| Japanese encephalitis virus (JEV), a number of the family Flaviviridae, is transmitted to humans by infected mosquitoes and causes acute viral encephalitic and neurologic disease manifestations that is a serious public health problem for humans, and JEV is also the agent of severe infectious disease in many domestic and wild animals, especially swine (viral associated abortions). At the same time, swine plays an important role in viremia-amplifying host. So, prevention of swine JE can benefit for both swine production and humans' health. This dissertation mainly targets exploring diagnostic method and vaccine for swine. The research results are summarized as following:1. The preparation of JEV SA14-14-2 attenuated live vaccine for swinePrimary hamster kidney (PHK) cells were used as culture substrate, a attenuated JEV SA14-14-2 as vaccine dependent viral strain. The liters of SA14-14-2 in PHK cell culture remain 10-7.2 TCID50/1.0mL and was more than 10-6.8 TCID50/1.0mL after lyophilization, loss was lower.Tests on mice and swine demonstrate that this vaccine have safety and immunogenicity. The vaccine can be kept under 4-8 and -20 for 18 months.Together, it will be a new safe, good immunogenic, large-scale production and practical vaccine for prevention of swine JE.2. The development and application of Dot Immune-gold Filtration Assay (DIGFA) for the detection of JEV antibody in swineThe concentrated viral antigen were absorbed onto the nitrocellulose filter membrane, then the tested sample (sera) were added, addition of rabbit-anti-swine IgG probe labeled with red colloidal gold particle ensued. A comparison was made in parallel with HI test established under the same conditions for 54 clinical sera. These two methods had a general coincidence rate of 81.4%, and the positive and negative rate were 71.4% and 65.5% respectively. The experimental results could be read with naked eyes within five minutes. This test shows that DIGFA is a rapid, easily performing, highly sensitive, highly specific and reproducible method. It is an efficient means, for rapid diagnosis and epidemiological investigation of JE for swine.3. Th cloning expression and gene immunity of prM,E and NS1 genes of JEV SA14-14-2The viral genomic RNA was abstracted from JEV attenuated live vaccine. Three pairs of primers were synthesized to amplify the cDNA fragments encoding JEVdominant immunogenic genes which is prM, NS1 and the pre one-third of E by one-step RT-PCR Their size were 558bp, 1146bp and 615bp respectively. Then they were cloned into pMD18-T, named pTprM, pTE and pTNSl respectively. After they were identified, they were subcloned into prokaryotic expression vectors. SDS-PAGE and Western-blot indicated that NS1 could be expressed highly in BL21 as a fusion protein and had biological activity.To assess the feasibility of DNA vaccination, in this study we constructed three recombinant eucaroytic expression plasmids, which were termed pcDNA3.1prM, pcDNA3.1E and pcDNA3.1NSl respectively. After they were expressed in BHK-21 cell, the immunogenicity of pcDNAS.lprM, pcDNA3.1E and pcDNA3.1NSl was characterized with mice as an animal model from two aspects of body fluid response and cell immunity response. Our results revealed that these three plasmids and their various Mix could induce special response, but the three-gene-combined immunity possessed the best effectiveness, NS1 is good among one-gene vaccination.4. The construction of recombinant virus strain TK7gGYNS1+One pair of primers was designed to subclone NS1 from pTNS1 into general transfer vector pUSK. A co-transfection experiment was carried out with the purified pUSK-NS1 and the genome of TK7gGYLacZ+ mutant of pseudorabies virus Ea strain in IBRS-2. By plaque purifications and PCR detection, we acquired recombinant virus TKVgGVNSl+. Western-blot and ELISA showed that NS1 protein could be expressed in PK-15 cell infected with TK-/gG-/NS1+.This recombinant virus strain can be used for the study of duel-valence vaccine of JEV and PRV. |
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