Abstract: In this study,the total RNA was isolated from Guangxi Bama mini-pig ovary tissue with RNA_(watson) extraction kit.The complementary DNA(cDNA) encoding Follistatin protein was ampilified by the reverse transcription polymerase chain reaction(RT-PCR) method and a pair of differential primers.The amplified cDNA fragment was inserted into pMD18-T vector.The recombined vectors were verified through the sequence survey.The results showed that the cloned Follistatin cDNA was highly homologous with the reported nucleotide encoding swine Follistatin in GenBank(99.4%).Meanwhile,the identified Follistatin cDNA was inserted in the multiple cloning sites of plasmid pET 32a+ to construct prokaryotic expression vector,the latter was transformed into the E.coli.BL_(21) induced by IPTG.Then a fusion protein was obtained(about 58.4 ku) in SDS-PAGE and Western blotting.These results showed that the Follistatin cDNA was expressed in prokaryotic cells.

Key words: Guangxi Bama minipig, Follistatin, Cloning, Prokaryotic expression

摘要： 目的通过RT-PCR扩增获得广西巴马小型猪Follistatin的cDNA全序列,经克隆测序分析其cDNA序列结构,进而构建pET-Fo原核表达质粒,使用IPTG诱导表达获得其融合蛋白,为将来进一步研究Follistatin对广西巴马小型猪肌肉生长和繁殖性能的影响奠定基础。方法从广西大学巴马小型猪卵巢中提取总RNA,并以其为模板,应用逆转录-聚合酶链式反应(RT-PCR),在合成的特异性引物引导下,扩增获得广西巴马小型猪卵泡抑素(Follistatin)cDNA的全序列,长度为1 035 bp。将PCR产物克隆到pMD18-T载体后进行序列测定及分析。结果广西巴马小型猪卵泡抑素cDNA序列与GenBank中已报道的家猪卵泡抑素同源性高达99.4%。同时,将目的基因插入到原核表达载体pET 32a+多克隆位点中,构建了Follistatin的原核表达载体,并转化于大肠杆菌BL21。转化菌经异丙基硫代半乳糖苷(IPTG)诱导,变性聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白质免疫印迹(Western blotting)分析。结论实验已成功地表达了Follistatin的融合蛋白(约58.4 ku)。

关键词: 广西巴马小型猪, 卵泡抑素, 克隆, 原核表达