Follistatin Gene CDNA Cloning And Eucaryotic Expression From Buffalo

Follistatin(FS) have abroadly biological functions, especially in regulatingthe reproductive action, muscle development and growth. There are some studiesabout the FS biological functions in several animal, however never in buffalo.The object of this study is to elucidate the effect of FS on buffalo reproductivemechanism and improve buffalo reproductive ability.There has not release buffalo FS cDNA sequence in public, so far.thereforethe primers were designed based on the Bos Taurus FS cDNA released at theGeneBank. The total RNA was extracted from the ovary tissue and FS cDNAsequence was amplified by the RT-PCR. The objective segment was subclonedinto pMD-18T vector after purification. The recombinants were identified withPCR, restrictive cleavage and sequencing, respectively.The results showed that buffalo FS cDNA was successfully cloned in thisstudy. The homologous analysis indicated that buffalo FS cDNA comparing withthe reported Bos Taurus, Equus cabaIlus, Homo sapiens, Sus scrofa were 98.8%,94.0%, 91.7% and 93.3% homologous,respectively. Comparing with Bos Taurus,12 point mutants were found in buffalo FS cDNA, among them, two pointmutant lead to amino acid alter. With the bFS cDNA sequence obtained from the first experiment,theeukaryotic expression vector pEGFP-bFS was constructed through enzymolysisdigested,ligation,transform E.coli,filtration,checkupand sequencing surveyetc,which has a full length of 5.7kb. pEGFP-bFS transfecting buffalo fibroblastcell and BHK cell line with Lipofectamine TM 2000. At 48 hours after culture,through examinations of RT-PCR and Western-blot,under phasecontrastfluorescence microscope. It is verified that the expression of pEGFP-bFS hasbeen successful in two kinds of cell.Therefore,the rerults in this study haveprovided more evidence for further studies in biological activities of FS andpreparation of engineering vaccines so as to improve reproductive ability andgrowth rate of buffalo.