结核杆菌抗原85A与小鼠白细胞介素21共表达DNA疫苗的构建及其免疫效应研究

目的:利用真核表达质粒pRSC,构建结核杆菌抗原85A(Ag85A)与小鼠白细胞介素21(mIL21)共表达重组体pRSC-mIL21-Ag85A,为研究新型结核杆菌DNA疫苗提供新的策略。方法:从质粒pcDNA3.1-mIL21中经PCR扩增出mIL21基因,并插入质粒pRSC中构成pRSC-mIL21;再从pIRES-Ag85A质粒中经PCR扩增出Ag85A基因,构建于pRSC-mIL21重组质粒上,成为共表达DNA疫苗pRSC-mIL21-Ag85A。结果:经酶切、基因测序证实,该疫苗构建正确并能成功表达目的基因。共表达DNA疫苗免疫小鼠后,CTL活性、特异性淋巴细胞增殖水平及小鼠血清特异性抗体均呈有意义的提高。结论:结核杆菌Ag85A与mIL21共表达DNA疫苗能诱导小鼠免疫反应,为进一步研究DNA疫苗抗结核杆菌攻击的免疫防护效应奠定了基础。

Construction and its Immune Effect of Co-Expression Mycobacterium tuberculosis DNA Vaccine Containing Antigen 85A and Mouse Interleukin 21

Objective: To construct an eukaryotic co-expression recombinant containing mouse interleukin 21(IL-21) and tuberculosis antigen 85A(Ag85A), it provides the new strategy for further development of new type anti-tuberculosis DNA vaccines. Methods: The gene of mIL-21 was amplified from plasmid pcDNA3.1-mIL21 by PCR and cloned directly into the plasmid pRSC, forming recombinant pRSC-mIL21. The gene of Ag85A was amplified from the plasmid pIRES-Ag85A by PCR and cloned directly into the recombinant pRSC-mIL21 again, forming co-expression DNA vaccine pRSC-mIL21-Ag85A finally. Results: It was identified by the endonuclease digestion and DNA sequencing. The results showed that the co-expression DNA vaccine had been constructed successfully and the target gene was expressed correctly. When mice were immunized with the pRSC-mIL21-Ag85A, the CTL activities, the spleen cell proliferation responses to Ag85A and the antibody titer of serum were significantly increased respectively compared with the control mice immunized with blank plasmid. Conclusion: The eukaryotic co-expression DNA vaccine pRSC-mIL21-Ag85A can induce immune responses in mice. This study has laid a foundation for further research about DNA vaccine protective function in immunized mice challenged by Mycobacterium tuberculosis.