| AimIn the present study, we described the construction of two kinds of recombi-nant plasmids to express prME and E proteins derived from JaGAr -01 strain of JEV (known as pJME and pJE) ; transfected pJME and pJE into HepG2, KN73 and COS -1 cell lines by the liposome method, respectively; compared the expression feature of prME and E protein in the transfected cells when both of re-combinant plasmids were transfected into mammalian cells; probed the relationship between the amount of recombinant plasmid for transfection and protein expression in transfected cells; delivered the plasmid DNA encoding prME and E proteins into mice by both intramuscular and gene gun injection respectively; and investigated the relationship between the level of protein expression encoded by pJME and pJE in transfected cells and DNA immunization by the way of analyzing neutralization antibody titers and protective immunity produced by pJME and pJE.Materials and methodsCells, virus, vector and animals; HepG2 and COS - 1 cell lines derived from human hepatoma and monkey kidney cells were grown at 37℃ in Dulbecco 'modified Eagle medium(D - MEM) supplemented with 10% fatal calf serum ( FCS). KN73 cells, derived from human liver cell origin were grown at 37℃ in RPMI1640 medium with 10% FCS. Three kinds of cells above were used for transfection experiments. Baby hamster kidney (BHK) cells were grown at 37 Tl in E - MEM with 1 % FCS, and used for 80% plaque reduction neutralizationtest (PRNT80). C6/36 cells, derived from mosquito cells were grown at 28℃ in Eagle medium with 10% FCS and amino acids, and used for JEV propagation. Prototype and neurovirulent JaGAr - 01 strains of JEV propagated in C6/ 36 cells were used for construction of plasmids, and viral challenge in mouse experiment. A eukaryotic vector pcDNA3 with the strong eukaryotic promoter derived from human cytomegalovirus and T7 bacteriaphage promoter was inserted FLAG gene between Hindlll and BamHI site in vector pcDNA3, known as pcD-NA(FLAG)3, then was used for plasmid construction. Female, 4 weeks old Balb/c mice, were directly used for the experiment of DNA immunization latter. The JEV - challenged mice were observed for symptoms of viral encephalitis and death every day for 21 days.Construction of plasmids expressing JEV prME and E protein; Viral RNA was isolated from JEV (JaGAr -01) -infected C6/36 cells. The cDNAs fragments of JEV prME and E (2001 and 1500 bps respectively) were generated by reverse transcription - PCR amplification of genomic RNA of strain JaGAr -01. Two different 5'- primers were used for prME or E genes construction during the PCR step: 5'- ATG AAG TTG TCA AAT TTC GAG GGGA, hybridizing to nu-cleotides (nt) 477 to 501 in the prME region, and 5'- TTT AAT TGT CTG GGA ATG GG, hybridizing to nt 978 to 997 in the E region. The same 3'primer was used for both construction in the first - strand cDNA synthesis step: 5' -AGC ATG CAC ATT GGT CGC TAA GAA, complementary to nt 2454 to 2477. For cloning of JEV prME and E genes, restriction enzyme site for BamHI was engineered at the 5' terminus of both sense primers, and EcoRI site was engineered at the 3'terminus of antisense primer. The RT - PCR products were purified using a QIAquick PCR purification kit ( Qiagen) as instructed by the manufacturer. Both RT - PCR products of JEV prME and E gene were sequenced by an ABI - PRSM-10 Genetic Analyzer ( Pekin - Elmer/Applied) , and compared to the published JaGAr - 01 virus sequence. Both constructions of plasmids and analysis were carried out using standard techniques. RT - PCR - amplified products of JEV prME and E regions were digested with restriction enzymes BamHI and EcoRI, and inserted into the pcDNA(FLAG)3 vector at theBamHI/EcoRI site between the strong eukaryotic promoter derived from human cytomegalovirus and the polyadenylation signal derived from the bovine growth hormone. The constructs containing prME or E regions of JEV were designated pJME and pJE respectively. Competent Escherichia coli JM109 cells were transformed and p...
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