| Epidemic Encephalitis B, caused by Japanese Encephalitis Virus (JEV), is a kind of acute contagious disease of center nerve systems. In mammals, the swine is easy to be infected with JEV. Now, the positive ratio of its serum is more than 90%. So Epidemic Encephalitis B is endangering to hoggery more and more. In this thesis, the JEV E gene is expressed highly in prokaryotic expression system, and then the method of indirect ELISA is established. The study includes:1. A pair of specific primers was designed according to the publisded sequences of JEV SAM-14-2 strain. By RT-PCR, cDNA fragments of JEV E genes are gotten. The cDNA fragments consist of 1,113 nucleotides, corresponding to the expected results. Compared with the published sequences of SA14-14-2 in China, the homology of the nucleotide sequences is 100%, and it indicates that the SA14-14-2 vaccine strain sequences have not mutated. Compared with other JEV strains' sequences, the homologies are all higher than 96%.2. The cDNA fragments of JEV E genes encode 371 ami no acids. Compared with the sequences of SA14, JaOArS982, Beijing-1 and Taiwan TC, TL, the homologies of the amino acids are 97.8%, 97.6%, 97.3% and 96.8%, 97.3% respectively. The results show that the E protein is conservative in different JEV strains, and the analysis of its antigenicity also indicates that the expressed protein is in common use.3. In order to testify the feasibility of the expression of JEV E gene in E.coli and the antigenicity of the expressed product, a recombinant expression plasmid of pET-E was constructed. The recombinant plasmid was transformed into E.coli BL21 (DE3) and then was highly expressed by inducing with IPTG The molecular weight of expressed protein is 62.3kD, corresponding to the expected results. The result of Western blotting indicated that the antigenicity of the protein was specific.4. The E.coli cells that had been broken were centrifugalized with 5,000g (15min), and then the supernate and the precipitate were collected. The result of SDS-PAGE showed that the expressed product was in the inclusion bodies mostly. The expressed protein was purified by His'Bind* Purification Kit, and the results of purification was tesitified to be fine by SDS-PAGE.5. The results of the phalanx titrimetry of indirect ELISA using OPD as substrate showed that the positive serum titrimetry is obvious. It indicated that the antigenicity of theexpressed protein was specific. When the antigen and the positive antibody were diluted by 1:1,280 and 1:320 respectively, the OD was 1.024. Compared with negative contrast (the antigen and the negative antibody are both diluted by 1:10 and the OD is 0.059), the ODp/N was also higer than 2 remarkably. The result showed that the study was feasible.6. The results of the substrate of OPD and TMB were contrasted in equal conditions. It showed that the positive serum titrimetries are both obvious, and the negative serum specimens almost have no reaction. When TMB was used as substrate, the result was fit to measure by eyes and qualitative study. But in quantitive study, the OD data of using TMB is much lower than using OPD, So OPD was used as substrate in this study finally.7. Through the phalanx titrimetry of indirect ELISA, the dilutability of the antigen and antibody were established as 1:1,500-1:3,000 and 1:200 respectively. The positive standard is as follows temporarily: ODTS>0.400, and ODTS/ODNS>2 (note: The TS represent tested serum, and the NS represents negative serum). According to the positive standard above, the tested serum specimens are all positive. The antibody levels of few pigs are higher or lower than whole antibody levels obviously from Dafeng and Puyang, etc. The results showed that a few of pigs have been infected with virulent JEV in these farms. The antibody levels of the other farms are balanceable relatively. It showed that the antibody was produced by vaccine.8. The results of Western blotting and indirect ELISA showed that the study could be used in Japanese encephalitis diagnos...
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