人regucalcin cDNA高表达重组质粒的构建及其在原核细胞中的表达

Construction of human regucalcin prokaryotic expression vectors and their expression

目的原核表达获得大量人regucalc in(RGN)蛋白。方法用逆转录聚合酶链反应(RT-PCR)技术从人肾皮质近曲小管上皮细胞(HK-2)中扩增RGN全长cDNA,将扩增的产物连接于测序载体pMD18-T,测序鉴定准确后,进行酶切,将正确的RGN全长cDNA与原核表达载体pET42b(+)构建成重组质粒pET42b(+)-RGN。将pET42b(+)-RGN先转化入大肠杆菌DH5α,提取质粒经双酶切鉴定正确后,再转化入表达宿主大肠杆菌DE3菌株。对转化菌株进行诱导、破菌,进行十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)与免疫印迹分析,鉴定RGN融合蛋白质表达的正确性。RGN重组融合蛋白经N i2+亲和层析纯化,达电泳纯。结果构建了重组质粒pET42b(+)-RGN并获得高效表达,RGN重组融合蛋白经异丙基硫化-β-D-半乳糖苷(IPTG)诱导后以包涵体形式存在,表达的蛋白质相对分子质量为34 000。结论人RGN蛋白在pET42b(+)原核表达载体可获得高效表达,为进一步了解RGN蛋白质的结构、功能及其抗体的制备等奠定了基础。

Objective To clone, identify and express human regucalcin （RGN） from human kidney-2 （HK-2） cell, Methods RGN cDNA full length was amplified with reverse transcription-polymerase chain reaction （RT-PCR） from HK-2 cell, inserted into pMD18-T and sequenced. Then the right RGN cDNA full length was cloned into pET42b （ ＋ ） prokaryotic expression vector to construct recombinant plasmid pET42b （ ＋ ） -RGN, then the recombinant was transformed into Escherichia coli （E. coli） DH5α, and plasmid DNA was extracted , identified with restriction endonucleases. Following transformation pET42b（ ＋ ）-RGN into DE3, the isopropyl-beta-D-thiogalactopyranoside （IPTG） induced cultures with the expression of the expected protein were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis （SDS-PAGE） and Western-blot. The Histidine-tagged RGN was purified with Ni2^＋ -NTA superflow affinity chromatography. Results Both cloning and expression of recombinants of RGN were obtained. The RGN was expressed an apparent MW of 34 000 by SDS-PAGE and Western-blot. Conclusions Human RGN protein can be successfully obtained from the pET42b（ ＋ ） system . It provides basis for further studying on the function of RGN and producing its monoclonal antibody.