Expression Of The E2 Gene Of Classical Swine Fever Virus And The Study On The Diagnosis Method Of Indirect ElISA

The 1.17kb fragments of E2 gene of seven strains of recent prevalent field virus that were collected from different provinces were amplified by RT-PCR and cloned into pGEM-T Easy vector. Their nucleotide sequences were determined and the amino acid sequences were deduced. Compared with the corresponding region of C, Alfort and Brescia strain of CSFV, the nucleotide sequence homologies were 91.6%~94.5%, 89.2%~92.7% and 85.9%~89.3% respectively, the deduced amino acid consistencies were 91.2%~95.8%, 88.9%~92.0% and 85.9%~89.3% respectively. But the homologies among the seven field strains were very high, their nucleotide sequence homologies and amino acid homologies were 95.8%~99.7%, 96.3%~99.1% respectively. The further studies of these field strains were done by the phylogenetic analysis, all the sequenced strains belong to the Group I of CSFV, and the seven strains can apparently be divided into two subgroups. What this paper showed does not accord with the prevalent opinion that the genetic variation of CSFV was diversity.The main antigen domains(A, B, C and D) of E2 gene of CSFV of C strain, Shimen strain and two prevalent virulent strains (Lintao strain and Xingjiang strain) were amplified by reverse transcription (RT) and the nested polymerase chain reaction (nPCR). The amplified E2 fragments of the four strains all were 561bp in length. And they were ligated with plasmid pGEM-T Easy vector. The recombinant plasmids and expression vector pGEX-4T-1 were digested by the same restriction endonucleases. These genes were ligated and transformed into Escherichia coli. The insert position, the size and the reading frame all were corrected by PCR, restriction digestion and the sequence analysis. The result showed that the prokaryotic expression vectors were constructed successfully. Then the recombinants were transformed into BL21 (DE3) for E2 expression with IPTG inducing. The expressed proteins were measured by SDS-PAGE and western-blotting. The results showed that the E2 genes can express successfully in E.coli. The western-blotting results indicated that the expressed protein be recognized by the CSFV positive serum. The rate of the expressed proteins in the induced bacteria protein was about 20%~30%.The inclusion bodies in E.coli with plasmid pGEX-C and pGEX-LT, which have the higher expression level, were obtained by sonication of the bacterial cells, from which the purified activative expressed recombinant E2 proteins were got by urea and TritonX-100 washing and by renaturing, refolding and dialyzing. The results of SDS-PAGE show that washing by urea and Triton X-100 can decrease the amounts of bacteria proteins in the inclusion body. The dissolving effect of urea for the inclusion body is good, which eventually resulted in 70% of purity of recombinant E2 protein. The reactivity of purified protein was increased 20 times in ELISA experiments comparing with the routine antigen. Using the purified recombinant proteins, an indirect ELISA for detection of anti-CSFV antibodies was developed and its optimal reactions were determined: coating antigen for 37℃ 1h and 4℃ overnight at a concentration of 8.7μg/ml of C strain and 11.7μg/ml of LT strain, serum sample (1:160) and HRP labeled anti-porcine IgG (1: 800) being incubated at 37℃ for 1h. The ELISA assay was confirmed to have a good reiterativity,specificity and sensitivity by repeated test, crossing test and blocking test. And this ELISA method was also compared with the established routine ELISA test kit and the indirect HA test with the whole virus, the specificity and sensitivity of the ELISA is 97.14% and 93.18% respectively. In addition, 148 serum samples collected from swine farms were detected by the developed assay, it was showed that the positive rate was 68.91% for antibody against CSFV.