Fusion Expression Of Epidemic Type B Encephalitis Virus E Major Domain And Porcine IgG₁-Fc Fragment And Its Application

Epidemic Encephalitis B, caused by Japanese Encephalitis Virus (JEV), is a kind of acute contagious disease of central nervous system. Now, Epidemic Encephalitis B exist all over the world and is endangering to hoggery and human more and more. Its diagnosis is done through clinical detection and epidemiology,but the final diagnosis result is decided by serodiagnosis and pathogenic diagnosis in the laboratory. At present,the therapy of Epidemic Encephalitis B isn' effective except for vaccine inoculation and exterminating mosquito. In this thesis, the JEV E gene and IgG1-Fc gene was expressed highly in E.coli prokaryotic expression system, and then the method of SPA Co-agglutination test was established by utilizing expression product. Meanwhile , the expression product was used to immunize the BALB/c mice, evaluating specific immune effect of the BALB/c mice induced by the fusion protein E-pFc.The study includes:1. A pair of specific primers was designed according to the publisded sequences of swine IgG₁ in the GenBank. By RT-PCR, cDNA fragments including the partial CH2 and the complete CH3 of IgG₁ genes are cloned. The sequencing result showed:the cDNA fragments consist of 469 nucleotides, corresponding to the expected results. Compared with the published sequences of IgG₁ IgG2a, IgG2b, IgG3, IgG4 in Swine, the homology of the nucleotide sequences is 99.2%,89.7%,89.7%,96.7% and 91.4%. The phylogenetic tree showed that the genetic distance between amplified fragment and swine IgG₁ is the nearest ,and they were in the same branch.these illustrated that the sequence of amplified fragment is subtype of swine IgG₁.2. The cDNA fragments of IgG₁-Fc genes encode 120 amino acids. Compared with the amino acid sequences of swine IgG, the homologies of the amino acids are higher than 97.2%,but lower than 68.3% with other animal IgG. The deducated amino acids show that the fragment contain Cys38 and Cys98. corresponding to the expected results.3. In order to construct the recombinated expression plasmid pET-E-pFc,the amplified fragment was conjuncted to the downstream of E gene in the expression vector pET-E ,and its correctness was verified through enzyme identification. Then the plasmid was transformed into host bacterium BL21 (DE3) .After IPTG inducing and spallation with...