阴道毛滴虫转染载体的构建及EGFP在阴道毛滴虫内的稳定表达

发布时间：2018-06-15 11:56

本文选题：阴道毛滴虫 + 转染载体　；参考：《吉林大学》2008年硕士论文

【摘要】： 本研究构建了阴道毛滴虫病毒转染载体和阴道毛滴虫DNA转染载体。阴道毛滴虫病毒转染载体的构建:根据我国阴道毛滴虫病毒基因组的序列特征,在本室构建阴道毛滴虫病毒瞬时表达载体的基础上,用新霉素磷酸转移酶(NEO)编码基因替换TVV全部编码区,同时引入多克隆酶切位点(MCS),构建阴道毛滴虫病毒转染载体。以增强型绿色荧光蛋白(EGFP)作为报告基因,经T7 RNA聚合酶体外转录成mRNA后,经电穿孔法转染阴道毛滴虫携病毒株细胞内,进行G418筛选,连续传代24次,荧光显微镜都可以观察到EGFP的表达,同时提取转染后虫体的总核酸,用RT-PCR方法检测到EGFP的mRNA,结果显示EGFP在阴道毛滴虫转染细胞内得到了稳定表达。阴道毛滴虫DNA转染载体构建:首先克隆阴道毛滴虫α-琥珀酰辅酶A合成酶(α-SCSB)全序列,并对其序列进行了分析;利用阴道毛滴虫α-琥珀酰辅酶A合成酶启动子序列,构建EGFP表达盒pα-SCSB5/EGFP/Eα-SCSB3,对α-SCSB转录活性进行了鉴定;构建NEO抗性盒pα-SCSB5/NEO /Nα-SCSB3,通过G418抗性筛选建立G418抗性虫株;最后将抗性盒和表达盒串联,构建阴道毛滴虫DNA稳定转染载体pNEO/EGFP,经电穿孔法转染阴道毛滴虫细胞内,进行G418筛选,连续传代17次,荧光显微镜都可以观察到EGFP的表达,成功实现了EGFP在阴道毛滴虫转染细胞内的稳定表达。本研究成功构建了阴道毛滴虫转染载体,介导了目的基因EGFP在阴道毛滴虫体内的稳定表达,为进一步研究阴道毛滴虫的分子生物学特性奠定基础。
[Abstract]:In this study, Trichomonas vaginalis vector and Trichomonas vaginalis DNA transfection vector were constructed.
Construction of transfection vector of Trichomonas vaginalis virus: Based on the sequence characteristics of Trichomonas vaginalis virus genome in our country, on the basis of constructing the transient expression vector of Trichomonas vaginalis virus in our room, the whole coding region of TVV was replaced with neomycin phosphonase (NEO) gene encoding gene, and the polyclonal site of MCS was introduced to construct the vagina Mao Dichong The virus transfection carrier, with enhanced green fluorescent protein (EGFP) as the reporter gene, was transcribed into mRNA by T7 RNA polymerase in vitro and transfected into the virus cell of Trichomonas vaginalis by electroporation and screened by G418 for 24 successive generations. The expression of EGFP could be observed by the fluorescence microscope, and the total nucleic acid of the insect body after transfection was extracted and R was extracted with R. The mRNA of EGFP was detected by T-PCR. The results showed that EGFP was stable in the transfected cells of Trichomonas vaginalis.
Construction of DNA transfection vector of Trichomonas vaginalis: first cloned the whole sequence of Trichomonas vaginalis alpha succinyl coenzyme A synthetase (alpha -SCSB), and analyzed its sequence. Using the promoter sequence of alpha succinyl coenzyme A synthetase of Trichomonas vaginalis, the EGFP expression box P alpha -SCSB5/EGFP/E alpha -SCSB3 was constructed, and the activity of alpha -SCSB transcriptional activity was identified, and NEO was constructed for NEO. Resistance box P alpha -SCSB5/NEO /N alpha -SCSB3 was selected to establish G418 resistant strain by G418 resistance screening. Finally, the resistance box and expression box were connected in series, and DNA stable transfection carrier pNEO/EGFP of Trichomonas vaginalis was constructed and transfected into Trichomonas vaginalis cells by electrical perforation, and G418 screening was performed for 17 times. The expression of EGFP was observed by fluorescence microscopy. The stable expression of EGFP in Trichomonas vaginalis transfected cells was achieved.
This study successfully constructed the transfection vector of Trichomonas vaginalis, which mediates the stable expression of the target gene EGFP in Trichomonas vaginalis, which lays the foundation for further study of the molecular biological characteristics of Trichomonas vaginalis.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R383

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