Expression Of The Genes Encoding The Main Antigenic Domains Of The E2 Envelope Glycoprotein Of CSFV In Pichia Pastoris And Preliminary Application

Classical swine fever virus(CSFV) is the etiological agent of CSF leading to substantial economical losses in pig husbandry,and successfully monitoring antibodies level of pigs against CSFV is premise to prevent this disease in pig herds.This study was carried out to develop a diagnostic method examining sera and following experiments were included:1. we designed two pairs of primers and amplified the specific genes encoding A/D and B/C antigenic domains of envelope glycoprotein E2 of CSFV ,and the PCR products of 373bp and 261bp were respectively inserted into secretory p PICZ. C P.pastoris expression vectors.Recombinant strains with specific genes encoding A/D (5 strains) or B/C (3 strains) antigenic domain were acquired and induced with methonl.SDS-PAGE and Western blot analysis showed that two genes could be expressed in P.pastoris and both products had good reacting ablility. Indirect ELISA suggested that capacity binding sera of protein E2AD expressed by recombinant with genes encoding A/D antigenic domain was higher than that of protein E2BC expressed by recombinant with genes encoding B/C antigenic domain.2.We studied the relations beween expression yield and growth conditions and found the optimal expression conditions of the recombinant E2 protein were: strains with high copies growed in BMMY medium of pH6.0 which was 4 times volume of BMGY with ODeoo being 3~-3.5,was incubated for 72h and added methanol to keep its concentration up to 0.5% ~ 1.0% between 24h. After induction under the optimal conditions, the concentration of protein E2AD was 275.38 g/mL,23.39 times of protein E2BC and 35.53% of protein E2HA which was prokaryotic expression product of gene encoding A/D antigenic domain. It was found that the supernatant should be conserved in -20 C after comparing effects of different conservation temperature on lysis of protein E2AD.3.An indirect ELISA using protein E2AD as coating antigen was established tomonitor antibodies level of pigs against CSFV and its operation methods were optimized. The optimal operation methods were: A protein concentration of 0.688 ug/uL was coated overnight at 4 C on 96-well microtiter plates and the plates were subsequently blocked 3h with 1% BSA ,and then incubated for 45 min with sera diluted by 1% BSA,after washing the plates,the anti-swine immunoglobulin conjuate was added and incubated for 30 min at 37*C or 45min at RT,and then the indicator TMB was added and incubated for 15 min at 37 "C, 0.15% HF was added before OD was measured at 630nm.30 pieces of sera from pigs infected by CSFV were examined and this method was compared with other methods including ELISA using protein E2BC or E2HA as coating antigen and HA test in the process of this.Experiments suggested that ELISA was more sensitive than HA test and among three ELISA methods ,the one using protein E2HA as coating antigen was most sensitive, followed by that using protein E2AD as coating antigen and ELISA coating antigen protein being E2BC was worst...