目的研究分枝杆菌热休克蛋白65(HSP65)重组质粒的构建与原核表达。方法 HSP65的开放阅读框经聚合酶链反应(PCR)扩增后,经NcoⅠ与NotⅠ内切酶依次酶切,连接至酶切后的原核表达载体PET-28a中,然后将重组质粒转入大肠杆菌BL-21中,PCR筛选阳性克隆,并经DNA测序进一步验证。阳性克隆进一步扩大培养,并进行乳糖诱导,十二烷基磺酸钠聚丙烯酰胺凝胶电泳对菌体蛋白进行检测。结果成功扩增出1 623 bp的目的片段HSP65;测序结果显示,该片段与目的序列完全相符,重组子PET-28a/HSP65构建成功;乳糖诱导后的HSP65在大肠杆菌中实现高表达。结论分枝杆菌HSP65蛋白的成功表达,为研究其在抗肿瘤疫苗中的应用奠定了基础。 Objective To study the construction and prokaryotic expression of mycobacterium heat shock protein 65 (HSP65) recombinant plasmid. Methods The open reading frame of HSP65 was amplified by polymerase chain reaction (PCR) and sequenced by Nco Ⅰ and Not Ⅰ endonucleases. The recombinant plasmid was ligated into the prokaryotic expression vector PET-28a and then transferred into the large intestine In Bacillus BL-21, positive clones were screened by PCR and further verified by DNA sequencing. The positive clones were further expanded and cultured, and lactose was induced. Sodium dodecyl sulfate polyacrylamide gel electrophoresis was used to detect the bacterial proteins. Results A fragment of 6265 bp was successfully amplified. Sequencing results showed that the fragment was completely consistent with the target sequence, and the recombinant PET-28a / HSP65 was successfully constructed. Lactose-induced HSP65 was highly expressed in E. coli. Conclusion The successful expression of mycobacterial HSP65 protein laid the foundation for the study of its application in anti-tumor vaccine.