Cloning and expression of extracellular region of human FSHR in E.coli
YAN Ping
SHEN Zigang
HE Wei
CHEN Zhengqiong
HE Haiyang
ZHANG Ji
YANG Xia
TANG Yan
WU Yuzhang
LIANG Zhiqing
LI Jintao
Abstract：Objective To construct a prokaryotic expression vector of extracellular region of human follicle stimulating hormone receptor (FSHR), purify this protein and prepare its antiserum. Methods Total RNA was isolated from human testis, while extracel-lular region gene of human FSHR was amplified with RT-PCR. The enzyme-digested target fragment was cloned into pET32 a (+) ex-pression vector, and then transferred into E. coli BL21 ( DE3 ), in which extracellular region of human FSHR was induced to express by IPTG. After the inclusion body protein was purified by Ni-NAT affinity chromatography, dialyzed and identified by Western blotting,male mice were immunized with the purified fusion protein for obtaining the antiserum. Results The gene fragment at length of 1 047 bp was obtained and cloned into pET32 a (+) successfully. SDS- PAGE showed a protein band with relative molecular weight of 58 000, which was consistent with the expectation, mainly existing in the inclusion body of E. coli. The titer of the antiserum to the pu-dried protein was 1 : 12800. Western blotting showed good antigenicity of the recombinant protein. Conclusion A recombinant extra-cellular region of FSHR protein and the specific polyclonal antibody have been obtained, which have some potential value in studying the function of FSHR protein.
Keywords：follicle stimulating hormone receptorRT-PCRprokaryotic expressioninclusion body protein
Publication Date：2009-01-01
Online Publishing Date：2025-08-15(First online date of this platform, not the publication date of the document)
Pages：5( 374-377,381 )