Construction, expression, putification and bioactivity analysis of a two functional domains containing small molecule CR1 derivative
- VernacularTitle:双功能域小分子补体受体1型衍生物的构建、表达、纯化及生物功能鉴定
- Author:
Yongtao YANG
;
Li HE
;
Gaoke LIU
;
Bing TAN
;
Zhengqing WANG
- Publication Type:Journal Article
- Keywords:
Complement receptor type 1;
SOE-PCR;
Prokaryotic expression;
Protein purifica-tion;
Bioactivity analysis
- From:
Chinese Journal of Microbiology and Immunology
2008;28(11):1044-1049
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct and express a small complement receptor type 1 (CR1) deriv-ative which contained two functional domains. Methods Total RNA was isolated from the peripheral blood mononuclear cell. The functional fragment Ⅰ of CR1 was amplified using the RT-PCR. The functional frag-ment Ⅱ was amplified with the plasmid of pET-32a-CR1-SCR15-18 as template which had been already con-structed in our laboratory. Then the chimeric gene that contained the two fragments was constructed with spli-cing overlap extention PCR. The chimeric gene was then inserted into the plasmid of pET-32a (+) and transformed into E. coli Rosetta(DE3). The inserted gene was verified by enzyme digestion and DNA se-quencing. After induced by IPTG, the expressed protein was analyzed by SDS-PAGE and Western blot. Then the fusion protein was purified by Ni-NTA affinity column and renatured through dialysis. The comple-ment inhibition activity was determined by CH50 method. Results The chimeric gene was successfully cloned into pET-32a(+). The result of SDS-PAGE and Western blot confirmed the expressed protein and showed that the molecule mass(Mr) of the expressed protein was 63×103. The purity of the recombinant protein was up to 92% after Ni-NTA column affinity chromatography. The bioactivity assay showed the fusion protein had a concentration-dependent complement inhibition activity within the concentration range of 0-200 μg/ml. Conclusion The two functional domains contained small CR1 derivative was successfully construc-ted and expressed in E. coli Rosetta. The fusion protein had a relative high bioactivity, providing a basis for further function experiment in vivo.
