Study On The Full-length CDNA Clone Of Hog Cholera Virus Lapinized Chinese C81/WHHV Strain And Expression Of Nonstructural Protein2

Classical swine fever (CSF) is a high contagious and often fatal viral disease of swine. Its characterization is high fever and haemorrhages. The disease is endemic in worldwide and classified as an OIE list A disease.Seven-pair primers covering the whole genome were devised according to published sequences of Chinese Lapnized attenuated strains. 7 cDNA fragments were amplified by RT-PCR from CSFV attenuated strain. The target fragment was further sequenced after being cloned into pMD18-T vector. The full genome cDNA sequence of C81/WHHV-strain was assembled (GenBank: AY663656). The genome has 12310bp and includes a big open reading frame coding a polyprotein of 3898 amino acids. Sequence analysis showed that the nucleotide sequences of Open Reading Frame (ORF) are 84.4%-99.6% and the amino acid sequences are 91.6%-99.4%. At the same time, the sequences of CSFV 5'-nontranslated region (5'-NTR) were compared and the functional domains of CSFV C81/WHHV polyprotein were predicted.With a serious of primers which were designed, different fragments in length of NS2 were amplified and cloned into the expression vector pGEX-KG to construct the recombinant plasmids pKG-NS2, pKG-NS876 and pKG-NS400. The recombinant plasmids transformed into E.coli BL21 (DE3) competent cells. Expression of the protein was induced with IPTG. SDS-PAGE results showed that the part protein of NS2 was highly expressed in E.coli. Molecular weight of the expressed protein was about 40 kDa. The expressed protein was specific to antisera against CSFV by Western-blot analysis, being presented as a single reaction band.Full-length NS2 was cloned into the expression vector pcDNA3.1 (+) to construct the recombinant plasmid pCD-NS2. The recombinant plasmid was transformed into cell lines PK-15 by lipid. The results by indirect immunofluorecence were showed that the nonstructural protein2 was expressed in cell members. This is a foundation for construction stable cell lines and the complement function of NS2 protein.A pair of primers of NS2 were devised according to yeast codon bias, amplified and cloned into yeast expression vector pPIC9K. Recombinant expression vector p9K-NS2 was constructed. Pichia pastoris strains GS115 was transformed by these recombinants respectively and the expression was induced by methanol. SDS-PAGE and Western-blot were performed to confirm the expression of NS2 gene. The result showed that NS2gene was not expressed in any strain of Pichia pastoris. That is probably because of codon bias in Pichia pastoris.