Cloning and expression of human laminin alpha4 LG3-4 module
- VernacularTitle:人层粘连蛋白?4链LG3-4组件的克隆和表达
- Author:
Xufang DAI
;
Jiqin LIAN
;
Yujing ZHANG
;
Zhongmin HU
- Publication Type:Journal Article
- Keywords:
hLN?4LG3-4;
cloning;
expression
- From:Journal of Third Military Medical University
2003;0(11):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective To express and detect the antigenicity of human laminin alpha4 LG3-4 module (hLN?4LG3-4) protein by gene engineering techniques. Methods The cDNA encoding hLN?4LG3-4 was amplified by RT-PCR from human placenta, then inserted into pMD-18T vector by T/A cloning and sequenced. Prokaryotic expression vector pET-28a-LG3-4 was constructed by recombinant DNA technique. The hLN?4LG3-4 fusion protein expressed in BL21(DE3)/pET system was identified by SDS-PAGE, purified by Ni-NTA resin, and assayed by Western blotting. Results The cDNA fragment of hLN?4LG3-4 was cloned successfully. While BL21 (DE3)/pET28a-LG3-4 bacterium was induced with IPTG, a new protein band with a relative molecular weight of 44 000 was shown on SDS-PAGE profile. hLN?4LG3-4 fusion protein of high purity (95%) was obtained and specific protein band was detected by Western blotting. Conclusion The hLN?4LG3-4 fusion protein was successfully expressed.
