RESUMO
In order to identify rat ovarian germ cells, we expressed and purified rat RVLG protein in Escherichia 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 double digested with the restriction endonucleases BamH I and EcoR 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 Escherichia coli BL21(DE3) for induction to express the GST-RVLG fusion protein by IPTG. The GST-RVLG fusion protein was expressed in Escherichia 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 ascetic 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 was 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 lay a foundation for our ongoing research on the specific expression of RVLG in rat ovary.
Assuntos
Animais , Feminino , Camundongos , Ratos , Anticorpos Monoclonais , Sequência de Bases , Clonagem Molecular , RNA Helicases DEAD-box , Genética , Alergia e Imunologia , DNA Complementar , Genética , Escherichia coli , Genética , Metabolismo , Dados de Sequência Molecular , Ovário , Biologia Celular , Metabolismo , RNA Mensageiro , Genética , Proteínas Recombinantes de Fusão , Genética , Alergia e ImunologiaRESUMO
Developing nationality medicine,to research the relationship between theory of Yi nationality medicine and correlated theories of traditional Chinese medicine. Panzhihua city is located in the juncture of Sichuan and Yunnan,and the Yi nationality inhabitants are more,so to understand and develop the Yi nationality medicine is very necessary.This question for discussion though the methods of investigating and studying on the spot and literatures collecting et al,found the characteristics and shortages of theory of Yi nationality medicine as well as the similarities and differences of theory of Yi nationality medicine and correlated theories of traditional Chinese medicine,and putted in discussion.Developing the Yi nationality medicine,in theory we need further excavation and unscrambling.