结核分枝杆菌Esat6与人集落刺激因子融合质粒的构建及表达

发布时间：2018-01-19 10:20

  本文关键词： 结核分枝杆菌 Esat6 人集落刺激因子 融合质粒 DNA疫苗　出处：《暨南大学》2007年硕士论文　论文类型：学位论文

【摘要】： 目的： 从质粒pVAE中克隆目的基因Esat6，与hGM-CSF一起构建双顺反子表达载体pIEG，进而转染Hela细胞，并进行鉴定。为进一步研究具有预防性和治疗性抗结核杆菌新型核酸疫苗奠定了基础。 方法： 1．应用分子克隆技术，，以质粒pVAE为模板PCR扩增出Esat6基因，与pIRES载体的MCS A进行重组，构建pIEsat6重组质粒。再以pORF-hGM-CSF为模板PCR扩增出hGM-CSF基因，与pIRES载体的MCS B进行重组，构建pIGM双顺反子重组质粒；2．上述两重组质粒经多种分子生物学方法进行鉴定后，再通过酶切、连接，将hGM-CSF构建于pIEsat6质粒中，形成双顺反子融合表达载体pIEG；3．将pIEG转染人宫颈癌细胞(Hela)；4．通过SDS-PAGE蛋白电泳鉴定细胞内Esat6基因的表达情况，并拍照；5．通过免疫组织化学方法检测细胞内Esat6基因的表达情况，并拍照；6．通过ELISA方法检测细胞中hGM-CSF基因的表达情况。 结果： 1．通过PCR可克隆得到相应大小的产物；酶切鉴定所切下的两片段大小约为0.29 kb以及0.47 kb，与预计相符；测序结果与报道一致；2．SDS-PAGE电泳能检测到细胞内有Esat6蛋白的表达；3．免疫组织化学方法能检测到表达Esat6的阳性细胞；4．ELISA方法能检测到细胞培养上清中hGM-CSF的量与阴性对照组存在显著性差异。 结论： 成功克隆并构建了含有结核分枝杆菌Esat6基因与hGM-CSF基因的双顺反子融合表达载体，并在Hela细胞中成功表达。为研制优于BCG的抗结核病的防治疗性DNA疫苗奠定了基础。
[Abstract]:Objective: The target gene Esat6 was cloned from the plasmid pVAE, and the double cistronic expression vector pIEG was constructed together with hGM-CSF, and then transfected into Hela cells. The results provide a basis for the further study of novel nucleic acid vaccine with preventive and therapeutic anti-tuberculosis. Methods: 1. Using molecular cloning technique, the Esat6 gene was amplified by PCR using plasmid pVAE as template, and was recombined with MCS A of pIRES vector. The pIEsat6 recombinant plasmid was constructed and the hGM-CSF gene was amplified by PCR using pORF-hGM-CSF as the template. The hGM-CSF gene was recombined with MCS B of pIRES vector. Construction of pIGM double cistron recombinant plasmid; 2.After the two recombinant plasmids were identified by many molecular biological methods, hGM-CSF was constructed into pIEsat6 plasmid by enzyme digestion and ligation. A double cistron fusion expression vector pIEG was formed. 3. Transfection of pIEG into human cervical carcinoma cell line HelaHN; 4. SDS-PAGE protein electrophoresis was used to identify the expression of Esat6 gene in the cells. 5. The expression of Esat6 gene in the cells was detected by immunohistochemical method, and the pictures were taken. 6. The expression of hGM-CSF gene was detected by ELISA. Results: 1. The products of corresponding size can be obtained by PCR cloning. The size of the two fragments was about 0.29 kb and 0.47 kb, which was consistent with the expected size. The results of sequencing were consistent with the report. 2. The expression of Esat6 protein was detected by SDS-PAGE. 3. The positive cells expressing Esat6 could be detected by immunohistochemistry. 4. The amount of hGM-CSF in the supernatant of cell culture was significantly different from that in the negative control group by Elisa. Conclusion: The expression vector containing Esat6 gene and hGM-CSF gene of Mycobacterium tuberculosis was cloned and constructed successfully. It was successfully expressed in Hela cells, which laid a foundation for the development of a therapeutic DNA vaccine against tuberculosis, which was superior to BCG.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392

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