DNA immunity by recombinant encoding prME protein derived from Japanese encephalitis virus and immunogiobulin G Fc of BALB/c mice
- VernacularTitle:乙脑病毒prME蛋白与BALB/c鼠IgG Fc段编码基因联合构建DNA免疫研究
- Author:
Ximei LI
;
Yan ZHOU
;
Yongzhen ZHAI
;
Li MA
;
Guohe FENG
- Publication Type:Journal Article
- Keywords:
Japanese encephalitis virus;
DNA vaccine;
IgG Fc;
CHO cell;
Neutralization test
- From:
Chinese Journal of Microbiology and Immunology
2008;28(7):634-638
- CountryChina
- Language:Chinese
-
Abstract:
Objective To study the effect of lgG Fc gene on JEV DNA vaccine immunity. Methods Gene encoding IgG Fc was amplified by nested-RT-PCR technique from BALB/c murine spleen cells. JEV prME protein gene was obtained with restriction endonuclease BamH Ⅰ/EcoR Ⅰ from the eukaryotic recombinant named after pJME, which was constructed by us before. Recombinant, named after pJME/IgG Fc, with above two genes encoding JEV prME protein and BALB/c murine IgG Fc was constructed, and was tested by restriction enzymes analysis and DNA sequencing, then was transfected into China hamster ovary (CHO) cells by Lipo-fectAMINE 2000. Distribution and expression of the fusion proteins encoded by JEV prME protein and BALB/c murine IgG Fc genes in transfected CHO cells were detected by immunofluorescence and Western blot. The BALB/c micc were vaccinated with pJME/IgG Fc via intramuscular injection. Then the cytotoxic T lymphocyt (CTL) activity were assessed by lactic dehydrogenase (LDH) and the neutralizing antibody titer were assessed by 80% plaque reduction neutralization test. Results Molecular weights (2001 bp, 2730 bp) of the two in- serts released from pJME/IgG Fc with two group of restriction analysis associated with BamH 1/EcoR I and BamH Ⅰ/Not Ⅰ were correlated to the expected theoretic results respectively. It was estimated that molecular weight (Mr) of the fusion protein was 101 x 103. The expression of the above fusion protein was mainly distribu- ted in endochylema of transfected CHO cells,and not much in membrane of transfected CHO cells. CHO cells transfected with pJME/IgG Fc could express the fusion protein at the 32th cell passage. After immunization, the CTL activity and the neutralizing antibody titer in the pJMF/IgG Fc vaccinated group increased significantly compared with other vaccinated groups(P <0.05). Conclusion The recombinant pJME/IgG Fc was construc- ted and transfected into CHO cells successfully, and CHO cellular lines expressed fusion protein encoded by JEV prME protein and BALB/c murine lgG Fc genes stably were obtained. IgG Fc gene could reinforce the cellular immunity and humoral immunity of JEV DNA vaccine.
