Cloning Of P80 Gene Of Classical Swine Fever Virus And Expression In E.coli.

The paper has two parts. One is nucleotide sequencing of p80 gene of the C-strain and the GXYL prevalent strain of classical swine fever virus(CSFV), the other is the expression of the NTPase/RNA helicase functional domain of p80 genes (sGXNSS and sCNS3) of the two CSFV strains in E.coli. The results are:1. Two pairs of primers were designed according to the published sequences of CSFV Alfort strain and Brescia strain .The p80 genes of C-strain derived from rabbit spleen tissue and GXYL strain were amplified by reverse transcription polymerase chain reaction (RT-PCR) and nested polymerase chain reaction (nPCR). The amplified p80 fragments of the two CSFV strains were all 942bp in length by agarose gel electrophoresis. Then the two fragments were cloned respectively into the pGEM-T Easy vector and were sequenced. Comparison on the nucleotide sequences and deduced amino acid sequences of the C-strain,GXYL strain, Alfort strain and Brescia strain showed that the homology of nucleotidc sequence was from 87.46% to 99.04%,and that of the amino acid sequence was from 94.27% to 98.09%.These prove again that the p80 gene is highly conserved in CSFV.2. Another pair of expression primers with digestion site BamH I and Xho I on 5' region were designed to amplify the NTPase/RNA helicase functional domain of p80 genes (sGXNS3 and sCNS3) of CSFV GXYL strain and C-strain by PCR on the basis of the two recombinant plasmids pGEM-GXNS3 and pGEM-CNS3 constructed in the former experiment. The recombinant plasmids pET-sGXNS3 and pET-sCNS3 were obtained after being cloned sGXNS3 and sCNS3 into expression vector pET-30a(+). The insert position, the orientation and the ORF were right by PCR,digestion and sequence analysis. The 26kd target proteins were produced by inducing with l.Ommol/L IPTG. Western blot showed that the expressed proteins could be recognized by CSFV positive serum. Thus, the recombinant p80 protein makes the basis for establishing ELISA method and other serum method in identifying the vaccinated pigs by attenuated vaccine and engenieering vaccine.