结核杆菌热休克蛋白65“自杀性”DNA疫苗的构建及其免疫效应研究

Construction of suicidal DNA vaccine expressing heat shock protein 65 of Mycobacterium Tuberculosis and its immune effects

目的构建表达结核杆菌热休克蛋白65(HSP65)的“自杀性”DNA疫苗。方法用聚合酶链反应,从我室构建的表达结核杆菌HSP65真核表达质粒中(pCHSP65),扩增出编码HSP65的基因,与“自杀性”DNA载体pSFV重组。经酶切、测序鉴定,间接免疫荧光(IF)证实其能表达HSP65后,将此“自杀性”DNA疫苗(pAHSP65)免疫小鼠。用ELISA、MTT法研究其免疫效应,并观察其对结核菌感染小鼠的保护作用。结果酶切、测序鉴定结果证实为结核杆菌HSP65基因,在BHK21细胞中经IF证实有HSP65表达。该疫苗接种能诱导小鼠产生特异性抗体,刺激淋巴细胞增殖,并能抵抗结核杆菌的感染。结论我们成功地构建了表达结核杆菌HSP65的“自杀性”DNA疫苗,该疫苗能诱导特异性免疫应答,并对结核菌感染的小鼠有保护作用。其免疫效应优于常规的HSP65DNA疫苗。

Objective To construct a suicidal DNA vaccine expressing heat shock protein 65 （HSP65） of Mycobactrium （Mt）. Methods The gene encoding HSP65 was amplified by PCR from an eukaryotic expression plasmid pCHSP65 constructed with the plasmid pcDNA3.1, and then recombined with suicidal DNA vector pSFV. After identification with restriction endonuclease digestion, sequencing, and indirect imnmnofluorescence （IF）, the suicidal DNA vaccine （pAHSP65） was inoculated into mice. The immune effect of pAHSP65 was explored by ELISA and MTr; protective effect of pAHSP65 in mouse model was observed. Results The DNA fragment inserted into the pSFV was the HSP65 gene of Mt. The pAHSP65 has ability to induce production of specific antibody, proliferation of lymphocyte, and protection against infection of Mt. Conclusion The suicidal DNA vaccine expressing Mt HSP65 is constructed successfully, which is able to elicit specific immune response and protective effects.