Cloning and Prokaryotic Expression of Rat RVLG and Preparation of Mouse Anti-RVLG Polyclonal Antibody

CHINESE JOURNAL OF BIOTECHNOLOGY Volume 24, Issue 11, November 2008 Online English edition of the Chinese language journal RESEARCH PAPER

Cite this article as: Chin J Biotech, 2008, 24(11), 1981í1987.

Cloning and Prokaryotic Expression of Rat RVLG and Preparation of Mouse Anti-RVLG Polyclonal Antibody Ping Zhang, Wanjin Xing, Xiaohong Bao, Zhida Liu, Lianqing Wang, Shunyao Li, and Riga Wu Department of Biology, School of Life Sciences, Inner Mongolia University, Hohhot 010021, China

Abstract:

In order to identify rat ovarian germ cells, the authors expressed and purified rat RVLG protein in E. coli cells and

prepared a mouse anti-rat RVLG polyclonal antibody. The rat RVLG cDNA was obtained from rat testicle tissue by RT-PCR and was cloned into the vector pMD19-T. Sequence analysis proves that the cloned RVLG cDNA fragment was 60 bp longer than that released in the GenBank (NM_001077647), resulting from an alternative splicing of the RVLG pre-mRNA. The RVLG cDNA was doubly digested with the restriction endonucleases BamHĉand EcoRĉ, and then was extracted from gel and inserted into the prokaryotic expression vector pGEX-4T-1. The recombinant expression plasmid pGEX-RVLG was verified for successful construction and then was transformed into E. coli BL21(DE3) for induction to express the GST-RVLG fusion protein by IPTG. The GST-RVLG fusion protein was expressed in E. coli BL21 (DE3) at a high level, which accounts for more than 10% of the total bacterial cellular protein. The purified RVLG protein was used as an antigen to immunize KM mouse for the production of polyclonal antibody in ascitic fluid followed by celiacly injecting the mouse with S180 cells. The mouse anti-rat RVLG antibody was analyzed by ELISA, Western blotting, and Immunohistochemistry for its specificity and titer. The antibody could recognize RVLG protein specifically and its titer is about 1:20 000. These results confirm that the mouse anti-rat RVLG polyclonal antibody with high affinity and specificity has been prepared successfully, and lays a foundation for the authors’ ongoing research on the specific expression of RVLG in rat ovary. Keywords: RVLG; prokaryotic expression; polyclonal antibody

Introduction DEAD(Asp-Glu-Ala-Asp)-box protein family plays a central role in intracellular RNA metabolism involved in the transcriptional regulation, pre-mRNA splicing, ribosomal assembly, RNA transportation, regulation of mRNA translation initiation, RNA degradation, and gene expression in organelles [1]. Vasa is an member of DEAD-box gene family that was first discovered in Drosophila [2], and its orthologs have been identified in Xenopus, mouse, hydra, silkworm, grasshopper, oyster,

chicken, human, swamp eel, goldfish, silver crucian carp, zebrafish, tilapia, as well as rat, in which it is named RVLG (rat vasa-like gene) [3]. Vasa is a germline-specific gene and is vital for gametogenesis. Vasa protein is an ATP-dependent RNA helicase [4], absence of which has been reported as resulting in oogenesis interruption in Drosophila and nematode [5,6], and spermatogenesis inhibition in mice [7,8]. In view of its tissue-specific expression and essential for gametogenesis, vasa has been regarded as a marker gene for germ cells. A long accepted dogma in the reproductive biology is that, unlike males

Received: May 20, 2008; Accepted: September 2, 2008. Corresponding author: Wanjin Xing. Tel: +86-471-4992944; E-mail: [email protected] Supported by: Higher Education Research Program of Inner Mongolia (No. NJ05026). Copyright © 2008, Institute of Microbiology, Chinese Academy of Sciences and Chinese Society for Microbiology. Published by Elsevier BV. All rights reserved.

Ping Zhang et al. / Chinese Journal of Biotechnology, 2008, 24(11): 1981–1987

who retain germline stem cells (GSCs) throughout their adult lives, oocyte production in females of most mammalian species ceases before birth. But recently, the debate about whether postnatal mammalian ovary contains germ stem cells (GSCs) and continues neo-oogenesis during adult life was reignited by Johnson et al [9] in 2004, and entangled many other researchers [10–12]. In order to verify the disputed issues, the authors cloned the RVLG cDNA, expressed RVLG protein in prokaryotic cells, and then prepared mouse anti-RVLG polyclonal antibody for identification of germ cells in adult rat ovary.

1

Materials and methods

1.1 Materials Bacterial strains, plasmids, and animals: E. coli DH5D cells and the pGEX-4T-1 plasmid were preserved in the authors’ Lab. E. coli BL21 (DE3) cells were a gift from Doctor Hou Xin. S-180 cells were kindly provided by Professor Wu Yingji of the Key Laboratory for Mammalian Reproductive Biology and Biotechnology, Ministry of Education, PR. China. Wistar rats and KM (Kunming) mice were purchased from the Experimental Animal Center of Inner Mongolia University. 1.2 Methods Primers and RVLG gene cloning: 1) Primers: Upstream primer:5c-GGATCCATGGGAGATGAAGATTGGGAG-3c with BamH I site(underlined) at 5c terminus, and downstream primers:5c-GAATTCCTTTCCAGAGCCTGT TACTAC-3c with EcoR I site (underlined) at 5c terminus, were designed according to the RVLG cDNA sequence (GenBank Accession No. NM_001077647). The expected amplified fragment is 639 bp in length. 2) RT-PCR amplifications: The total RNA from testicular tissue of Wista rats (53 days) was isolated by the TaKaRa RNAiso Reagent (TaKaRa Biotechnology, Dalian) according to the manufacturer’s instructions and converted into singlestranded cDNA by RNA PCR Kit (AMV) (TaKaRa Biotechnology, Dalian) according to the manufacturer’s instructions. PCR was carried out using the rTaq DNA polymerase (TaKaRa Biotechnology, Dalian) at a thermal cycle ramping consisting of 35 denaturing cycles of denaturation at 94°C for 1 min, annealing at 57°C for 30 s and extension at 72°C for 1 min with a final extension at 72°C for 10 min. Construction of recombinant cloning vector and Prokaryotic expression vector: The amplified PCR products were purified using the AxyPrep DNA Gel Extraction Kit (Axygen, USA) and ligated with the pMD19-T vector (TaKaRa Biotechnology, Dalian). Three positive recombinant cloning vectors, pMD-RVLG, were screened by PCR, restriction enzyme digestion, and gene sequencing. The correct pMD-RVLG and the prokaryotic expression

vector pGEX-4T-1, which has a GST tag for purification of GST fusion protein, were digested with restriction enzyme EcoR I and BamH I, and the RVLG DNA fragment was isolated and ligated with linearized pGEX-4T-1 vector for constructing the recombinant expression vector pGEX-RVLG. The pGEX- RVLG was verified by sequencing for correct open reading frame and was transformed into the competent E. coli BL21(DE3) for expression of GST-RVLG fusion protein. Expression of the GST-RVLG fusion protein and SDS-PAGE analysis: Fresh overnight culture of transformant E. coli BL21(DE3) was diluted in LuriaBertani (LB) medium supplemented with 100 ȝg/mL ampicillin (Sigma, USA) and grown at 37oC at a stirring speed of 250 r/min. When cell density reached 0.4/0.6 of OD600, 100 Pg/mL IPTG was added for 1–5 h to induce expression of GST-RVLG fusion protein. After induction, the cells were harvested by centrifugation and then disrupted by sonication in 2×sample buffer (0.1 mol/L TrisHCl (pH 6.8), 10% E-2-mercaptoethanol, 4% SDS, 20% glycerol, and 0.1% bromophenol blue) on ice, boiled for 5 min at 100°C. Then the cell debris was removed by centrifugation, the supernatants were analyzed by 12% SDS-PAGE and UVI Band software. Identification of GST-RVLG fusion protein: The protein bands in the SDS polyacrylamide gel were first transferred electrophoretically onto a PVDF (polyvinylidene difluoride, Solarbio) membrane. After blocking in 5% fat-free milk in TBST (TBS-0.05% Tween-20) for 1 h at 37oC, the membrane was incubated in the anti-GST monoclonal antibody (TIANGEN, Beijing) at a dilution of 1:500 overnight at 4oC, and was washed thrice with TBST, 10 min each time. Then the membrane was treated with horseradish peroxidase- conjugated goat anti-mouse IgG (Bios, Beijing) diluted at 1:1000 in blocking buffer for 1 h at room temperature (RT) followed by washing thrice as mentioned earlier. The immunoreactive protein bands were visualized in DAB (diaminobenzidine) (Sangon, Shanghai). Purification of GST-RVLG fusion protein: 100~500 mg of bacteria cells harboring pGEX-RVLG were harvested by centrifugation at 12 000 r/min for 2 min at 4oC after induction with IPTG. The cell pellets were resuspended with Extraction buffer (Clontech) and lysed by repeated sonication on ice (10–15 cycles of 6 s on/24 s off). Then the GST-RVLG protein was purified from the lysates according to the GST Purification Kit (Clontech) protocol and separated by 12% SDS-PAGE. Gel fraction containing the GST-RVLG fusion protein was cut in precooled 0.5 mol/L KCl and sliced into pieces to undergo horizontal electrophoresis in dialysis bag under the conditions of 60 V overnight or 100 V for 3 h, with a final reverse electrophoresis for 5 min. The gel debris was discarded and the supernatant was dialysed in PBS (0.01 mol/L, Ph 8.0)

Ping Zhang et al. / Chinese Journal of Biotechnology, 2008, 24(11): 1981–1987

for one day. Preparation of ascites polyclonal antibodies: The KM mice (6–8 weeks old, male) were subcutaneously inoculated by four injections at 10–15 days intervals with each injection consisting of 15–20 Pg GST-RVLG in 70 PL PBS emulsified with an equal volume of complete (first injection) or incomplete (three booster injections) Freund’s adjuvant. Five days after the fourth injection, GST-RVLG fusion protein in PBS without Freund’s adjuvant was intraperitoneally injected to enhance the response to the immunogen. Anti- GST-RVLG antibodies from immunized mice serum were detected by indirect Enzyme-linked Immunosorbent Assay (ELISA) five days after the final injection. Ascitic fluid was harvested when the mouse abdomen became distended after intraperitoneal injection of S-180 cells (1×106/mL) in 200 PL PBS (0.01 mol/L, pH 7.2) and was subjected to centrifugation at 2000 r/min for five minutes at 4oC to collect the supernatant. The control mice (6–8 weeks old, male) were immunized with Freund’s adjuvant containing PBS under the same procedures described earlier. Detection of specificity of the ascitic polyclonal antibody: 1) Western blotting analysis: Cryopreserved Wistar rat testis, muscle, liver, and lung tissues were homogenized in liquid nitrogen for total protein extraction by KEYGEN Total Protein Extraction kit (KEYGEN, Nanjing) following the instructions provided by the manufacturer. Then western blotting was carried out based on the standard protocol described earlier except for using ascitic anti-GST-RVLG polyclonal antibody (1:80) as primary antibody. (2) Immunohistochemistry: After being ¿xed in 4% neutral-buffered paraformaldehyde for 16–24 h, rat ovary tissues were subsequently dehydrated through graded alcohols, cleared in xylene and embedded in paraffin wax. Serial sections were cut into 5 Pm thickness and placed on polylysine treated slides. Paraffin sections were dewaxed, rehydrated, and washed in 0.01 mol/L PBST (0.01 mol/L PBS with 0.05% Tween-20, pH 7.2–7.4). Antigen retrieval was carried out by high temperature in 10 mmol/L sodium citrate (pH 6.0) for 15–20 min using a microwave. Immunostaining was performed using the UltraSensitive S-P Kit (Maixin, Fuzhou) following the instructions provided by the manufacturer. Firstly, endogenous peroxidase activity was inactivated by incubation in peroxidase blocking agent, and nonspecific binding sites were blocked using normal nonimmune goat serum from the same source as the second antibody for 20 min at room temperature. Then sections were incubated with the anti-GST-RVLG ascetic fluid (1:100) overnight at 4°C. The primary antibodies were detected using biotinylated goat anti-mouse IgG, followed by streptomyces antibiotin peroxidase linkage technique (Technique SP). Finally, sections were visualized with the chromogen AEC

(showing red signals, Maixin), and examined under light microscope (WPI). Negative controls were performed by replacing anti-GST-RVLG ascetic fluid with ascitic fluid prepared from unimmunized mice. Measurement of the titer of mouse anti-GST-RVLG polyclonal antibody: The titer of mouse anti-GST-RVLG polyclonal antibody was measured by indirect ELISA as following. The purified GST-RVLG fusion protein (final concentration is 5 mg/L) was incubated overnight at 4oC in carbonate-buffered saline (0.05 mol/L, pH 9.6) in ninetysix-well polystyrene plate and was washed thrice with PBST (0.01 mol/L PBS-0.05% Tween-20). The remaining binding sites were blocked in 5% BSA-PBST for 1 h at 37°C. Then appropriate anti-GST-RVLG ascetic fluid and control ascites dilutions in 1% BSA-PBST were added and incubated for 1 h at 37°C. Plates were washed again, and incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (Bios, Beijing) (1:1500) for 1 h at 37°C. Horseradish peroxidase was detected by addition of the tetramethyl benzidine (TMB, Sigma) substrate solution. The enzyme reaction was stopped with 2 mol/L H2SO4 and absorbance of the yellow color representing oxidized TMB was measured at 450 nm. The OD value of positive/negative > 2.1 was regarded as the optimal dilution titer of anti-GST- RVLG antibody [13].

2

Results

2.1 RVLG cDNA cloning RVLG cDNA RT-PCR product of about 700 bp was detected by agarose gel electrophoresis (Fig. 1, lane 2) and its size is longer than expected of 639 bp. Digestion of the recombinant plasmid pMD-RVLG by restriction endonucleases BamHĉand EcoRĉproved successful insertion of PCR product (Fig. 1, lane 4). As there is a Hind III site in the expected PCR product, Hind III was also applied to cut the pMD-RVLG, and a fragment of about 500 bp was released (Fig. 1, lane 3) confirming that the PCR product is RVLG fragment. Sequencing of three pMD-RVLG clones all showed a RVLG fragment of 687 bp (Fig. 2), which was 60 bp longer than that in GenBank (Accession No. NM_001077647), encoding a peptide with calculated molecular weight of 24.447 kD, was obtained. Of the extra 60 bp, 45 bp are inserted before the second base of the codon GAT downstream of the 456 bp of the NM_001077647, adding 15 amino acid residues (GSFRGCRGGFGLGRP) and altering the Asp (D) to Asn168 (N); the other 15 bp are inserted before the second base of the codon GGC downstream of the following 582 bp, adding 5 amino acid residues (VQCFT) but causing no amino acid mutation. The rest 627 bp are nearly the same as those of the NM_001077647 except for three mutant base pairs (453, 635, and 644) causing two Ala to Gly212

Ping Zhang et al. / Chinese Journal of Biotechnology, 2008, 24(11): 1981–1987

Fig. 1 RT-PCR product of RVLG and restriction enzyme digestion of the recombinant vector 1: DNA marker II; 2: RT-PCR product (699 bp);3: pMD-RVLG/Hind III(452 bp); 4: pMD-RVLG/EcoR I+BamH I(2.69 kb and 699 bp); 5: recombinant plasmid pGEX-RVLG/EcoR I (5589 bp); 6: recombinant plasmid pGEX-RVLG/EcoR I+BamH I (4.89 kb and 699 bp)

and Gly 215 alterations (Fig. 2). Compared with the rat genomic DNA in the chromosome 2 (ftp://ftp.ncbi.nih.gov/genomes/R_norvegicus/), the authors found that the extra 45 bp and 15 bp of cloned RVLG cDNA fragment are of parts of two introns of the RVLG gene (Fig. 3). The authors found that there are six canonical splicing sites (GT-AG) in the genomic DNA region containing the extra 45 bp and 15 bp. The mRNA sequence NM_001077647 is a splicing result at the GT1-AG1 and GT3-AG3. The cloned RVLG cDNA fragment is a result of alternative splicing pattern at GT2-AG1 and GT3-AG2. 2.2 Construction of expression vector The result that pGEX-RVLG recombinant vector were digested by BamHĉand EcoRĉ (Fig. 1, lane 6) confirms that the inserted fragment is identical in size to the RT-PCR product, and further sequencing result shows the codons of inserted RVLG fragment are successive following the open reading frame (ORF) of the glutathione transferase (GST) tag. 2.3 RVLG expression in E. coli After inducing by IPTG for 1–5 h, recombinant expression vector pGEX-RVLG expressed GST-RVLG fusion protein of about 50 kD, which is in consistent with the predicted molecular weight of 50.4 kD (26 kD GST plus 24.4 kD RVLG), in E. coli BL21(DE3) at a high level (Fig. 4, lanes 6-9; Fig. 5, lane 2). The recombinant fusion protein content reached the maximum peak when induced for 4 h by adding IPTG (final concentration is 100 mg/L) at 0.513 of OD600, accounting for 13% of total supernatant in BL21(DE3). 2.4 Purification of the GST-RVLG fusion protein The GST-RVLG fusion protein purified by the GST purification column is still accompanied by some undesired proteins unveiled by SDS-PAGE analysis (Fig. 6, lane 4).

After repurification by cutting the objective band and undergoing horizontal electrophoresis, the undesired proteins were removed (Fig. 6, lane 5) leaving the GST-RVLG fusion protein qualified to be immunogen. 2.5 Detection of mouse anti-RVLG polyclonal antibody Western blotting analysis confirmed that the ascitic mouse anti-RVLG polyclonal antibody can recognize both the GST-RVLG fusion protein and the GST (Fig.7, lanes 2 and 3), indicating that the mouse ascetic fluid contains the anti-GST antibody. As the RVLG expresses specifically in germline cells, the authors used the mouse ascetic fluid to recognize the total protein extracted from rat testis, muscle, liver, and lung tissues by western blotting. The results prove that it can also recognize a testicular protein with molecular weight about 80 kD (Fig. 8, lane 2), but not any proteins in muscle, liver, and lung tissues (Fig.8, lanes 3–5). The Immunohistochemistry analysis on the adult ratovary sections also shows that the ascitic anti-RVLG polyclonal antibody can recognize RVLG-expressing germ cellscumulus oocyte complex (Fig. 9A, arrowhead). These results demonstrate that the mouse ascitic anti-RVLG polyclonal antibody has both immunoreactive efficiency and high specificity. 2.6 Measurement of the titer of anti-GST-RVLG polyclonal antibody Results of indirect ELISA suggest that the anti-GSTRVLG polyclonal antibody can recognize purified GSTRVLG fusion protein under various dilutions from 1:100 to 1:20 000 (Table 1). Its titer is about 1:20 000.

3

Discussion

The full-length cDNA sequence of the rat RVLG is 3.03 kb in chromosome 2 (GenBank Accession No. NM_001077647), including eight conservative frame of DEAD protein family, encoding 713 amino acids which exhibits 52.0% amino acid identity and 85.0% similarity with the that encoded by Xenopus laevis vasa gene (XVLGI)[8]. Antigen epitope analysis indicates that its N-terminus possesses antigenic determinants. Thus the authors designed specific primers for cloning the 687 bp at the 5’ terminus of RVLG ORF. Sequencing results show that the cloned RVLG is 60 bp longer than the corresponding region of NM_001077647, of which two fragments of 45 bp and 15 bp are inserted into two codons of the NM_001077647 without causing amino acid shift Table 1 Measurement of the titer of anti-GST-RVLG polyclonal antibody by indirect ELISA Antibody concentration 1:100 1.223

1:500 1.213

1:1000 1:5000 1:10000 1:20000 0.974

0.664

0.339

0.225

Negative control

Null

0.091

0.001

Ping Zhang et al. / Chinese Journal of Biotechnology, 2008, 24(11): 1981–1987

Fig. 2 Sequence of rat RVLG fragment at 5c of ORF Two primers were underlined. Hind III site was in bold form. Two excess fragments were boxed

Fig. 3 Partial sequence of rat RVLG DNA in chromosome 2 The introns were in bold form and splicing boundary sequences GT/AG were boxed. Two fragments underlined were the same as the excess 60 bp cloned in this paper. Dots represented nucleotides omitted

Fig. 4 Expression of recombinant expression vector pGEX-RVLG in E. coli BL21(DE3) 1: low molecular weight protein marker; 2: supernatant of BL21 (DE3); 3: supernatant of BL21 (DE3) transformed with pGEX-4T-1 induced by IPTG; 4–9: supernatant of BL21(DE3) transformed with pGEX-RVLG induced by IPTG for 0, 1, 2, 3, 4 and 5 h, respectively

Fig. 5 Western blotting analysis of recombinant protein using anti-GST monoclonal antibody 1: prestained protein marker; 2: total supernatant of BL21(DE3) transformed with pGEX-RVLG induced by IPTG; 3: total supernatant of BL21(DE3) transformed with pGEX-4T-1 induced by IPTG

Ping Zhang et al. / Chinese Journal of Biotechnology, 2008, 24(11): 1981–1987

Fig. 8

High-specificity of RVLG polyclonal antibody

1: prestained protein marker; 2,3,4,5: total protein extracts of rat testis, muscle, liver and lung tissue, respectively

Fig. 6 Analysis of purified recombinant GST-RVLG protein by SDS-PAGE

A

1: protein molecular weight marker (low); 2: unpurified total protein of recombinant strain with induction by IPTG; 3: soluble fraction of recombinant strain with induction by IPTG; 4: first-step purified protein of GST-RVLG; 5: second-step purified protein of GST-RVLG

Fig. 9 Immunohistochemical analysis of mouse anti-RVLG polyclonal antibody (×400) A: analysis of polyclonal antibody against RVLG in 60 days’ rat ovary (×400); B: negative control of 60 days’ rat ovary (×400)

Fig. 7 Western blotting analysis of recombinant protein using polyclonal antibody against GST-RVLG 1: prestained marker; 2: purified protein of GST-RVLG; 3: total supernatant of BL21 (DE3) transformed with pGEX-4T-1 induced by IPTG

mutation, except for only one Asp to Asn alteration (Fig. 2). Sequence alignment of cloned RVLG fragment with the rat genomic DNA sequence in chromosome 2 displays that the extra 45 bp and 15 bp are intrinsic sequences of the RVLG genomic DNA, and this to clone the full-length of RVLG cDNA sequence to further not remove the GST tag from the fusion protein. The Immunohistochemistry and Western blotting analysis prove that the authors’ anti-GST-RVLG polyclonal antibody about GST in the mammalian ovary, and the authors did indeed has high immunoreactive efficiency and specificity region accommodates more than three pairs of canonical splicing sites making it obvious that the authors’ cloned longer RVLG fragment results from an alternative splicing of RVLG pre-mRNA (Fig. 2). The alternative splicing of RVLG mRNA has not been reported, triggering the authors’ study the alternative splicing pattern in the whole ORF. Preparation of high efficient antibody needs antigen with high purity. Prokaryotic expression system has the advantage of collecting and purifying recombinant protein. The pGEX-4T-1 is a widely used commercial prokaryotic expression vector which efficiently expresses a soluble GST fusion protein that can be easily purified by reduced glutathione columns, and the target protein can be harvested by excising the GST tag using site-specific

cleavage by thrombin. In this study, the authors expressed and collected the soluble GST-RVLG fusion protein successfully with a small amount of undesired bacterial proteins. To further purify the fusion protein, the authors separated the collected sample with SDS-PAGE, then cut the objective band and succeeded in extracting higher purified GST-RVLG fusion protein. In view of theanti-GST antibody in the final anti-GST-RVLG polyclonal antibody, this will not disturb the observation of against the endogenous RVLG (Figs. 8 and 9), these results also confirm the previous reports that the Vasa protein is specifically expressed in germline cells[14]. S180-cells have been used to prepare polyclonal antibody in many laboratories because they can stimulate organisms to produce mass ascites by rapid breeding in mouse abdominal cavity[15]. In this study, the authors obtained mouse anti-GST-RVLG ascitic polyclonal antibodies successfully with a titer of 1:20 000. The polyclonal antibodies can be used for the ongoing study on whether neo-oogenesis exists in the postnatal rat ovary.

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