Cloning and high level expression of gene encoding human sCR1 binding domain in E.coli
- VernacularTitle:人sCR1结合C3b结构域基因的克隆及在大肠杆菌中高效表达
- Author:
Xue LUO
;
Zhengqing WANG
- Publication Type:Journal Article
- Keywords:
soluble complement receptor 1;
reverse transcription-polymerase chain reaction;
complementary DNA clone;
protein expression
- From:Journal of Third Military Medical University
2003;0(15):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective To clone and express the gene encoding the binding domain of human soluble complement receptor type 1 (sCR1). Methods sCR1-SCR15-18 cDNA was amplified using RT-PCR from human monocytes of peripheral blood and sequenced using vector pMD-18T. Recombinant pET32-sCR1-SCR15-18 was constructed using prokaryotic expression vector pET32 and transformed into bacterium BL21. IPTG was used to induce gene expression and the obtained expression product was identified by immunoblotting. Results The gene segment that specifically encodes sCR1 was synthesized, the sequence of which was consistent with that of sCR1-SCR15-18 cDNA as registered at GenBank. A prokaryotic expression recombinant pET32-sCR1-SCR15-18 was constructed. The amount of target protein accounted for 40% of the total bacterial proteins and inclusion bodies were present in the bacteria. Immunoblotting showed a single positive band at the site of 43?10~(3). Conclusion The gene encoding sCR1-SCR15-18 was cloned from human monocytes and efficiently expressed in E.coli.
