Expression of Cyanovirin-N in E.coli and preparation of polyclonal antibody to Cyanovirin-N
- VernacularTitle:蓝藻抗病毒蛋白-N在大肠杆菌中的表达及其抗体的制备
- Author:
Xiaohua HUANG
;
Jun ZHANG
- Publication Type:Journal Article
- Keywords:
Cyanovirin-N;
Expression;
ELISA;
Western blot
- From:
Chinese Journal of Immunology
1999;0(12):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To construct Cyanovirin-N(CV-N) expression plasmid pET-CVN and express the protein in E.coli, and to prepare specific antibody against Cyanovirin-N.Methods:In this study, the encoding sequence of Cyanovirin-N was obtained by PCR. The expression plasmid pET-CVN was constructed by inserting the encoding sequence of CV-N into pET-His containing T7 promoter and then BL21(DE3)strain was transformed. Expression of CV-N was induced by IPTG at 30℃ for 8 h. Cyanovirin-N was refolded by using dialysis method. The target protein was purified through Ni2+-NTA Agarose Fast Flow. The purified Cyanovirin-N was used as an antigen to immunize rats,and the polycolonal antibody to Cyanovirin-N was obtained.Results:Cyanovirin-N was cloned and expressed in E.coli successfully. SDS-PAGE analysis showed that the Cyanovirin-N was expressed in the form of insoluble inclusion body,and the expression amount of Cyanovirin-N was about 42.08% of total protein. With ELISA detection the titer of antibody was more than 1∶8 000, Western blot analysis showed that the antibody can bind with Cyanovirin-N specifically.Conclusion:Cyanovirin-N expression plasmid pET-CVN has been constructed, Cyanovirin-N with bioactivity has been obtained. The prepared antibody against Cyanovirin-N has well specificity and reactivity and can be used to detect the expression products by recombinant strains of E.coli.The work lays a foundation further study of its functions.
