pVAX1-Ag85B质粒的构建及其免疫原性的可视化研究

Study on the Construction and Immunogenicity of Recombinant DNA Vaccine pVAX1-Ag85B

构建以Ag85B基因为基础的DNA疫苗,并用活体红色荧光成像探讨其在动物体内免疫原性。方法:以pET28a-Ag85B基因组DNA为模板,用PCR扩增获得Ag85B全长基因;将PCR产物构建成pUCm-Ag85B亚克隆;经限制性内切酶消化后克隆入pVAX1载体中构建pVAX1-Ag85B,酶切、DNA测序鉴定。并将构建的DNA疫苗经肌肉免疫可视化膀胱癌移植瘤模型,通过活体红色荧光成像,从整体上直接、可视地观测Ag85B的免疫原性。结果:经NheⅠ和HindⅢ双酶切、DNA测序鉴定后证实,Ag85B基因定向克隆入pVAX1载体,碱基无突变,序列完全正确。免疫小鼠后,pVAX1-Ag85B组和BCG组均可提高淋巴细胞增殖活性,但效果不及卡介苗。结论:成功地构建了pVAX1-Ag85B质粒,为膀胱肿瘤的基因免疫治疗提供可靠的实验方法。免疫小鼠后,可提高膀胱癌小鼠免疫水平,但效果不及卡介苗。

Objective：To construct nucleotide vaccine based on Ag85B gene of Mycobacterium tuberculosis,and to explore its immunogenicity. Methods：The Ag85B gene was amplified by PCR from genome＇of pET28a-Ag85B, and inserted into pUCm-T vector after endonuclease digestion, and then was subcloned to Corresponding sites cut with Nhe I plus HindⅢ of eukaryotic expression vector pVAX1. 615 mice were immunized with the recombinant DNA vaccine,and Level of serum specific antibody and Proliferative response of spleen lymphocyte were measured and analyzed. Results： The Ag85B gene was Cloned into pVAX1 correctly after endonuclease digestion, and no mutation was observed, after treatment, pVAX1-Ag85B group can improve the Proliferative response of spleen lymphocyte as BCG,but poorer than BCG. The serum specific antibody titer of mice immunized with pVAX1-Ag85B is 1 ： 400,and that of mice immunized with BCG is 1 ： 800. Conclusions：The successful construction of recombinant eu- karyotic expression vector pVAX1-Ag85B lays the foundation for bladder tomolar genic immunotherapy, pVAX1- Ag85B can improve the immune response of mouse,but couldnt come up to BCG level.