Cloning and Prokaryotic Expression of Rat RVLG and Preparation of Mouse Anti-RVLG Polyclonal Antibody

Abstract In order to identify rat ovarian germ cells, the authors expressed and purified rat RVLG protein in E. coli cells and prepared a mouse anti-rat RVLG polyclonal antibody. The rat RVLG cDNA was obtained from rat testicle tissue by RT-PCR and was cloned into the vector pMD19-T. Sequence analysis proves that the cloned RVLG cDNA fragment was 60 bp longer than that released in the GenBank (NM_001077647), resulting from an alternative splicing of the RVLG pre-mRNA. The RVLG cDNA was doubly digested with the restriction endonucleases Bam H I and Eco R I, and then was extracted from gel and inserted into the prokaryotic expression vector pGEX-4T-1. The recombinant expression plasmid pGEX- RVLG was verified for successful construction and then was transformed into E. coli BL21(DE3) for induction to express the GST-RVLG fusion protein by IPTG. The GST-RVLG fusion protein was expressed in E. coli BL21 (DE3) at a high level, which accounts for more than 10% of the total bacterial cellular protein. The purified RVLG protein was used as an antigen to immunize KM mouse for the production of polyclonal antibody in ascitic fluid followed by celiacly injecting the mouse with S180 cells. The mouse anti-rat RVLG antibody was analyzed by ELISA, Western blotting, and Immunohistochemistry for its specificity and titer. The antibody could recognize RVLG protein specifically and its titer is about 1:20 000. These results confirm that the mouse anti-rat RVLG polyclonal antibody with high affinity and specificity has been prepared successfully, and lays a foundation for the authors' ongoing research on the specific expression of RVLG in rat ovary.