Cloning And Expression Of The PrM-E Gene Of Japanese Encephalitis Virus And DNA Immunization Of PrM-E Gene And E Gene

Japanese encephalitis virus was the main causes of infectious reproductive failure in swine, and it was economically important diseases. Meanwhile, JEV was transmitted to humans by infected mosquitoes and caused acute viral encephalitic and neurologic disease manifestations that was a serious public health problem for humans. Therefore, establishing a secure, simple and economical technique of producing JEV diagnosis antigen was imperative under the situation. And now it had been a main study trend of JEV control questing a steady-going, secure, new-style JEV gene-engineering vaccine. Referred to the reported referenes, two pairs of oligonucleotide primers were designed and synthesized, prM-E and E of JEV SA14-14-2 strain were amplified by RT-PCR. The purified DNA fragment of prM-E was amplified and cloned into pUC19 vector. And transformed into E.coli. JM109 successfully. The specific recombinant plasmid was identified by molecular weight and restriction endonuclease analysis. The results indicated the resultant construct contained the gene of prM-E at right orientation of the insert. The prM-E was cloned into prokaryotic expression vector PET-32a. The recombinant expressing plasmid (PET-prME) was identified by restriction enzymes analysis. The results indicated the fragment was correctly inserted into the PET-32a vector and conformed to the reading frame 89KDa, 53KDa and 36KDa protein were produced in E. coli BL21(DE3) under the induction of IPTG. And the expression quantity and immunoreactivity of the protein were detected by SDS-PAGE electrophoresis and Western -blotting. It provided the basis for further studying on anti-single special antibody.Furthermore, the prM-E and E were cloned into eukaryotic vector pcDNA3.1(+). The two recombinant expressing plasmids(pcDNA-prME,pcDNA-E) were identified by restriction enzymes analysis, and the result indicate the recombinant vectors conformed to a reading frame. DNA immunization of Balb/c mice: Groups of five 4 week old and female mice were used for evaluating induction of antibody. For intramuscular DNA immunization, all mice were immunized at the left thigh with 50μl of immunogens diluted in phosphate-buffered -saline(PBS)at one site. Immunogens and doses were pcDNA-ME and pcDNA-E at doses of 50μg respectively, pcDNA3.1 vectors at a dose 50μg, and 50μl of PBS alone. All mice were injected 2 times at 2 week intervals. Two weeks after the second immunization, mice of each group (5/5) were alive healthily. Blood of each group was collected and serum was extracted. The result of neutralization tests indicated neutralization titer of pcDNA-prME and pcDNA-E were 1:35 and 1:20 respectively. Came to a conclusion that the mice by intramuscular injection with pcDNA-prME and pcDNA-E can cause neutralization antibody. Titers of neutralization antibody caused by pcDNA-prME are higher than those caused by pcDNA-E. Nowadays, vaccine of flaviviruses almost contained neutralizing antigen epitope of envelope protein E, and prM protein was indispensable to maintain the structure of E protein. Therefore the groups of DNA immunization with pcDNA-prME and pcDNA-E were established. Comparing the DNA immunization effect caused by pcDNA-prME with those caused by pcDNA-E, it can provided date and basis for future study on DNA Vaccine against JEV.