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当前位置: 首页 > 文章 > 版纳微型猪近交系性别决定基因(SRY)原核表达载体的构建及蛋白高效表达 云南农业大学学报(自然科学) 2012,27 (2) 198-202
Position: Home > Articles > Construction of Prokaryotic Expression Vector for Sex-determining Gene(SRY) of Banna Mini-pig Inbred Line and Its High Efficient Expression Journal of Yunnan Agricultural University(Natural Science) 2012,27 (2) 198-202

版纳微型猪近交系性别决定基因(SRY)原核表达载体的构建及蛋白高效表达

作  者：
霍金龙;王配;成文敏;王淑燕;曾养志
单  位：
云南农业大学
关键词：
版纳微型猪近交系;Y染色体性别决定基因;SRY;原核表达
摘  要：
为了构建版纳微型猪近交系SRY的原核表达载体pET-32a(+)-SRY,并通过诱导使其在大肠杆菌中获得高效表达,研究采用添加限制性内切酶位点的引物特异性扩增SRY,连入pMD19-T simple载体,转化入大肠杆菌DH5α,克隆后提取pMD19-T-SRY阳性重组质粒,使用相同的内切酶同时对pMD19-T-SRY质粒和原核表达pET-32a(+)载体进行酶切,连接后使SRY定向克隆到pET-32a(+)表达载体中。经PCR、酶切和测序鉴定后,重组质粒转化大肠杆菌感受态DH5α,提取质粒后再次转化大肠杆菌Rosetta(DE3),用不同浓度的异丙基硫代半乳糖苷(IPTG)诱导表达,并通过15%SDS-PAGE鉴定。结果显示,不同浓度IPTG诱导的SRY均在大肠杆菌中进行了高效特异性融合表达。
译  名：
Construction of Prokaryotic Expression Vector for Sex-determining Gene(SRY) of Banna Mini-pig Inbred Line and Its High Efficient Expression
作  者：
HUO Jin-long,WANG Pei,CHENG Wen-min,WANG Shu-yan,ZENG Yang-zhi(1.Key Laboratory of Banna Mini-pig Inbred Line of Yunnan,Yunnan Agricultural University,Kunming 650201,China; 2.College of Animal Science and Technology,Yunnan Agricultural University,Kunming 650201,China)
关键词：
Banna mini-pig inbred line(BMI);sex-determining gene of Y chromosome;SRY;prokaryotic expression
摘  要：
To construct prokaryotic expression vector pET-32a(+)-SRY of Banna mini-pig inbred line(BMI) and induce it to express efficiently in E.coli cell,SRY was amplified by primers with restriction enzyme sites,and it was inserted into pMD19-T simple vector and transferred into the bacterium DH5α.After replication,the pMD19-T-SRY recombinant plasmids were extracted,then pMD19-T-SRY and pET-32a(+)were digested by the same restriction enzymes at the same time.By this directed cloning technique,the SRY was inserted into pET-32a(+) expression plasmid.After PCR identifying,restriction enzyme digestion and sequencing testing,the pMD19-T-SRY recombinant plasmids were transformed into competent cell DH5α.The pMD19-T-SRY plasmids were extracted and transformed to competent cell Rosetta(DE3).pMD19-T-SRY was induced with different concentrations of isopropyl-β-D-thiogalactopyranoside(IPTG) and identified on 15% SDS-PAGE.The results showed that the fusion protein had high efficient expression in SDS-PAGE when induced by different concentrations of IPTG.

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