pVAX1-Ag85B的构建及其免疫原性的初步探索

目的构建以Ag85B基因为基础的DNA疫苗,并对其在小鼠体内的免疫原性进行初步分析。方法以pET28a-Ag85B基因组DNA为模板,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载体中构建真核表达质粒pVAX1-Ag85B,酶切、DNA测序鉴定。并将构建的DNA疫苗经肌肉免疫荷瘤小鼠,检测血清特异性抗体水平和脾淋巴细胞的增殖活性,并与BCG组、空白质粒组、生理盐水组相比较。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。免疫荷瘤小鼠后,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;pVAX1-Ag85B组的抗体滴度为1∶400,BCG免疫组的抗体滴度为1∶800。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依据。免疫小鼠后,可提高小鼠免疫水平,但效果不及卡介苗。

Study on the construction and immunogenicity of recombinant DNA vaccine pVAX1-Ag85B

Objective mycobacterium tuberculosis,and to explore its immunogenicity.Methods The Ag85B gene was amplified by PCR from genome of pET28a-Ag85B,inserted into pUCm-T vector after endonuclease digestion,and then subcloned to corresponding sites cut with NheⅠplus HindⅢ of eukaryotic expression vector pVAX 1.615 mice were immunized with the recombinant DNA vaccine,and their 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.Both pVAX1-Ag85B and BCG improved the proliferative response of spleen lymphocyte,but pVAX1-Ag85B was not as effective as BCG.The serum specific antibody titer of mice immunized with pVAX1-Ag85B and BCG was 1∶400 and 1∶800 respectively.Conclusions The successful construction of recombinant eukaryotic expression vector pVAX1-Ag85B lays the foundation for bladder tumor genic immunotherapy.pVAX1-Ag85B can improve the immune response of mouse,but not as effectively as BCG.