电穿孔转化沙眼衣原体建立衣原体载体实验研究

发布时间：2018-09-18 16:59

【摘要】： 目的:通过电穿孔将重组质粒导入到沙眼衣原体(CT)中,并由CT携带进入真核宿主细胞中,观察重组质粒在宿主细胞中的遗传、表达,探索建立沙眼衣原体载体的方法和思路,为疾病的基因治疗奠定基础。方法:分别用2.5kV和1.5kV电压电击经电击感受态制备的CT和重组pECFP-C1/U6-2质粒的混悬液,扩增电击过的CT,观察重组pECFP-C1/U6-2质粒标记基因产物——绿色荧光蛋白(GFP)的表达,并用PCR方法检测重组基因插入序列在是否在经电击的CT中存在。结果:经电击的CT扩增培养48h后,倒置荧光显微镜观察未见在宿主细胞中有绿色荧光。提取经电击转化CT的基因组,酶切后PCR扩增重组的pECFP-C1质粒插入序列,可见2.5kV电击组中质粒插入序列存在。结论:CT能够耐受电击感受态制备及电击过程,2.5kV电压电击转化可将外源质粒导入电击感受态的CT中,进入CT的外源质粒随CT一起进行遗传复制,但未发现外源质粒在CT中表达的证据。为制备以CT为基础的载体系统提供了试验依据和初步的试验方法,并建立了将外源性大分子物质导入CT的方法模型。
[Abstract]:Objective: to introduce the recombinant plasmid into Chlamydia trachomatis (CT) by electroporation, and carry it into eukaryotic host cells by CT, observe the inheritance and expression of recombinant plasmid in the host cell, and explore the method and thought of establishing Chlamydia trachomatis vector. To lay the foundation for gene therapy of disease. Methods: the suspension of CT and recombinant pECFP-C1/U6-2 plasmids prepared by 2.5kV and 1.5kV voltage shock were used to detect the expression of green fluorescent protein (GFP) (GFP), which was labeled by recombinant pECFP-C1/U6-2 plasmid. PCR method was used to detect whether the inserted sequence of the recombinant gene existed in the electrocuted CT. Results: after 48 hours of CT amplification, no green fluorescence was observed in host cells by inverted fluorescence microscope. The genome of CT was extracted and transformed into CT by electroporation. The inserted sequence of recombinant pECFP-C1 plasmid was amplified by PCR after restriction endonuclease digestion. It can be seen that the inserted sequence of plasmid existed in 2.5kV shock group. Conclusion\% CT can be used to prepare the electroshock receptive state and to transform the electric shock into the CT with 2.5 kV voltage shock. The exogenous plasmids entering into the CT can be copied along with the CT, and the exogenous plasmids can be copied along with the CT. However, no evidence of the expression of foreign plasmids in CT was found. The experimental basis and preliminary test method were provided for the preparation of carrier system based on CT, and the method model of introducing exogenous macromolecular material into CT was established.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R374

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