Cloning and characterization of a cDNA clone encoding troponin T from tick Haemaphysalis qinghaiensis (Acari: Ixodidae)

Comparative Biochemistry and Physiology, Part B 151 (2008) 323–329

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Comparative Biochemistry and Physiology, Part B j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c b p b

Cloning and characterization of a cDNA clone encoding troponin T from tick Haemaphysalis qinghaiensis (Acari: Ixodidae) Jinliang Gao a,b, Jianxun Luo a, Ruiquan Fan a, Guiquan Guan a, Volker Fingerle b, Chihiro Sugimoto c, Noboru Inoue c, Hong Yin a,⁎ a

Key Laboratory of Veterinary Parasitology of Gansu Province, State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China Max von Pettenkofer-Institut für Medizinische Mikrobiologie und Hygiene der Ludwig-Maximilians-Universität München, D-80336 Munich, Germany c The National Research Center for Protozoan Disease, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555, Japan b

a r t i c l e

i n f o

Article history: Received 17 June 2008 Received in revised form 27 July 2008 Accepted 28 July 2008 Available online 3 August 2008 Keywords: Tick Haemaphysalis qinghaiensis Troponin T Vaccination

a b s t r a c t Troponin T (TnT) is a key protein for Ca2+-sensitive molecular switching of muscle contraction. The entire cDNA sequence of Haemaphysalis qinghaiensis troponin T gene (HqTnT) from the hard tick was cloned here. The cDNA sequence of HqTnT possesses an ORF of 1170 bp coding for a mature protein with 389 amino acid residues and a molecular mass of 46.5 kDa. Search of the cloned sequence in GenBank revealed that HqTnT gene shared some homology with TnT genes of other organisms. By sequence comparison, two conserved domains were identified in the middle (residues 54–78) and close to the carboxyl-terminal end (residues 181–229) of the amino acid sequence of HqTnT. These conserved domains might be responsible for the interaction with tick tropomyosin and troponin I subunit, respectively. Reverse transcription-polymerase chain reaction showed a ubiquitous expression of HqTnT gene at different developmental stages and in different tissues of the tick. The HqTnT was expressed as glutathione S-transferase fused protein in a prokaryotic system. Though rHqTnT could induce specific antibodies in sheep, the antibodies could not protect sheep from the infestation of ticks. Therefore, recombinantly produced HqTnT could not be a candidate antigen for developing anti-tick vaccine. To our knowledge, this is the first report of tick TnT gene. © 2008 Elsevier Inc. All rights reserved.

1. Introduction Ticks are not only important vectors of human diseases but also the most important arthropods transmitting pathogens to domestic animals, causing economic losses for animal husbandry. Haemaphysalis qinghaiensis, a distinctive tick species of China, transmits at least three species of Theileria, namely, Theileria luwenshuni, Theileria uilenbergi and Theileria sinense. T. luwenshuni and T. uilenbergi are infective to sheep and goats but not to yaks while T. sinense is infective to yaks and cattle but not to sheep and goats. Further, the tick is also a vector of Babesia motasi (Yin and Luo, 2007a; Yin et al., 2007b). Theileria and Babesia, the causative agents of theileriosis and babesiosis, respectively, are tick-borne parasitic protozoa, and a number of them are highly pathogenic for cattle, sheep and goats. The economic losses caused by theileriosis and babesiosis are enormous in tropical and subtropical areas (Mehlhorn and Schein, 1984; Mehlhorn et al., 1994). H. qinghaiensis and the protozoal diseases transmitted by the tick are a major economic burden to animal husbandry in China. Extensive usage of chemical acaricides in tick control is problematic because of the selection of acaricide-resistant ticks, environ⁎ Corresponding author. Tel.: +86 931 8342515; fax: +86 931 8340977. E-mail address: [email protected] (H. Yin). 1096-4959/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpb.2008.07.016

mental contamination and pollution of milk and meat products with drug residues (Graf et al., 2004; Davey et al., 2006; Chevillon et al., 2007). Controlling of tick infestations through immunization of host with selected tick antigens has already demonstrated to be feasible in other tick species (Willadsen, 2006; Willadsen et al., 1989; Rand et al., 1989; de la Fuente and Kocan, 2003; de la Fuente et al., 2007). In the latter control approach, identification of tick-protective antigens is a key step for the success of the development of effective tick vaccines. Muscle-associated molecules play very important roles for moving the coxae of the appendages, retracting the chelicerae, controlling pharyngeal action, and other necessary functions during blood sucking of ticks (Sonenshine, 1991). There are some reports describing actin, myosin alkali light chain, paramyosin and troponin I of ticks (da Silva et al., 2005; Horigane et al., 2007; Gao et al., 2007a; Ferreira et al., 2002a; You et al., 2001), while descriptions on other tick fiber related molecules such as tropomyosin, troponin C and troponin T and their interactions are still not available. Several studies suggested that muscle-associated molecules could induce protective immunity against respective parasites (Jenkins et al., 1998; Hartmann et al., 1997; You, 2004). Previously we have identified a myosin alkali light chain protein by immunoscreening of a cDNA expression library of H. qinghaiensis and the potential application of the recombinant protein in tick control was also evaluated (Gao et al., 2007a). Later, a troponin

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T-like clone with partial sequence was cloned from the cDNA library of H. qinghaiensis and named as Hq08 following the other positive clones of the tick (Gao et al., 2007b). Troponin is located on the thin filament and consists of three components: the tropomyosin-binding subunit, troponin T (TnT), the inhibitory subunit, troponin I (TnI), and the Ca2+-binding subunit, troponin C (TnC) (Ebashi et al., 1968; Ohtsuki et al., 1986; Zot et al., 1987). TnT genes are best characterized in mammals and birds, where three TnT genes have been identified which are restricted in expression to either slow (red) skeletal muscle (sTnT), fast (white) skeletal muscle (fTnT) or cardiac muscle (cTnT), respectively (Perry, 1998). Some researchers have given direct evidence for the involvement of TnT in invertebrate muscle contraction (Perry, 1998; Bullard et al., 1988; Fyrberg et al., 1990; Marden et al., 1999; Marden et al., 2001). To our knowledge, there has been no report on troponin T of blood sucking arthropods including ticks. Here, the entire cDNA sequence of Hq08 was cloned from H. qinghaiensis and designated HqTnT. Two conserved domains in the deduced amino acid sequence of HqTnT were identified by sequence comparison with TnTs from both invertebrates and vertebrates. Anti-tick immunity effect of recombinant HqTnT is also evaluated.

2.4. Expression analysis by reverse transcription PCR To determine the expression profile of HqTnT gene in adult tick tissues and different developmental stages, reverse transcriptase PCR (RT-PCR) was preformed as we described elsewhere (Gao et al., 2007a, b). Positive control PCR reaction was carried out using the cloned HqTnT cDNA as template. Negative PCR control and negative RT control reactions were also included. The gene-specific primers were as follows: sense primer, 5′-ATGACGGAGGCTGAGATGCT-3′ and antisense primer, 5′-GGTGTAAGAGCGATCGTGGAGT-3′. 2.5. In vitro expression

2. Materials and methods

Recombinant HqTnT protein (rHqTnT) fused with glutathione Stransferase (GST) was expressed in Escherichia coli BL21(DE3) pLysE (Novagen, USA) using the same expression system as described previously (Gao et al., 2008). Gene-specific primers with added restriction enzyme sites for unidirectional cloning were as follows: forward primer, 5′-TACCCGGGTATGACGGAGGCTGAGATGCT-3′ (the italicized nucleotides were SmaI site) and reverse primer, 5′TAAAGCGGCCGCGGTGTAAGAGCGATCGTGGAGT-3′ (the italicized part was Not I 0 site). Cell pellet of the culture was lysed and recombinant protein in the supernatant was purified by GST•Bind Resin (Novagen, USA) affinity chromatography as described elsewhere (Gao et al., 2008).

2.1. Ticks and tissue collection

2.6. SDS-PAGE and western blot analysis

Eggs, larvae, nymphs and adults of H. qinghaiensis (reared in our laboratory) were maintained in an incubator at 28 °C and 85% relative humidity, and their life cycle was completed in rabbit or sheep. Dissections of ticks were performed as described elsewhere (Gao et al., 2007b).

SDS-PAGE was performed by standard techniques (Sambrook and Russel, 2001). Twenty micrograms of crude recombinant proteins per lane were resolved on a 12% SDS-PAGE gel. The amount of the purified rHqTnT per lane was 2 μg. For western blot analysis, about 2 μg each of the purified GST-fused rHqTnT protein and GST were resolved on a 12% SDS-PAGE gel and transferred to nitrocellulose (NC) membrane. After the membrane was incubated with sheep anti-rHqTnT serum (preparation of immuno-serum will be described in Section 2.7, Immunization and challenge infestation) at room temperature for 1 h, the membrane was washed three times with PBST. Subsequently, the membrane was incubated with peroxidase-conjugated rabbit antigoat immunoglobulin G at a dilution of 1:10,000 at room temperature for 1 h, and washed with PBST for 3 times. Positive signals were visualized by using 3′3-diaminobenzidine tetrahydrochloride and cobalt chloride as substrate. Prior to the incubation with the antigens, sheep anti-rHqTnT serum was diluted in an E. coli BL21 strain lysate expressing the pGEX-4T1 vector and incubated for 1 h at 37 °C for absorption of anti-E. coli and anti-vector derived protein antibodies (Ferreira et al., 2002b).

2.2. 5′ Rapid amplification of cDNA ends Previously, a cDNA library in λScreen vector (Novagen, USA) was constructed from H. qinghaiensis mRNA that was extracted from the dissected tissues such as salivary glands, Malpighian tubules and ovaries of partially fed female adult ticks. A troponin Tlike clone with partial sequence was screened from the library and named as Hq08 following the other positive clones of the tick (Gao et al., 2007a,b). Here, 5′ rapid amplification cDNA ends (RACE) was performed using a BD SMART™ RACE cDNA amplification kit (BD Biosciences Clontech, USA). Gene-specific anti-sense primer (5′CTGATTCGCTGCTCCTCTTGCTT-3′) designed from the primary cDNA sequence of Hq08 was used in 5′RACE. Amplified polymerase chain reaction (PCR) fragment was cloned into pGEM-T vector (Promega, USA) and nucleotide sequence was determined as described elsewhere (Gao et al., 2007b). 2.3. Bioinformatic analysis Sequence analysis was carried out using the Lasergene software package for Windows (DNASTAR, Madison, WI, USA). Database searches were performed with BLASTP programs on the NCBI non-redundant database. To predict the location of coiled coil regions in amino acid sequences, the online version of the COILS Prediction of Coiled Coil Regions in Proteins program (Lupas et al., 1991) was used (http://www.ch.embnet.org/software/COILS_form.html). Multiple alignments were performed using CLUSTAL W and phylogenetic analysis was conducted using the neighbor-joining method as implemented in the CLUSTAL W program (Thompson et al., 1994). Hq08 containing full cDNA coding region sharing some homology with TnTs of other organisms was designated as HqTnT.

2.7. Immunization and challenge infestation Six sheep purchased from a H. qinghaiensis free area, 6 months old, were used for the immunization and challenge experiment. They were divided into two groups of three sheep each. Sheep of the first group were immunized with rHqTnT, and those of the second group were offered with GST protein as a control. About 500 μg of the recombinant fusion protein emulsified in Freund's complete adjuvant was subcutaneously inoculated into each sheep. Half amount of the same antigen in Freun's incomplete adjuvant was given for each sheep on days 30 and 60 after the first inoculation. Sera from the rHqTnT and GST-immunized sheep were collected 30 days after the last booster and defined as sheep anti-rHqTnT and sheep anti-GST serum, respectively. For challenge infestation, 200 adult (100 male and 100 female) and 100 nymphs per animal were introduced and maintained on sheep backs with the help of bags pasted onto sites where hair had been removed, 30 days after the last booster injection. To evaluate the effects of recombinant

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Fig. 1. Nucleotide sequence of the cloned cDNA coding for the H. qinghaiensis tick TnT and its deduced amino acid sequence. The start codon (ATG), the stop codon (TAA) and the polyadenylation signal (AATAAA) are indicated in bold letters. The two conserved domains are underlined and conserved hydrophobic residues in positions “a” and “d” of the HR units (abcdefg) in the second domain are shaded. The proline rich region is framed.

protein-induced anti-tick immunity, parameters such as mortality rates, engorgement weights, oviposition rates, egg weight per engorged tick and hatchability were analyzed. Mortality of ticks in this study was confirmed by observing all detached and moribund partially fed or engorged ticks at room temperature for about 10– 20 min. Within this period, any tick that did not show any mobility was confirmed dead (Imamura et al., 2005). All data were presented as means ± standard error or percentages where applicable, and differences were considered to be statistically significant if the P value was less than 0.05 in Student’s t-test. 2.8. Animal care and manipulations Conduct of animal experiments in this research was in accordance with the “Animal House of Lanzhou Veterinary Research Institute”

instructions and ordinances on Animal Welfare and adhered to the “Guide for the Care and Use of Laboratory Animals”. 3. Results and discussion 3.1. Cloning and characterization of HqTnT The entire cDNA sequence of Hq08 gene with a length of 1502 bp was obtained by using 5′RACE. Hq08 cDNA possessed a 106 bp 5′UTR region and a 226 bp 3′UTR (GenBank accession number AY962876). Thus the cloned cDNA had ORF of 1170 bp coding for a putative protein of 389 amino acids with a predicted molecular mass of 46.5 kDa and an isoelectric point of 4.86. The only polyadenylation consensus signal was located 17 bp from the poly (A) tail. A proline rich region with three repeats of “PPXPX” is also found near a poly-glutamate tail (Fig. 1).

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The amino acid sequence of Hq08 was submitted to NCBI protein– protein blast (http://www.ncbi.nlm.nih.gov/BLAST) for sequence comparison with known proteins in the GenBank. Results showed that the amino acid sequence of Hq08 shared 54% identity with TnTs of both Aedes aegypti (EAT46401) and Drosophila melanogaster (AAU09446), and 24–26% identities with Homo sapiens TnTs (cTnT, NP_000355; fTnT, AAV38800; and sTnT, AAB30272). This suggested

that the cloned Hq08 gene was a TnT gene of H. qinghaiensis, namely, HqTnT. Amino acid sequence alignment of HqTnT and TnTs from both vertebrates and invertebrates showed that all the compared sequences shared the same major motifs. These included an acidic N-terminus, a pattern of charged residues in the middle predicted to interact with Tm, a domain believed to form a coiled coil with TnI

Fig. 2. Multiple alignment analysis. (a) deduced amino acid sequence of HqTnT was aligned with five other TnTs from both vertebrates and invertebrates. Residues conserved in majority were marked with gray shadow. Tm- and TnI-binding sites were underlined. Conserved Leu-s or hydrophobic residues in positions “a” and “d” of the HR units (abcdefg) were shaded with dark gray. GenBank accession numbers (from top to bottom): AY962876,EAT46401,AAU09446,NP_000355,AAV38800 and AAB30272. (b) amino acid sequence alignment of the HR in TnI polypeptides. Conserved hydrophobic residues in positions “a” and “d” of each repeat are shaded. GenBank accession numbers (from top to bottom): BAB55451, ABB89211, XP001661109, NP_728142, P02644, P19429 and P23693.

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Fig. 3. Phylogenetic analysis of HqTnT and TnTs from other organisms. The dendrogram was generated by the MEGALIGN program of the DNASTAR package using the Clustal W method. With the balanced display, MEGALIGN averaged the distances between ancestors in the tree. The dotted lines indicated a negative branch length introduced by averaging the tree. The GenBank accession numbers were: Mizuhopecten yessoensis, BAA22850; Chlamys nipponensis, BAA13610; Periplaneta Americana, AAD33603; Apis mellifera, DAA05518; Bombyx mori, ABD36267; Drosophila melanogaster, AAU09446; Aedes aegypti, EAT46401; Gallus gallus cTnT, NP_990780; Gallus gallus fTnT 1, AAA49100; Gallus gallus sTnT, NP_990445; Homo sapiens cTnT, NP_000355; Homo sapiens fTnT, AAV38800; Homo sapiens sTnT, AAB30272; Rattus norvegicus fTnT, P09739; Rattus norvegicus sTnT, AAQ19259; and Rattus norvegicus cTnT, NP_036808.

close to the carboxyl-terminal end, and an extremely acidic polyglutamate tail in invertebrates (Fig. 2a) Functions of some of the domains had already been characterized in some organisms (Perry, 1998; Stefancsik et al., 1998). The N-terminal segments of all the compared TnTs were highly variable and rich in acidic amino acids. The acidic N-terminus has been proposed to weaken the binding affinity between TnT and tropomyosin, and thereby reduces the inhibition of myosin cross-bridge attachment (Pan et al., 1991; Chandra et al., 1999; Ogut and Jin, 2000). The TnT interaction domains with tropomysoin or with TnI and TnC had been identified in two different regions, located in the middle and close to the carboxyl-terminal end (Stefancsik et al., 1998; Ohtsuki and Nagano, 1982; White et al., 1987; Cabral-Lilly et al., 1997; Potter et al., 1995; Jha et al., 1996; Jin et al., 2000; Hinkle and Tobacman, 2003; Takeda et al., 2003). Two conserved domains were identified in the middle and close to the carboxyl-terminal end of the amino acid sequence of HqTnT by sequence comparison. The most important feature of the domain close to carboxyl-terminal end was the presence of heptad repeat (HR) motifs with periodic occurrence of hydrophobic residues in positions “a” and “d” in a seven amino acid repeat unit “abcdefg” (Figs. 2a, and 1). This type of structural motif can, in principle, form an α-helical coiled coil with another protein containing HR (Stefancsik et al., 1998). By using the online coiled coil prediction program, HqTnT sequence also showed very high coiled coil forming probability in the region (residues 181–229). The interaction of TnT and TnI is mediated by HR motifs through the formation of αhelical coiled coils in other organisms (Stefancsik et al., 1998; Takeda et al., 2003). Comparison of tick TnIs (from H. longicornis and Rhipicephalus haemaphysaloides haemaphysaloides) with TnIs of other organisms revealed conserved domains characterized with HR motifs in the middle of selected sequences (Fig. 2b). The presence of highly

conserved domains containing HR motifs close to the carboxylterminal end of HqTnT and in the middle of tick TnIs suggested that tick TnT might bind to TnI also through the formation of α-helical coiled coils, in the same manner as in the known vertebrates and invertebrates. The central conserved domain of HqTnT also comprised a highly conserved subregion (residues 54–78) corresponding to residues 112–136 of TnTs from other organisms (Fig. 2a), which had been proposed to be the critical element for the interaction between tropomyosin and troponin (Hinkle and Tobacman, 2003). Therefore, we speculated that residues 54–78 of HqTnT should be responsible for tropomyosin binding. Amino acid residues between those two conserved domains of the compared TnTs were highly variable. Compared with other TnT amino acid sequences, there was a unique insertion, GGSALGSSGFDKFAN, in the variable region of HqTnT (Fig. 2a). Compared with vertebrate TnT isoforms, invertebrate TnTs possessed an additional C-terminal extension of approximately 120 residues with an extremely acidic poly-glutamate tail. The extension on the invertebrate isoform had no known function, but being rich in acidic and basic residues, the extension may play a role in protein– protein interactions (Bullard et al., 1988). Apart from this, a proline rich region with three repeats of “PPXPX”, which was absent in all the other TnTs, was found near the poly-glutamate tail of HqTnT (Figs. 2a, and 1). At present, the functional significance of these regions remains largely unknown. A phylogenetic tree based on the predicted amino acid sequence of HqTnT and TnT sequences from other organisms divided TnTs into two big groups, vertebrate and invertebrate. Even within the invertebrate group, HqTnT clustered into an independent branch of a subclade (Fig. 3). This further indicated HqTnT was quite different from TnTs from other organisms. Moreover, we did not find any TnT sequences

Fig. 4. Expression analysis of HqTnT mRNA by using RT-PCR. Lanes: M, maker; E, egg; L, larvae; N, nympha; A, adult; SG, salivary glands; MG, midgut; C, carcasses (remnants after removal of salivary glands and midguts); +, positive control (plasmid containing the HqTnT full-length cDNA); −, negative controls (negative PCR control and negative RT control).

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J. Gao et al. / Comparative Biochemistry and Physiology, Part B 151 (2008) 323–329 Table 1 Vaccine effect of rHqTnT on tick feeding

Mortality during and after feeding (%) Engorged mass, mean (mg)a Oviposition rate (%)b Egg mass/engorged tick (mg) Hatchability (%)c

Adult Adult Nymph

Immunized with rGST

Immunized with rHqTnT

38.7 ± 7.4 187.3 ± 1.0 3.01 ± 0.11 87.1 ± 2.6 38.1 ± 2.6 57.2 ± 4.9

39.0 ± 6.3 182.8 ± 3.9 3.18 ± 0.06 87.5 ± 4.2 50.3 ± 2.5 73.9 ± 8.3

All value were expressed as follows: average ± standard error. a Calculated as batch average for live ticks only (batch weight/number of live ticks, ticks collected from one sheep were regarded as a batch). b Calculated as number of ticks which had laid eggs/number of live ticks. c Calculated as batch of eggs which developed into larvae/number of ticks collected from one sheep which could lay eggs (all the eggs laid by one tick were regarded as a batch).

Fig. 5. SDS-PAGE analysis of recombinant HqTnT expressed in E. coli. M, the molecular weight marker; Lanes from left to right, Lane 1, GST protein; Lane 2, fusion protein expressed by recombinant plasmid of HqTnT gene; and Lane3, purified fusion protein.

from any ticks after scanning the GenBank. So this is the first time the TnT gene in ticks has been identified. 3.2. Expression analysis by RT-PCR Total RNA extracted from eggs, larvae, nymphs, adults and dissected salivary glands, midguts and carcasses (remnants of the tick body after removal of salivary glands and midguts) of the partially engorged female ticks was subjected to RT-PCR analysis. Result summarized in Fig. 4 showed that HqTnT gene was transcripted ubiquitously. 3.3. In vitro expression The expression product GST-HqTnT was a soluble protein. As shown in the SDS-PAGE gel, the expressed product of HqTnT was an

85 kDa fusion protein, 11 kDa bigger than the predicted one (Fig. 5). Being rich in strongly acidic amino acids (D, E), especially in the Cterminal of the protein, could explain the cause of the anomalous migration in SDS-PAGE (You et al., 2001; Alderuccio et al., 1991). The control protein, a 28-kDa GST, was produced from the expression vector. Both protein expressions were induced with isopropyl β-D-1-thiogalactopyranoside. 3.4. Vaccine effects Previous studies suggested that muscle-associated molecules, such as tropomyosin, troponin I, could induce protective immunity against respective parasites (Jenkins et al., 1998; Hartmann et al., 1997; You, 2004). On western blot analysis, sera collected from sheep immunized with rHqTnT 30 days after the last booster could react with the recombinant HqTnT protein, this suggested rHqTnT could induce good humoral response in sheep (Fig. 6). Since rHqTnT used here to immunize sheep was fused with GST, sheep anti-GST antibody might also be generated. Therefore, before usage of sheep anti-rHqTnT serum, any antibodies might be induced by GST protein were absorbed by E. coli BL21 strain lysate expressing GST protein. Tick challenge infestation on recombinant protein immunized sheep showed that rHqTnT could not generate any anti-tick immunity in sheep among the parameters observed (P N 0.05 in Student’s t-test) (Table 1). All these suggested, though rHqTnT could induce specific antibodies in sheep, the antibodies could not protect sheep from the infestation of ticks. Therefore, recombinantly produced HqTnT could not be a candidate antigen for developing anti-tick vaccine. In summary, for the first time we have identified a cDNA encoding tick troponin T subunit comprising two conserved domains in the middle and close to the carboxyl-terminal end of the amino acid sequence, which might interact with tick tropomyosin and troponin I subunit respectively. And these findings support the idea that the interaction domains of TnT that allow the whole tropomyosin– troponin complex to be bound to the thin filament are fully conserved during evolution, whereas in the other domains there is important variation. Acknowledgments

Fig. 6. Western blot analysis of recombinant HqTnT. Purified rHqTnT and GST proteins were resolved on a SDS-PAGE gel and transferred to NC membrane. The membrane was incubated with a sera collected from an rHqTnT immunized sheep. Prior to use the sera, antibodies against E. coli extract and GST protein were incubated with both E. coli extract and GST protein at 37 °C. M, a pre-stained molecular weight marker; lane 1, GST protein and 2, rHqTnT.

This work was financially supported by National Natural Science Foundation of China (NSFC) (Contract no. 30270992 and 30571097), Hi-Tech Research and Development Program of China (863 Program: 2006AA10A207), Specific Fund for Sino-Europe Cooperation, MOST, China, the Outstanding Research Fellowship of CAAS and the ICTTD-3 Coordination Action Project No. 510561 (the INCO programme of the European Commission) as well. The author also would like to thank the special PhD- Programme of the China Scholarship Council in cooperation with the Ludwig-Maximilian University of Munich (CSC 2006, No. 6011). We are grateful to Dr. Baiker Amin and Dr. Vaibhav

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