Application Research Of Recombination E2 Protein In Detection And Immunity Of Classical Swine Fever

Vaccination and detection of CSFV antibody in pigs is very important. The main antigen domains(A, B, C and D) of E2 gene of CSFV of C strain was amplified by reverse transcription (RT) and the nested polymerase chain reaction (nPCR). It was ligated with plasmid pGEM-T Easy vector. The recombinant plasmid and expression vector pGEX-4T-l were digested by the same restriction endonucleases. It is 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 low consistence IPTG inducing and at low temperature. The expressed dissolubility E2 protein was measured by SDS-PAGE and western-blotting. The western-blotting results indicated that the expressed protein be recognized by the CSFV positive serum. The rate of the expressed dissolubility E2 protein in the induced bacteria protein was about 40%.The MagneGST? Protein Purification System provides a simple, rapid and reliable method for the purification of glutathione-S-transferase (GST) fusion proteins with high efficiency and low background. Purification of the GST-fusion protein starting from cell pellets can be accomplished in less than 2 hours from either crude or cleared lysates with high yield and purity. The rate of the purification from crude and cleared bacterial lysate( E2 protein) in all proteins was about 85%, and concentration was 4~5 mg/mL.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 °C 1h and 4°C overnight at a concentration of 8.91μg/ml, serum sample (1:80) and HRP labeled anti-porcine IgG (1: 1000) being incubated at 37°C for 45min. 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, the specificity and sensitivity of the ELISA is 95.3% and 98.6% respectively. In addition, 500 serum samples collected from swine farms were detected by the developed assay, it was showed that the positive rate was 86.4% for antibody against CSFV.Using the purified recombinant proteins, 7 pigs were immunized. 21 days after immunization, we get serum from pigs. After 21 days of second immunization, CSFV Ab's level got high enough to resist affection of Shimen strain. It was proved that E2 protein had assured immunization effect.