Construction of pGEX-KG/mTSARG3 recombinant vector and preparation of anti-mTSARG3 polyclonal antibody / 南方医科大学学报

ABSTRACT

<p><b>OBJECTIVE</b>To construct pGEX-KG/mTSARG3 recombinant vector and prepare the fusion protein to obtain rabbit polyclonal antibody against mTSARG3.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>