Construction and immunogenicity of fusion protein containing respiratory syncytial virus M2:81-95 and heat shock protein 70L1
10.3760/cma.j.issn.1000-6680.2009.01.004
- VernacularTitle:呼吸道合胞病毒M2:81-95与热休克蛋白70L1融合蛋白的制备及其免疫原性
- Author:
Ruihong ZENG
;
Xingguo MEI
;
Xiaowen QI
;
Zhenya ZHANG
;
Lin WEI
- Publication Type:Journal Article
- Keywords:
Respiratory syncytial viruses;
T-lymphocytes,cytotoxic;
Epitopes,T-lymphocytcs;
Heat-shock proteins;
Recombinant fusion protcins;
T-lymphocyte subsets
- From:
Chinese Journal of Infectious Diseases
2009;27(1):11-15
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct the recombinant plasmid of fusion protein containing respiratory syncytial virus (RSV) cytotoxicity T lymphocyte (CTL) epitope M2:81-95 and heat shock protein (HSP) 70L1, and to investigate its immunogenicity after prokaryotic expression. Methods HSP70L1 gone was cloned from SMMC7721 cells. The M2:81-95 gene fragment was amplified by polymerase chain reaction (PCR) method. Plasmid pET-HSP70L1-M2 : 81-95 (pET-HSP70L1-M2) was constructed, identified and transferred into E. coli BL21 (DE3). Expression of HSP70L1-M2 : 81-95(HSP70L1-M2) was induced by isopropy-β-D-thiogalaetosidc (IPTG). The expressed protein was purified by affinity chromatography and renatured by gradient dialysis. The BALB/c mice were immunized with this fusion protein. IgG antibodies and the subtypes were detected by enzyme-linked immunosorbent assay (ELISA), and CTL responses were determined by methyl thiazolyl tetrazolium (MTT). Results The recombinant plasmid pET-HSP70L1-M2 was successfully constructed. The fusion protein HSP70L1-M2 was expressed in E. coll. The purified protein induced strong RSV-and CTL epitope-specific CTL responses and high titer of protein specific lgG antibody 4.87±0.35. The subtypes were IgG1 (5.53±0.28) and lgG2a (4.40±0.21) and IgG1/ IgG2a ratio was balanced. The titers of lgG, IgG1 and IgG2a in PBS control group were 0.33±0.17, 0.51±0.21 and 0, respectively, which werc significantly lower than those in immunized group (t = 3.512, 3.681, 5.856; P<0.05). Conclusions The recombinant plasmid pET-HSP70L1-M2 is successfully constructed and the fusion protein is expressed and purified. HSP70L1-M2 induced strong RSV-and CTL epitope-specific CTL responses and mixed T helper cell (Th)1/Th2 response in BALB/c mice.
