[Construction of pGEX-KG/mTSARG3 recombinant vector and preparation of anti-mTSARG3 polyclonal antibody].
OBJECTIVE
To construct pGEX-KG/mTSARG3 recombinant vector and prepare the fusion protein to obtain rabbit polyclonal antibody against mTSARG3.


METHODS
The open reading frame (ORF) of mTSARG3 gene was amplified from mouse testis cDNA library by PCR. The products were cloned into pGEM-T Easy vectors and sequenced. The recombinant plasmids were digested by EcoRI and SalI and subcloned into PGEX-KG vector. After identification by DNA sequence analysis, the recombinant plasmids were transformed into component E.coli BL21 cells, and the GST/mTSARG3 fusion protein was expressed with IPTG induction. The anti-mTSARG3 polyclonal antibody was produced by immunization of rabbits with the fusion protein. The resultant antibody was purified by antigen column and used for Western blotting for detecting mTSARG3 expression.


RESULTS
The recombinant vector pGEX-KG/mTSARG3 was successfully constructed. GST/mTSARG3 fusion protein was expressed abundantly at 4 h after IPTG induction and polyclonal antibodies were obtained. Western blot analysis demonstrated that the antibodies specifically detected mTSARG3 expression in E.coli BL21.


CONCLUSIONS
We have successfully constructed pGEX-KG/mTSARG3 recombinant vector and obtained rabbit polyclonal antibody, which may facilitate further investigation of the role of mTSARG3 in spermatogenesis.
Liu
Gang
G
Institute of Reproduction and Stem Cell Engineering, Central South University, Changsha 410078, China. lg5809cn@yahoo.com.cn
Lu
Guang-xiu
GX
D000818:Animals; D000906:Antibodies / Q000276:immunology*; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics*; D006360:Heat-Shock Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology*; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D018832:Molecular Chaperones; D051017:Apoptosis Regulatory Proteins
D000906:Antibodies; D006360:Heat-Shock Proteins; D011993:Recombinant Fusion Proteins; C531170:DNAJB13 protein, mouse; D018832:Molecular Chaperones; D051017:Apoptosis Regulatory Proteins