Expression Chicken Anemia Virus VP1 And VP2 Gene In Pichia Pastoris And Protection Research

Two pairs of primers were designed by Oligo6.0 according to isolated CAV VP1 and CAV VP2 gene sequences which were accepted by Genebank, amplified the VP1 and VP2 genes from the vector pBluemCAV which was constructed and the PCR products were inserted into secretory pPIC9K P.pastoris expression vectors.Recombinant strains with VP1 and VP2 gene were acquired and induced with methanol. The expressioning plasmid pPIC9K-VP1 and pPIC9K-VP2 was linearized with SalⅠ, then they were transformed into Pichia pastoris SMD1168 by electroporation. After the electroporatic transfornants were selected by PCR, the positive recombination yeast were obtained. They were induced with methanol at 30℃for target protein expression. CAV VP1 and CAV VP2 protein were analyzed by SDS-PAGE, Dot-Elisa and Weetern blot assay. The results showed that CAV VP1 gene was expressed successfully in Pichia pastoris and product was about 50KD which could act with the positive serum of CAV VP1. CAV VP2 protein was obtained by crushing Pichia pastoris cells, and product was about 30KD which could act with the positive serum of CAV VP2. BALb/c mice were immunized with expressed protein and specific antibodies were produced in Elisa and IFA detection test. The present study lay on foundation for the vaccine research of CAV.We studied the relations between expression yield and fermentation conditions and found the optimal expression conditions of the recombinant CAV VP1 and CAV VP2 proteins were: 30℃,pH 6.0, DO 40%, air flow rate 1.25vvm. When make sure glycerol are exhausted, add methanol in batch, at last OD600 can reach 250 or more. It was found that the supernatant should be conserved in -20℃after comparing effects of different conservation temperature on lysis of protein VP1 and VP2.The occup before did good groundwork for large scale fermentation and application of CAV VP1 and CAV VP2.Quantitate the recombinant protein CAV VP1 and CAV VP2 by thin plastic scanning, quantitative UV spectrophotometer, then phacoemulsificated with the same volume FREUND'SADJUVANT (SIGMA). Divide CAV negative SPF 10-day-old chicks into control group, CAV VP1 and CAV VP1+ CAV VP2 three groups, whom were injected in the breast. Blood both before and after immunization, then separate serum. Elisa test showed that immune after three weeks CAV VP1+CAV VP2 group can be detected by CAV antibody , until four weeks later CAV VP1+CAV VP2 group's antibody titers has increased, however the highest titer appear in the fifth week, six weeks later antibody titer showed a stable trend, CAV VP1 group was detected antibodies after three weeks, which level is much lower and there was no higher antibody levels afterward. Control group was always negative reaction; IFA test results show that CAV VP1+CAV VP2 group three weeks after immunization could be detected by antibodies, four weeks later strong fluorescence appeared. We did not see the fluorescence in CAV VP1 group and the control group. At the same time, divided breeders into control group, CAV VP1 and CAV VP1+CAV VP2 three groups which were injected by recombinant protein CAV VP1 and CAV VP2, and detected the yolk of serum antibody titers. Elisa showed that CAV VP1+CAV VP2 group five weeks later antibodies in serum and egg yolk reach the highest level at the same time, and then come to stable age. Yolk and serum antibody levels of CAV VP1 group and the control group were no significant fluctuations. After 1-day-old chicks were challenged with CAV for about two weeks, weights of control group had gone up slowly. In 7,14 and 21 days after dissection, thought histopathological section examination, we found that CAV VP1 test group and control group thymocyte had necrosised and reduced, accompanied by bleeding. Medulla pulp had also preciped; Liver cell vacuolus had degened, small vascular had been congested; splenic sheath artery hyperplasia, a small amount of peripheral lymphocytes around. Immune .CAV VP1+CAV VP2 test group had no significant change.