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Construction of the New JEV Replicon Vectors and Research the Immunogenicity of JEV Replicative DNA Vector Expressing PA from Bacillus Anthracis

Author: YangPeng
Tutor: YuZuoYuan
School: PLA Military Academy of Medical Sciences
Course: Genetics
Keywords: Japanese encephalitis virus Replicon Protective antigen Domain III
CLC: R392
Type: Master's thesis
Year: 2009
Downloads: 72
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Abstract

Flavivirus genus includes more than 70 kinds of viruses, the vast majority of arthropod-borne human pathogen. Japanese encephalitis (JE) is a mosquito-borne viral disease, the annual incidence of about one hundred thousandth fatality rate of about 5-40%, while 20-40% will cause severe neurological diseases and sequelae . China except Xinjiang, Tibet, Qinghai, have the disease, and now there are still thousands of patients each year, causing great harm to people's health, the Ministry of Health as a Category B infectious diseases. A significant characteristic of the flavivirus genome, the RNA having autonomous replication ability, while having a septic, bare RNA to enter sensitive cells, it is possible to a large number of independent replication and further translation into the corresponding virus protein, the intracellular packaging of the complete virus particles . Flavivirus replication sub usually refers to the genome structural gene portion cut down, and retained a complete non-structural protein gene, such subunit genomic RNA retained the autonomous replication ability, not only able to expression of non-structural protein, but able expression reservations structural protein and / or added exogenous gene. In theory, this subgenomic replication capacity equivalent to the complete genome, positive strand RNA increased exponentially. Such replicons insert a foreign gene, due to the large amplification of the replicon RNA in the cell cytoplasm, such that the exogenous gene expressed efficiently, therefore, the replicon RNA can be used as the expression of exogenous gene good carrier. Flavivirus of KUN the DEN and YFV replicon has been reported to be used as a potentially useful tool for the development of new vaccines. In the present study, we hope to build JEV subgenomic replicon basis to JEV SA14-14-2 Zhu and further establishing a JEV replicon vector system for vaccine research. We have to build a series of JEV pMW-G2R ~ pMW-G4R replicon. These replicon main difference is that the structural protein coding sequence deleted different ways, such as the MCS of FMDV-2A sequence added. The replicons stability is a major problem of constructing a replicon, we have chosen a different plasmid vector was used as a backbone vector replicon, in order to promote the stability of the replicon, eventually we use an extremely low copy but stability good bacterial plasmid vector pMW-118. In addition, convenient for subsequent experimental operation, and to improve the effectiveness of the expression of the replicon vector, the CMV promoter sequence, added to the replicon vector replicon vector constructed pCMW-2M, several sequencing proved JEV replicon pCMW-2M at 30 ℃ in E. coli Top10 breeding process is very stable and no mutations, insertions or deletions. We analyzed by indirect immunofluorescence assay (IFA) the JEV replication sub-transfected BHK-21 cells the expression of the viral proteins. The antigen was mainly localized in the cytoplasm. These antigen-positive cells with the control cell shape, the same size. JEV antigen positive cells was not observed any significant cytopathic effect (CPE). the pMW-G2R ~ pMW-G4R pCMW-2M replicon transfected cells IFA positive rates vary widely, ranging from 1% to 30%, indicating that the integrity of the C protein, the presence of the CMV promoter and non-structural protein gene The correct expression of the ability of the virus protein expression replicon vector is essential. Respectively the report of the green fluorescent protein (EGFP), and luciferase (Luc) gene insert the replicon vector pCMW-2M. p2MEGFP transfected BHK-21 cells 24 hours after the observation of EGFP expression. Positive cells shape, size, there is no difference with the control BHK-21 cells, also did not observe cytopathic effect. EGFP-positive cells at 24 hours after transfection, rarely, in the 72 hours after transfection. And the expression of EGFP was observed similar to the expression of the luciferase gene in the 7 days after transfection still able to detect the luciferase signal at 24 hours after transfection, the exponential growth to 96 hours. These results indicate that the insertion of exogenous gene of JEV replication sub-expression of exogenous genes can be efficiently. We immunized mice to study the the JEV replication the sub pCMW-2M and pCMW-G2R immunogenicity. Per mice 50μg plasmid pCMW of - 2M dose intramuscular injection of 6-week-old female of BALB / c mice, induced by anti-JEV antibody titers after the third immunization of 1:1270, the neutralizing antibody titer of 1:3 (250% plaque reduction experiments), slightly higher than pCMW-G2R group. The group of pCMW-2M T-lymphocyte stimulation index (SI) of 2.8, slightly higher than the pCMW-G2R group. These results prove that the the the JEV replicon vector constructed pCMW-2M and pCMW-G2R mice can induce both humoral and cellular immunity against JEV. In addition, pCMW-2M pCMW-G2R immune serum with better protective effect on JEV virus attacks, up to about 70% survival. Bacillus anthracis (Bacillus anthracis) is a Gram-positive bacteria form spores, by its infection caused a fatal human anthrax is a serious hazard of major infectious diseases, but it also is a potential biological warfare agents and bioterrorism agents. PA is the current application of the vaccine main component and cause immune response of the most effective immunogen. To the development of safe, effective anthrax vaccines, DNA vaccines is an important direction. JEV replicon PA a fourth structural domain of the antigen gene is inserted into the 2M of the JEV replicon pCMW-, this inserted PA4 gene to induce good humoral and cellular immune response, in the form of DNA immunized mice after immunized three times antibody titer of 1:36200 T lymphocyte stimulation index of 2.6, which is a preliminary exploration at home and abroad for the first time JEV-replicating DNA vector construct expression anthrax PA. In addition, we have expressed and purified the yellow fever virus YFV envelope protein (E protein) domain III, to study the possibility of its prevention as a subunit vaccine against YFV, JEV infection. YFD Ⅲ protein Efficient soluble expression in E. coli, the expression of the total bacterial proteins 50%. The YFD Ⅲ protein purified immunize New Zealand rabbits and BALB / C mice. Western Blot analysis and ELISA showed that the purified expression product has good antigenicity and immunogenicity. Utilize the YFD Ⅲ purified protein was used to immunize New Zealand rabbits to obtain anti-YFV antibody titers of 1:4 × 105 and 1:2 × 104 anti-JEV antibody titer; YFD Ⅲ using purified protein to immunize BALB / C mice, 1 : 7 × 104 titer anti YFV antibody and 1:2 × 103 titer of anti-JEV antibody. That produce confrontation YFV and JEV antibodies, antigenicity and immunogenicity. The experimental results show that of the protective effect of the anti-YFV immune serum the YFV attack on neonatal rat poison YFD Ⅲ protein immune serum YFV virus attacks better protective effect, these results suggest that the YFD Ⅲ protein might have developed into a potential subunit vaccine This is the first use of Escherichia coli efficiently soluble expression the YFD Ⅲ proteins and to demonstrate that their good immunogenicity and protective efficacy. In summary, we successfully constructed a new JEV replicon vector, at home and abroad for the first time based on the JEV attenuated live vaccine strain SA14-14-2 replicon vector, to express themselves and the ability of the exogenous protein immunogen other aspects of the system studies prove the the JEV replicon has potential as a good carrier for delivery of exogenous gene, and accumulated important information for the study of JEV structural proteins and non-structural proteins in the viral replication, packaging, and for further study of JEV immunogenicity and protective efficacy of the non-structural proteins (NS1 protein) to provide help, laid the foundation to develop a new type of expression anthrax PA JEV replicating DNA vector or even double-or multivalent replicating DNA vector, but also to take advantage of JEV replicon vector as a vaccine vector for further development of other novel DNA vaccine a valuable technology platform.

Construction of the New JEV Replicon Vectors and Research the Immunogenicity of JEV Replicative DNA Vector Expressing PA from Bacillus Anthracis

Abstract

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