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

 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。

Construction of Prokaryotic Expression Vector for Sex determining Gene(SRY) of Banna Mini pig Inbred Line and Its High Efficient Expression

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 βD 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.