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目的:克隆人分泌型IL-6 cDNA,构建IL-16的原核表达质粒,并观察其在大肠杆菌中的表达。方法:采用PCR技术从健康成人外周血单个核细胞(PBMC)总cDNA文库中扩增人分泌型IL-16 DNA片段;PCR产物分离纯化后,将其克隆至pUC18-T载体中,经PCR、酶切鉴定及DNA测序确证后,将该基因定向插入原核表达载体pMAL-C2,获得重组质粒pMAL-IL-16,然后将其转化至E.coli DH5α,经IPTG诱导表达,采用SDS-PAGE及Western blot法鉴定表达产物。结果:获得了编码人分泌型IL-16的cDNA,经测序证明其长度为393 bp,编码130个氨基酸;构建了IL-16原核表达载体,并应用E.coli DH5α表达,得到1个相对分子质量(Mr)约56 000的IL-16重组融合蛋白,与理论大小相符,且经SDS-PAGE及Western blot法验证。表明人分泌型IL-16原核表达载体构建并表达成功。结论:本实验成功克隆了人分泌型IL-16 cDNA,构建了IL-16原核表达载体,并在大肠杆菌中获得表达,为IL-16的纯化奠定了基础。
OBJECTIVE: To clone human secreted IL-6 cDNA and construct prokaryotic expression plasmid of IL-16 and observe its expression in E. coli. Methods: The human secretory IL-16 DNA fragment was amplified from the total cDNA library of healthy adult peripheral blood mononuclear cells (PBMCs) by PCR. The PCR product was isolated and purified and cloned into pUC18-T vector. The recombinant plasmid pMAL-IL-16 was inserted into the prokaryotic expression vector pMAL-C2 to obtain the recombinant plasmid pMAL-IL-16. The recombinant plasmid pMAL-IL-16 was transformed into E. coli DH5α and induced by IPTG. SDS- Western blot method to identify the expression product. Results: The cDNA encoding human secreted IL-16 was obtained and sequenced. The length of the cDNA was 393 bp and encoded a polypeptide of 130 amino acids. The prokaryotic expression vector of IL-16 was constructed and expressed by E. coli DH5α. One relative molecule The IL-16 recombinant fusion protein with a mass of Mr about 56,000 was in agreement with the theoretical size and was verified by SDS-PAGE and Western blot. Prokaryotic expression of human IL-16 prokaryotic expression vector was constructed and expressed successfully. Conclusion: The recombinant human IL-16 cDNA was successfully cloned and the prokaryotic expression vector of IL-16 was constructed and expressed in Escherichia coli, which laid the foundation for the purification of IL-16.