Expression, purification and immunogenicity analysis of recombinant MPB64 using baculovirus expression system
10.3969/j.issn.1000-484X.2014.07.013
- VernacularTitle:MPB64在杆状病毒系统中的表达纯化及免疫原性鉴定
- Author:
Dan XU
;
Chao JIANG
;
Yu CHEN
;
Jun AI
;
Xiaoyan WANG
;
Xiaokun LI
- Publication Type:Journal Article
- Keywords:
MPB64;
Baculovirus expression vector system;
Sf 9 insect cells;
Mycobacterium tuberculosis
- From:
Chinese Journal of Immunology
2014;(7):921-926
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To express mycobacterium tuberculosis protein MPB 64 in baculovirus insect cell expression system ,and identify its immunogenicity.Methods:The target gene MPB64 was connected to pFastBac vector ,then the pFastBac-MPB64 plasmid which was harvested would transformed to DH10Bac competent,and the target gene was transposition into Bacmid by Tn7 transposase fragment,therefore Bacmid-MPB64 Shuttle vector was obtained.The shuttle vector was packaged by liposomes and transfected Sf 9 cells to harvest P1-generation virus ,then high titers of P4 generation virus was harvested by repeat transfected Sf 9 cells three times.The target protein MPB64 was purified from the supernatant by Q Sepharose FF and Ni affine chromatography ,which were used to immunize BALB/c mice.Antibody changes in serum would be detected ,and the proliferation of immunized mice spleen cells would be detected by MTT,detected the IFN-γsecretion by MPB64 stimulated spleen cells by ELISA method.Results: MPB64 successfully expressed in insect cells.The purity of target protein was over 90% and yield up to 35 mg/L after purification.Purified protein can effectively stimulate BALB/c mice to produce antibodies , increase the content of IFN-γmedium in mice spleen cells ,and significantly promoting proliferation in spleen cells between 0.2-100 μg/ml.Conclusion: MPB64 which has immunogenicity was successfully expressed in baculovirus insect cell expression system ,that open a new avenue for tuberculosis vaccine production.
