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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。
The DNA vaccine based on Ag85B gene was constructed and its immunogenicity in animals was investigated by living red fluorescence imaging. Methods: The full length of Ag85B gene was amplified by PCR using pET28a-Ag85B genomic DNA as a template. The PCR product was subcloned into pUCm-Ag85B and cloned into pVAX1 vector for restriction enzyme digestion to construct pVAX1-Ag85B. Digestion, DNA sequencing identification. The immunogenicity of Ag85B was visualized directly and visually by in vivo red fluorescence imaging using the constructed DNA vaccine to visualize the bladder cancer xenograft model by muscle immunization. Results: Nhe Ⅰ and Hind Ⅲ double enzyme digestion, DNA sequencing identified confirmed Ag85B gene cloned into pVAX1 vector, the base without mutation, the sequence is correct. Immunized mice, pVAX1-Ag85B group and BCG group can increase lymphocyte proliferation activity, but the effect is less than BCG. Conclusion: The plasmid pVAX1-Ag85B has been constructed successfully and provides a reliable experimental method for gene therapy of bladder cancer. Immune mice, can improve the immune level of bladder cancer mice, but the effect is less than BCG.