The Clone And Expression Analysis Of Rabbit IFRG Gene

Objective To construct prokaryotic expression vector carrying rabbit ovary IFRG gene and express it in E.coli BL21(DE3) in that recombinant IFRG protein is very critical to the study on the function of interferon in early embryo development. Last, we analysised the expression of IFRG gene in seven different rabbit tissue by RT-PCR. Methods1. Total RNA was isolated with RNAprep pure Tissue Kit from fresh rabbit ovary,and the isolated total mRNA was used for preparation of IFRG cDNA RT-PCR.2. Total RNA was isolated with RNAprep pure Tissue Kit from rabbit heart. Liver,spleen,lung,ovary,cerebra,kidney,and the total RNA was used for RT-PCR.3. The IFRG gene was cloned by insertion of IFRG cDNA into vector pGEM-T using T/A clone and the recombinant pGEM-T/IFRG was amplified by expansion of E.coli DH5a transformed with the recombinant pGEM-T/IFRG.The pGEM-T/IFRG was identified by digestion with the enzyme EcoRl and enzyme Xhol,and sequence analysis.4. A prokaryotic expression system,pET-41(c)-IFRG,was constructed with the cloned IFRG gene and pET-41(c),and it was used to transform E.coli BL21(DE3).The fusion protein expressed in transformed E.coli BL21(DE3) was identified by SDS-PAGE.Rsults1. The IFRG cDNA produced by RT-PCR was found to be 416bp in size as demonstrated by agarose electrophoresis.2. The resul of rabbit heart,Liver,spleen,lung,ovary,cerebra,kidney by RT-PCR displays that IFRG gene has different degree expression in seven tissues.3. The IFRG cDNA was used to clone IFRG gene by insertion it into vectors pGEM-T.endonucleases digestion showed that the size of cloned IFRG gene was founed to be the same as the IFRG cDNA.Sequencing demonstrated the cloned gene was 99.7% identity to the IFRG sequence from the database of GeneBank.these results indicated that the gene cloned in the expression was IFRG gene.4. The IFRG DNA contained in pET-41(c)-IFRG was also of 99.7% identity to the IFRG sequence from the database of GeneBank.When transformed with pET-41(c)-IFRG,expressed by the transformed E.coli BL21(DE3) expressed a fusion protein of 41KD as indicated by SDS-PAGE.Conclusion1. The gene cloned in the present study is rabbit ovary IFRG gene.2. The result by RT-PCR displays that the IFRG gene has expression in seven tissues,but high expression in heart,liver and ovary,low expression in cerebra and lidney.3. The recombinant plasmid pET-41(c)-IFRG constructed in the present study is a functional prokaryotic expression system.4. The fusion protein expressed in E.coli BL21(DE3) transformed with pET-41(c)-IFRG is IFRG protein.