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)。
In this study, the total RNA was isolated from Guangxi Bama mini-pig ovary tissue with RNAwatson 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 reeombined 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. BLj induced by IPTG. Then a fusion protein was obtained （about 58.4 ku） in SDS-PAGE and Western blotting. These results showed that the Follistatiu cDNA was expressed in prokaITotic cells.
出处
《实验动物科学》
2007年第6期26-29,5,共5页
Laboratory Animal Science
基金
国家科技攻关计划(2004BA717B-01-03)
广西科学研究与技术开发计划项目(桂科能0630006-6B)
广西自然科学基金项目(桂科自0542025)
关键词
广西巴马小型猪
卵泡抑素
克隆
原核表达
Guangxi Bama minipig
Follistatin
Cloning
Prokaryotic expression
