Cloning, Expression And Detection Of The GD Gene Of Porcine Pseudorabies Virus Ja Strain

The study shows the genomic DNA of PRV was extracted through three different methods. Referred to the reported referenes, a pair of primers was designed and synthesized. The gD gene of PRV Ja strain was amplified by PCR. The PCR products were checked by agarose gel electrophoresis and purified by Agarose Gel DNA Purification. The purified products were cloned into pUC18 vector successfully and the cloned plasmids were transformed into E. coli JM109.The specific recombinant plasmid was identified by PCR and restriction endonuclease analysis. The results indicated that the recombinant vector contained the gene of gD at right orientation of the insert. The positive cloned was sequenced by TaKaRa. The results demonstrated the size of gD gene was 1197bp in length and included an open reading frame encoding a protein of 398 amino acid residues. The homologies of nucleotide and amino acid sequences were compared between PRV Ja strain and other five strains-YangSan, Ea, Fa, Ka and Min-A . The result indicated that not only Ja strain has thirteen spot mutations and one defect mutation but also PRV Ja strain has one C(A)GGCCC repeat variability in gD 816nt-831nt which corresponds with Arg-Pro repeat variability in gD 268-275 amino acid sequence. The DNA of gD gene from Ja strain shares a high identity between 98.8%~99.7% with that of reported gD genes. The amino acid residues predicted shares an identity between 77.9%~79.5% with that of other strains. The results showed that the homologies of nucleotide which origin from different PRV strains were higher and the homologies of amino acid sequences between PRV Ja strain and other five strains varied to a great extent. A phylogenetic tree based on the published sequences of PRV reference strains was constructed. It is proved that obvious relative connection lies between PRV Ja strain and Min-A strain .Furthermore, the fragment encoding gD was excised from the positive clone pUC1.2 with EcoR I and BamH I enzyme, and purified by Agarose Gel DNA Purification. Then the fragment was cloned into the pGEX-4T-3 expressing vector digested by EcoR I and BamH I restriction enzyme. The recombinant expressing vector conformed to a reading frame. The fusion protein was expressed in E.coli BL21(DE3) with predicted molecular weight of 53KDa. Peaks of target protein was achieved at 6h after adding IPTG(1mmol/L). The target protein presented in the SDS-PAGE and reacted with rabbit anti-PRV serum in Western blotting, suggested that the protein could be used as one of candidated antigens. It provided the basis for further studying on anti-single special antibody.