Cloning of Bmi1 cDNA from mouse testis and its expression in E. coli BL21.
- Author:
Shi-qing ZHANG
1
;
De-xue LI
;
En-zhong LI
;
Chang-yong WANG
;
Xue-ming ZHANG
;
Jing-yan LU
Author Information
- Publication Type:Journal Article
- MeSH: Animals; Antibodies, Monoclonal; immunology; Cloning, Molecular; DNA, Complementary; genetics; Escherichia coli; genetics; Gene Expression; Male; Mice; Nuclear Proteins; biosynthesis; genetics; immunology; Polycomb Repressive Complex 1; Proto-Oncogene Proteins; biosynthesis; genetics; immunology; Recombinant Proteins; biosynthesis; immunology; Repressor Proteins; biosynthesis; genetics; immunology; Testis; metabolism
- From: National Journal of Andrology 2006;12(4):308-314
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo produce BMI1 polyclonal antibody, mouse Bmi1 cDNA was cloned from mouse testis and expressed in E. coli BL21.
METHODSBmi1 gene was amplified from mouse testis by RT-PCR and inserted into the prokaryotic expression vector pET-28c(+). Subsequently the recombined vector was transformed and expressed in E. coli BL21 (DE3) and the immunogenicity of recombined protein BMI1 (rBMI1) was tested by Western blot.
RESULTSMouse Bmi1 cDNA of 975 bp was successfully cloned and recombined. E. coli BL21 strains expressed rBMI1 were screened. The expression protein amounted to 12% of the total bacterial protein after induced with IPTG, which included inclusion body and soluble protein. Inclusion body was the major pattern of the expression that amounted to 71% of the insoluble protein. Western blot analysis showed that rBMI1 could be specially recognized by mouse monoclonal IgG1 anti-BMI1 and His-tag antibody.
CONCLUSIONThere was expression of Bmi1 gene in mouse testis. Mouse Bmi1 cDNA was successfully cloned and expressed prokaryoticly.
