Cloning,Prokaryotic Expression And Polyclonal Antibody Preparation Of Chicken CVH

CVH gene is chicken VASA homolog gene, its production has ATP-dependent RNA helicase due to the protein contains DEAD-box motif. The Cvh proteins specifically exist in embryonic stem cells (ESs) and primordial germ cells (PGCs) which are necessary to evolution and differentiation of germ line cells. Thence, as a marker in germ line cells, Cvh has been used to identify and screen ESs and PGCs.The purpose of this study was to clone a part of coding sequence of CVH gene and prepare polyclonal anti-Cvh antibody using purified His-Cvh fusion proteins by Ni-NTA agarose matrices. Total RNA was extracted from the testis of chick at age of 3 weeks, and cDNA was obtained by RT-PCR. Encoding 213 amino acids and locating in the up-stream of CVH, and the cDNA fragment was ligated to pMD18-T vector to construct recombinant plasmid pMD18-T-CVH. Confirmed by sequencing, the target gene was subcloned to pET-30a vector to construct prokaryotic expression vector (pET-30a-CVH). transformed with pET-30a-CVH, E.coli BL21(DE3) were induced to expression His-Cvh protein with 1 mmol/L Isopropyl-?-D-thiogalactopyranoside (IPTG) at 37 ?, which was subsequently detected by SDS-PAGE and Western blot assays. The target protein was purtified by Ni-NTA agarose matrices under denaturing condition (dissolved in 8 mol/L urea). As an antigen, His-Cvh fusion protein mixed with Freund's adjuvant and emulsificed, and subcutaneously injected New Zealand white rabbits for three times at an interval of 3 weeks. On the 7th day after the last injection, blood was collected from the heart, and serum was isolated by centrifugation. This serum was namely the polyclonal anti-Cvh antibody, and subsequently used to detect endogenous Cvh expression in the genital ridge of chick at age of 5.5 days.The results show that:(1) Part of open reading frame (ORF) of CVH gene was cloned that composed of 640 bp nucleotides, which share respectively 99.5% and 99.2% homology with the homologs of CVH. (2) Recombinant pMD18-T-CVH and pET-30a-CVH had been constructed, and His-Cvh fusion protein expressed efficiently in the E.coli BL 21 cells under the optimal condition. (3) Western blot asssy showed that anti-His-Tag mouse monoclonal antibody (1:5,000 dilution) could specifically bind to the purified His-Cvh protein. (4) The polyclonal anti-CVH antibody (1:10,000 dilution) could also specifically bind to both of the purified His-Cvh fusion protein and the endogenous Cvh in the genital ridge of 5.5-day chicks.In conclusion, part of CVH gene was successfully cloned, and His-Cvh fusion protein was obtained by utilizing prokaryotic expression system, and polyclonal anti-Cvh antibody were prepared. The present study will lead to and identification of PGCs and functional re research of CVH.