新基因BC047440原核表达载体构建及其重组蛋白的表达纯化

Constructing prokaryotic vector of BC047440 gene expression and purification of its recombinant protein

目的构建肝癌相关新基因BC047440原核表达载体，表达并纯化出其重组蛋白。方法从HepG2细胞中提取总RNA，经逆转录-聚合酶链式反应（RT-PCR）扩增BC047440cDNA，克隆进质粒pMD19-T，转化E．coliDH5a，测序正确后，酶切目的基因片段，插入质粒pET-28a（＋），转化E．coliBL21，IPTG诱导蛋白表达，Ni—NTA柱亲和层析纯化重组蛋白。结果电泳证实RT-PCR扩增产物与预期目的基因BC047440长度一致。RT-PCR纯化产物与载体pMD19-T连接后，经蓝白斑筛选，挑出5个白色菌落做酶切鉴定，其中2个菌落被证实为阳性克隆。测定其基因序列，结果显示与Genbank公布的BC047440基因序列完全一致。将BC047440cDNA插入质粒pET一28a（＋），转化E．coliBL21，培养过夜后，挑选7个白色菌落做酶切鉴定，证实均为阳性克隆。IPTG诱导其中一个克隆表达蛋白，SDS-PAGE电泳分析表明，在相对分子质量23000左右出现新的蛋白表达条带，诱导4h后表达蛋白量最多，占菌体总蛋白的22．3％。将诱导3h的菌体超声破碎后，Ni柱亲和层析，50和125mmol／L咪唑洗脱液SDS-PAGE电泳显示出清晰的单一条带。结论成功构建BC047440基因原核表达载体，表达并纯化出重组蛋白，为深入研究其临床应用打下基础。

Objective To construct prokaryotic expression vector of BC047440 gene to express and purify its recombinant protein. Methods The total RNA was extracted from HepG2 cells. The BC047440 gene fragment was amplified from the total RNA by RT-PCR. The resulting product was cloned into pMD19-T vector and sequenced. Then the confirmed BC047440 cDNA was cloned into plasmid pET-28a（ ＋ ） and transformed into E. coil BL21 where it was induced to express proteins by i- sopropyl-l-thio-b-Dgalactopyranoside （IPTG）. The expression of protein was analyzed through SDS- PAGE cells induced for 3 h by IPTG harvesting. Then it was sonicated briefly and the proteins were purified through affinity chromatography. Results The target sequences were specifically amplified through RT-PCR, cloned into pMD19-T vector, and then transformed into E. coli DHSa. Five white colonies were selected and cut by BamHI and EcoRI separately. The plasmids of two white colonies showed positive results and was sequenced, demonstrating to be the same as that of BC047440 gene in GenBank. Then BC047440 cDNA was cut down from pMD19-T, ligated into the vector pET-28a（＋） and then transformed into E. coli BL21. After an overnight incubation, seven white colonies were picked and showed positive results after being digested by both BamHI and EcoRI. After IPTG induc- tion of one positive colony, a new protein band about Mr 23000 showed on SDS-PAGE. The percentage of expressed product over total bacterial proteins after 4 hours of induction was 22.3%. After affinity chromatography, SDS-PAGE showed only one clear band existed in 50 mmol/L or 125 mmol/L imidizone elution. Conclusion The prokaryotic expression vector of BC047440 gene has been successfully constructed. Meanwhile, purified recombinant proteins are obtained through affinity chromatography, laying foundation for further study of its clinical use.