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。
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.
出处
《云南农业大学学报（自然科学版）》
CAS
CSCD
北大核心
2012年第2期198-202,共5页
Journal of Yunnan Agricultural University:Natural Science
基金
国家自然科学基金项目(31160439)
云南省教育厅科学研究基金项目(2010C253)
云南农业大学动物科学技术学院科学研究基金项目(YNDKKY1005)
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
Banna mini-pig inbred line（BMI）
sex-determining gene of Y chromosome
SRY
prokaryotic expression
