Construction and expression of procaryotic expression vector of a chimeric GPI-B7-1 cDNA
10.3760/cma.j.issn.1007-631X.2009.01.014
- VernacularTitle:人B7-1基因与GPI信号肽序列融和基因的原核表达载体的构建及表达
- Author:
Jianwei WANG
;
Yingbin LIU
;
Yanjing CAO
;
Xuan WANG
;
Jiangtao LI
;
Ying KONG
;
Xiaoming MA
;
Shuyou PENG
- Publication Type:Journal Article
- Keywords:
Gene expression;
Glycosylphos phatidylinositols
- From:
Chinese Journal of General Surgery
2009;24(1):38-41
- CountryChina
- Language:Chinese
-
Abstract:
Objective Through constructing prokaryotic expression vector pET-30a-GPI-B7-1, to gain purified GPI-B7-1 fusion protein so as to confirm the tumor immune effect. Methods The DNA fragment encoding the signal for GPI-anchor attachment of hPLAP-1 and the cDNA encoding the human costimulatory molecule CD80 ( BT-1 ) were cloned from fresh placenta and human peripheral blood monocytes (PBMC) respectively. The two fragment were annealed to form a fusion gene (GPI-BT-1) by PCR. Then the fusion gene was inserted into the prokaryotic expression vector pET-30a, resulting in pET-30a-GPI-BT-1. Transfer to E. coli BL21, purified fusion protein were analysed by SDS-PAGE and Western blot. Results Agarose gel electrophoresis map of GPI and BT-1 PCR products show that GPI goal gene strap was seen at 133bp region and BT-1 goal gene strap at 792 region. Identification of recombinant pET-30a-GPI-B7-1 by restriction enzyme and PCR illustrate two goal fragment for 5000 bp and 900 bp, to realize the expression of fusion gene ( GPI-B7-1 ) at the E. coli BL21. The fusion protein was successfully produced in the pET expression system induced by IPTG and purified by Ni2 + -NTA agarose column. By SDS-PAGE and Western blot analysis, the observed molecular weight of the fusion protein was 38 kDa. Conclusion The purified GPI-B7-1 fusion protein can be obtained from E. coli BL21 transfered by prokaryotic expression vector pET-30a-GPI-B7-1, which will prove useful tool for the study of tumor immune therapy.
