Study of fusion expression effect on conformation and immune activity of polyepitope protein,M.RCAg-1
10.3760/cma.j.issn.0254-5101.2012.01.013
- VernacularTitle:融合表达对人工重组多表位蛋白M.RCAg-1构象及免疫效能影响的研究
- Author:
Jian WANG
;
Yahui LIN
;
Xiangpeng CHEN
;
Lili LI
;
Jun LI
;
Heng WANG
;
Zhenlong ZHANG
- Publication Type:Journal Article
- Keywords:
Polyepitope protein;
Vector peptide;
Fusion expression
- From:
Chinese Journal of Microbiology and Immunology
2012;32(1):58-64
- CountryChina
- Language:Chinese
-
Abstract:
ObjectiveTo explore the effects of vector fusion peptides on the conformation and immune reactivity of recombinant polyepitope antigens,M.RCAg-1.MethodsWe subcloned polyepitope artificial antigen gene,M.RCAg-1,into prokaryotic expression vectors,pDS-ex,that contain different fusion tags at the N-terminus or no any tag by different restriction enzyme cutting site.Three recombinant proteins expressed by these vectors,named P312-1,P312-2,and P312-3,were purified and purity is greater than 95％.Then BALB/c mice were vaccinated with the three proteins formulated with Freund's adjuvant through intranasal.Serum were collected to detect specific antibodies of M.RCAg-1 and individual epitope by ELISA ; natural parasite antigen was recognized by indirect immunofluorescence assay; mouse specific T lymphocyte activation was detected by enzyme-linked immunosorbent spot test (ELISPOT) ; and the growth of Plasmodium falciparum in vitro to evaluate by growth inhibition assay(GIA),and analyze secondary and tertiary structures of recombinant proteins from different expression vectors by bioinformatics and circular dichroism technique.ResultsThe P312-1,P312-2 have almost the same amino acid sequence,and the three proteins have the same immunogenicity in animal models(P＞0.05),however,the different proteins elicited various T-cell responses,the rabbit antibody induced by these proteins showed diverse efficacy in malaria parasite growth inhibition assaysin vitro ( respectively,93.9％,14.7％,54.3％ ).The significant differences of secondary and tertiary structures were shown in recombinant proteins from different expression vectors,analyzed by bioinformatics and circular dichroism technique,which demonstrated the change of protein molecule spaces conformation,and the obviously change of some epitope locations.ConclusionThese results suggest that the expressed polyepitope artificial antigens originating from the different vector fusion peptides indeed affect the protein folding and,subsequently,the epitope exposure.Thus,these proteins are able to induce both distinct humoral and cellular immune responses in animal models,and they affect the efficacy of immune inhibition against the parasite,the enhancement or suppresses.
