基因佐剂结核杆菌HSP70增强HCA661基因疫苗免疫应答

Mycobacterium tuberculosis heat shock protein 70 enhances immunization respond of the gene vaccine against hepatocellular carcinoma-associated antigens 661

目的:评价结核分枝杆菌HSP70(Mycobacterium tuberculosis heat shock protein 70,mtHSP70)羧基端作为基因佐剂对HCA661(Hepatocellular carcinoma associated antigens 661)基因疫苗免疫效果的影响。方法:将HCA661及与mtHSP70羧基端的串联体分别克隆到pCI-neo中构建基因疫苗;将eGFP及与mtHSP70羧基端的串联体分别克隆到pCI-neo,体外转染φA细胞,根据eGFP的表达评价重组质粒中靶基因的转录表达效率。基因疫苗免疫BALB/c小鼠,SABC、Western blot检测免疫血清中特异性HCA661抗体;流式细胞术、ELISA法检测免疫血清中特异性HCA661抗体水平。结果:重组质粒构建成功;80%φA细胞转染eGFP后呈现绿色荧光;单基因和融合基因疫苗组动物均产生了针对HCA661的特异性抗体,融合疫苗组诱导的抗体水平较单基因疫苗组显著增高,融合基因疫苗组未产生针对mtHSP70的抗体。结论:mtHSP70羧基端作为基因佐剂增强机体对HCA661融合基因疫苗的特异性免疫应答。

Objective： To evaluate the immunizating response of the gene vaccine against hepatocellular carcinoma-associated antigens 661 （HCA661） enhanced by C-terminal of Myeobacterium tuberculosis HSP70 （mtHSP70）. Methods： The C-terminal of mtHSP70 gene and HCA661 gene were amplified to construct gene vaccine pCI-HCA661 and pCI-HCA661-mtHSP70. The eGFP gene was amplified to construct pCI-eGFP and pCI-HCA661-eGFP, which were transfected into φA cells. Then green fluorescence was observed in order to evaluate whether the target gene subcloned into these recombinant plasmids could transcript and translate effectively. BALB/e mice were immunized with the gene vaccine.The polyclonal antibodies recognizing HCA661 or mtHSP70 in antiserum were detected by Western blot and SABC.The level of polyclonal antibodies recognizing HCA661 was detected by flow cytometry and ELISA. Results： All recombinant plasmids were constructed successfully. More than 80% cells transfected with eGFP expressed green fluorescence protein. Although HCA661 specific antibodies existed in the antiserum of pCI-HCA661 or pCI-HCA661-mtHSP70C group,the level of the antibodies in the latter was increased significantly（ P 〈0.05）,and there were no mtHSP70 antibodies in the latter. Conclusion： The polypeptide carrier and immunologic adjuvant of the C-terminal of mtHSP70 enhances the immune respond to the fused antigen in the vaccinated animals.