结核分枝杆菌Ag85B基因的克隆及真核表达载体的构建

Cloning of Ag85B gene of Mycobacterium tuberculosis and its construction of eukaryotic expression vector

目的构建以结核分枝杆菌分泌蛋白Ag85B基因为基础的核酸疫苗。方法采用聚合酶链反应从结核杆菌H37Ra株基因组中,扩增出分泌蛋白Ag85B的成熟蛋白的编码基因 ,用限制性内切酶消化后,插入克隆载体 pUC19中。经酶切鉴定与序列测定证实后 ,以亚克隆法构建于真核表达载体 pcDNA3的相应酶切位点。结果结核分枝杆菌H37Ra株Ag85B成熟蛋白的编码基因经序列测定证实 ,与Erdman株完全一致 ;用EcoRⅠ和HindⅢ双酶切鉴定证实 ,克隆基因正确插入载体pcDNA3。结论以Ag85B成熟蛋白的编码基因为基础的真核表达载体的构建成功 ,为进一步研究其在结核病防治中的作用奠定了基础。

Aim To construct polynucleotide vaccine based on Ag85B gene of Mycobacterium tuberculosis. Methods The gene encoding Ag85B mature protein was amplified by polymerase chain reaction (PCR) from genome of Mycobacterium tuberculosis H37Ra strain, and inserted into cloning vector pUC19 after restriction endonuclease digestion. Gene fragment encoding Ag85B mature protein was correctly inserted into the vector, which was confirmed by partial nucleotide sequencing and restriction endonuclease digestion, and then was subcloned to Corresponding sites cut with HindⅢplus EcoRⅠof eukaryotic expression vector pcDNA3. Results The gene encoding Ag85B mature form of Mycobacterium tuberculosis H37Ra strain is identical with that of Erdman strain. The cloned gene was correctly inserted into the vector pcDNA3, which was confirmed by restriction endonuclease digestion. Conclusion The successful construction of recombinant eukaryotic expression vector based on the gene encoding Ag85B mature protein of Mycobacterium tuberculosis, lays the foundation for further researching prevention and treatment of tuberculosis.