, D., Das, A., Sharma, A.K., Gupta, P.K. 2014. Sequence-based appraisal of the genes encoding conglutinin neck and carbohydrate recognition

domain in blackbuck (Antilope cervicapra) and goat (Capra hircus). BioMed Research International. Hindawi Publishing Corporation. Volume 2014, Article ID 389150, 10 pages. 26. Chung, C.T., Niamela, S.L., Miller, R.H., 1989. One step preparation of competent Escherichia coli transformation and storage of bacterial cells in the same solution. Proceedings of the National Academy of Sciences USA 86, 2172-2175. 27. Laemmli, U.K., 1970. Cleavage of structural protein during the assembly of the head of bacteriophage T4. Nature 227, 680-695.

of

28. Sambrook, J., Russell, D.W., 2010. Molecular Cloning: A Laboratory Manual, Third Ed. Cold Spring Harbor Press, Cold Spring Harbor, New York.

29. Towbin, H., Staehelin, T., Gordon, J., 1979. Electrophoretic transfer of proteins from

the National Academy of Sciences USA 76, 4350-4354.

ro

polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proceedings of

-p

30. Matthew J. Cotter, Keith E. Norman,Paul G. Hellewell, and Victoria C. Ridger. 2001. A Novel Method for Isolation of Neutrophils from Murine Blood Using Negative Immunomagnetic

re

Separation. American Journal of Pathology 159(2): 473–481.

31. Pick, E. & Mizel, D. 1981. Rapid microassays for measurement of superoxide and hydrogen-

lP

peroxide production by macrophages in culture using an automatic enzyme-immunoassay reader. Journal of immunological Methods 46,211-226. 32. Pick, E., 1986. Microassays for superoxide and hydrogen peroxide production and nitroblue

ur na

tetrazolium reduction using an enzyme immunoassay microplate reader. Methods in Enzymology 132, 407-421.

33. Pugine, S. M. P.; Faria, M. F.; Maia, A. A. M.; Valle, C. R.; Boschini, C.; Poleti, M. D.; Silva, M. R. M. & de Melo, M. P., 2005. Effect of Cysticercus cellulosae fractions on the respiratory burst of pig neutrophils. Revista do Instituto de Medicina Tropical de Sao Paulo 47(2), 91-94.

Jo

34. Chandra Mohan S, Ramesh, D., Shynu, M., Saini, M., Das, A., Sharma, A.K., Gupta, P.K. 2013. Molecularcharacterization and in-silico analysis of the cDNA encoding conglutinin from sheep liver. Indian Journal of Animal Sciences 83, 600-603.

35. Gupta, K., Bind, R.B., Walunj, S.S., Saini, M. 2005. Cloning, characterization and expression of bovine (Bos indicus) tumour necrosis factor-α. Indian Journal of Biotechnology 4, 363-366. 36. Drickamer, K. 1994. Binding of sugar ligands to Ca2+-dependent animal lectins-I. Analysis

of mannose binding by site-directed mutagenesis and NMR. The Journal of Biological Chemistry 269, 15505– 15511. 37. Hoppe, H.J., Barlow, P.N. and Reid, K.B.M. 1994. A parallel three stranded a-helical bundle at the nucleation site of collagen triple-helix formation. FEBS Letters 344, 191-195. 38. Holmskov, U., Fischer P.B., Rothmann A., Hojrup P. (1996). Affinity and kinetic analysis of the bovine plasma C-type lectin collectin-43 (CL-43) interacting with mannan. FEBS Letters 393, 314-316. 39. Kalina, M., Blau, H., Riklis, S., Kravtsov, V. 1995. Interaction of surfactant protein A with

of

bacterial lipopolysaccharide may affect some biological functions. American Journal of Physiology 268, 144-51.

40. Yokota, S., Amano, K., Nishitani, C., Ariki, S., Kuroki, Y., Fujiia, N. 2012. Implication of

ro

antigenic conversion of Helicobacter pylori lipopolysaccharides that involve interaction with Surfactant Protein D. Infection and Immunity 80, 2956-2962

-p

41. Kuan, S., Rust, K., Crouch, E. 1992. Interactions of surfactant protein D with bacterial lipopolysaccahrides. Surfactant protein D is an Escherichia coli-binding protein in

re

bronchoalveolar lavage. Journal of Clinical Investigation 90, 97-106. 42. Van de Wetering J. K., van Golde L. M. G., Batenburg J. J. 2004. Collectins: players of the

lP

innate immune system. European Journal of Biochemistry 271, 1229-1249. 43. Dec, M., Wernicki, A. 2006. Conglutinin, CL-43 and CL-46—three bovine collectins. Polish Journal of Veterinary Sciences 9, 265-275.

ur na

44. Friis-Christiansen, P., Thiel, S., Svehag, S.E., Dessau, R., Svendsen, P., Andersen, O., Laursen, S.B., Jensenius, J.C. 1990. In vivo and in vitro antibacterial activity of conglutinin, a mammalian plasma lectin. Scandinavian Journal of Immunology 31, 453-460. 45. Friis, P., Svehag, S.E., Andersen, O., Gahrn-Hansen, B., Leslie, R.G.Q. 1991. Conglutinin exhibits a complement-dependent enhancement of the respiratory burst of phagocytes

Jo

stimulated by E. coli. Immunology 74, 680-684.

46. Mehrzad, J., Dosogne, H., Vangroenweghe, F., Burvenich, C. 2001. A comparative study of bovine blood and milk neutrophil functions with luminol-dependent chemiluminescence. Luminescence 16, 343-356. 47. Muñoz, M., Cedeño, R., Rodríguez, J., Van-der-Knaap, W.P.W., Mialhe, E., Bachère, E. 2000. Measurement of reactive oxygen intermediate production in haemocytes of the penaeid shrimp, Penaeus vannamei. Aquaculture 191, 89-107.

48. Uemura, K., Yamamoto, H., Nakagawa, T., Nakamura, K., Kawasaki, N., Oka, S., Ma, B.Y., Kawasaki, T. 2004. Superoxide production from human polymorphonuclear leukocytes by human mannan-binding protein (MBP). Glycoconjugate Journal 21, 79-84. 49. Madan, T., Eggleton, P., Kishore, U., Strong, P., Aggrawal, S.S., Sarma, P.U., Reid, K.B.M. 1997. Binding of pulmonary surfactant proteins A and D to Aspergillus fumigatus conidia enhances phagocytosis and killing by human neutrophils and alveolar macrophages. Infection and Immunity 65, 3171-3179.

of

50. Holmskov, U., Thiel, S., Jensenius, J.C., 2003. Collectins and ficolins: humoral lectins of the innate immune defense. Annual Review of Immunology 21, 47–578.

51. Kania, P.W., Sorensen, R.R., Koch, C., Brandt, J., Kliem, A., Vitved, L., Hansen, S., Skjodt, K.

ro

2010. Evolutionary conservation of mannan-binding lectin (MBL) in bony fish: identification, characterization and expression analysis of three bona fide collectin

-p

homologues of MBL in the rainbow trout (Onchorhynchus mykiss). Fish and Shellfish Immunology 29, 910–920.

animal sera. Immunology 25, 237-240.

re

52. Mittal, K.R. and Jaiswal, T.N. 1973. Conglutinating activity in apparently healthy human and

lP

53. Akiyama, K., Sugii, S., Hirota, Y., 1992. Development of enzyme-linked immunosorbent assays for conglutinin, mannan-binding protein, and serum myloid-P component in bovine sera. American Journal of Veterinary Research 53, 2102-2104.

ur na

54. Holmskov, U., Jensenius, J.C., Tornoe, I., Lovendahl, P. 1998. The plasma levels of conglutinin are heritable in cattle and low levels predispose to infection. Immunlogy 93, 431-436. 55. Mehmood, A., Kouser, L., Kaur, A., Holmskov, U., Al-Ahdal, M.N., Sim, R.B., Kishore, U. and Tsolaki, A.G. 2018. Complement dependent and independent interaction between bovine conglutinin and Mycobacterium bovis BCG: Implications in bovine tuberculosis. Front.

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Immunol. 9:3159. doi: 10.3389/fimmu.2018.03159.

56. Fraser, R.S., Lumsden, J.S. and Lillie, B.N. 2018. Identification of polymorphisms in the bovine collagenous lectins and their association with infectious diseases in cattle. Immunogenetics. 70:533–546. 57. Majee, S.B. and Biswas, G.R. 2013. Exploring plant lectins in diagnosis, prophylaxis and therapy. Journal of Medicinal Plants Research 7(47), 3444-3451.

58. Laursen, S.B. and Nielsen, O.L. 2000. Mannan-binding lectin (MBL) in chickens: molecular and functional aspects. Developmental and Comparative Immunology 24,

Jo

ur na

lP

re

-p

ro

of

85-101.

Legends to Figures Figure 1: Characterization of goat recombinant plasmid (pRSET-GCGN) using restriction enzymes Pvu I, Nhe I and Pst I. (M(left): λ-DNA Eco RI+Hind III digested marker; M(right): 100 bp DNA ladder; 1: Pvu I digestion yielding 1.039 bp and 2.3 kb; Lane

Figure 2:

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and 3.1 kb fragments)

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2: Nhe I digestion yielding 289 bp and 3.072 kb; 3: Pst I digestion yielding 233 bp

Expression kinetics of recombinant goat conglutinin (rGCGN) upon 1 mM IPTG

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induction in E.coli BL21 cells as analyzed in 12 % SDS-PAGE. (M: Prestained protein marker (Fermentas); 1: Un-induced cell lysate; 2: 1 h induced cell lysate; 3: 2

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h induced cell lysate; 4: 3 h induced cell lysate; 5: 4 h induced cell lysate)

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Figure 3:

(A) Reactivity of recombinant goat conglutinin in Direct ELISA with poultry antirecombinant buffalo conglutinin polyclonal serum. (rGCGN: Mean absorbance of 4 wells coated with recombinant goat conglutinin in PBS at the concentration of 4 μg/ml. Control: Mean absorbance of 4 wells coated with PBS alone) (B) Western blot analysis of purified recombinant goat conglutinin using poultry anti-recombinant buffalo conglutinin polyclonal serum. (1: cell lysate prior loading to the Ni2+-NTA agarose affinity column; 2: fraction collected by washing the column with wash weight

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buffer. M: Prestained protein molecular

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#SM1811);

3:

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(Fermentas,

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recombinant protein renatured by dialysis.)

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27 kDa

(A)

(B)

Figure 4:

(A) Sugar binding activity of rGCGN at different concentrations against coated yeast mannan using ELISA method. Activity as mean absorbance of quadruplicate wells coated with yeast mannan at 0.1 to 1000 ng/well protein concentration. (Binding assay was performed in the presence of Calcium ions, 10 mM EDTA and 20 mM Nacetyl D-glucosamine.) The data were expressed as mean of three independent

the unpaired one-way ANOVA test (***p<0.0001) (n=4).

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experiments carried out in tripquadruplates±SEM. Significanc was de termined using

(B) LPS binding activity of rGCGN at different concentrations against coated

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yeast mannan using ELISA method. Activity as mean absorbance of quadruplicate wells coated with yeast mannan at 0.1 to 1000 ng/well

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protein concentration. (Binding assay was performed in the presence of Calcium ions, 10 mM EDTA and 20 mM N-acetyl D-glucosamine.) The

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data were expressed as mean of three independent experiments carried out in tripquadruplates±SEM. Significanc was de termined using the

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(C)

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unpaired one-way ANOVA test (***p<0.0001) (n=4).

(D)

(E) (F)

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(G)

(I)

Figure 5:

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(H)

(A) The effect of rGCGN on superoxide radical production by goat neutophils as measured by an NBT assay. 300µg/ml of rGCGN was added to a cell suspension in mannan plated wells. Control wells received HBSS and positive control wells

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contained PMA, Zymosan, LPS. The results are presented as mean ± SD for fourindependent experiments, each done in triplicate. * * *p < 0.0001 vs. the control. (B) The effect of rGCGN on peroxide radical production by Goat neutophils as measured by PR assay. 300µg/ml of rGCGN was added to a cell suspension in mannan plated wells. Control wells received HBSS and positive control wells contained PMA, Zymosan, LPS. The results are presented as mean ± SD for fourindependent experiments, each done in triplicate. * * *p < 0.0001 vs. the control.

(B)

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Figure 6:

(A) Standardization of sandwich ELISA using rGCGN as antigen. Standard curve was generated with rGCGN as antigen at different concentrations from 0ng/100µl to 1600ng/100µl. (B) Straght line obtained in the range of 200ng/100µl to 1000ng/100µl was analysed to generate equation which can be used to quantify native serum conglutinin concentration. (C) Graphical representation of estimated conglutinin

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concentration in goat serum samples.

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(B)