【作者】 李亚明；

【导师】 杨增岐； 韩凌霞； 曲连东；

【作者基本信息】 西北农林科技大学 ， 细胞生物学， 2007， 硕士

【摘要】 马传染性贫血病毒(Equine Infectious Anemia Virus, EIAV)是一种非灵长类慢病毒,而且是基因组结构最简单的慢病毒,可以将前病毒基因组持久地整合到感染细胞的染色体上并在感染细胞和子代细胞中表达病毒基因,可作为将外源基因引入细胞的载体。孙成群利用我国EIAV的研究成果,在中国EIAV驴白细胞弱毒疫苗株(EIAV-DLA)感染性克隆(pOK8266)的基础上,采用反向遗传学技术,构建了包括EIAV载体质粒、EIAV-gag/pol/rev包装质粒和VSV-G囊膜表达质粒的EIAV三质粒基因转移载体系统。本研究是孙成群开发的中国EIAV三质粒基因转移载体系统的继续,将对其进行一系列优化和改造,主要目的在于进一步提高EIAV基因转移载体的转基因效率,同时为四质粒基因转移载体系统的构建奠定基础。研究内容及结果如下:1.利用土拨鼠肝炎病毒转录后调控元件(WPRE)对EIAV基因转移载体质粒进行修饰,构建了WPRE正向插入pcPPTPRE(+)的EIAV载体质粒pcPPTWPRE。转染实验表明,在体外传代细胞系HEK293中,WPRE可以显著提高EIAV基因转移载体的外源蛋白表达能力;而在DF-1细胞中,载体pcPPTWPRE表达外源蛋白的能力略高于pcPPTPRE(+),虽然两者的表达量均不高。2.利用杂合启动子人巨细胞病毒立即早期增强子/鸡β-肌动蛋白启动子(CAG启动子)构建EIAV基因转移载体。PCR扩增CAG核心启动子(不含鸡β-肌动蛋白第一内含子),将其替换EIAV载体质粒pcPPTWPRE中的内部启动子人巨细胞病毒立即早期增强子/启动子(CMVIE启动子)构建了EIAV载体质粒pcPPTEIACAGp。体外转染实验表明,CAG核心启动子在鸡体外培养细胞DF-1中启动外源基因表达的能力明显高于CMVIE启动子;而在HEK293细胞中,两者启动外源基因表达的能力相当。3.在CAG启动子的基础上,利用酶切的方式分别构建了含部分鸡β-肌动蛋白第一内含子的EIAV载体质粒pWCAGP0.8和含全长内含子的EIAV载体质粒pWCAGP1.6,研究鸡β-肌动蛋白第一内含子对EIAV基因转移载体质粒外源蛋白表达能力的影响。体外转染实验结果表明,不含内含子的EIAV载体质粒pcPPTEIACAGp表达外源蛋白的能力优于含部分或全长内含子的载体pWCAGP0.8和pWCAGP1.6,鸡β-肌动蛋白第一内含子并没有提高EIAV载体质粒的外源蛋白表达能力,反而降低了其表达水平。4. PCR扩增rev反应元件RRE序列和EIAV 5′端R-U5序列,将其依次亚克隆入EIAV蛋白表达载体pCDGP中,借此研究RRE序列和5′端R-U5区对EIAV结构蛋白表达的影响。体外表达实验表明,EIAV-DLA基因组5`端的R区和U5区对EIAV结构蛋白的转录起决定性作用;Rev蛋白及其反应元件RRE序列对gag/pol蛋白表达载体的表达是必不可少的。改造之后的gag/pol表达载体pSRU5GPR与rev表达载体共转染HEK293,IFA检测获得了明显的蛋白表达。上述研究结果将为中国马传染性贫血病毒四质粒基因转移载体系统的构建提供理论基础和技术支持,也将为人类的基因治疗、转基因动物的制备及结合RNAi技术研究基因功能提供一个新的方法。更多还原

【Abstract】 Equine infectious anemia virus (EIAV) is a non-primate and the genetically simplest lentivirus, which has the abilities to integrate efficiently their genome into the chromosomal DNA of host cells and be stably replicated and transmitted to all of the progeny of these cells offering the potential for long-term expression, can be as gene transfer vectors to introduce the foreign target gene into host cells.Chinese equine infectious anemia virus donkey leukocyte attenuated strain (EIAV-DLA) is a unique lentiviral vaccine strain used widely for prevention and control of equine infectious anemia (EIA) in China up to now. A series of replication-deficient EIAV gene transfer vector plasmids, envelope gene-deleted gag/pol expression plasmids and VSV-G gene expression plasmids have been constructed and the gene transfer vector systems of these three plasmids were established based on an infectious molecular clone, pOK8266, derived from the EIAV-DLA.This study was a continuous job after the construction of EIAV three-plasmid gene transfer vector systems. A series of optimization and reformation methods were done to improve the gene transfer efficiency, trying to construct EIAV four-plasmid gene transfer vector systems. The results were as follows:1.The EIAV vector pcPPTWPRE was constructed by inserting the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) into pcPPTPRE(+) by sense. Results of transfection showed that the pcPPTWPRE could express the EGFP more efficiently than pcPPTPRE(+) in HEK293 cells, while a little efficiently in DF-1 cells.2.The core CAG promoter was amplified by polymerase chain reaction(PCR)from the plasmid pCAGG-MCS and subcloned into pcPPTWPRE to obtain recombinant plasmid pcPPTEIACAGP. Results of transfection showed that the core CAG promoter could express the EGFP more efficiently than CMVIE promoter in DF-1 cell,while the equal efficiency with CMVIE promoter in HEK293 cell.3.The EIAV vectors pWCAGP0.8 containing part of chickenβ-actin intron 1 and pWCAGP1.6 containing the full intron were constructed by Restriction Enzyme digestion to investigate the influence of the intron on the EGFP expression of EIAV vectors. Results of transfection showed that the chickenβ-actin intron 1 could not increase the EGFP expression, but decrease the expression in HEK293 cell and DF-1 cells. 4.The sequences of RRE and RU5 were amplified by PCR from the plasmid pOK8266T and subcloned into the EIAV gag/pol eukaryotic expression vector pCDGP in order. It was found that the R-U5 area of the EIAV 5`-LTR was critical to the transcription of the gag/pol gene. It was also demonstrated that the Rev protein and Rev-response element (RRE) were essential to the expression of the EIAV gag/pol expression vectors. The reformed EIAV vector pSRU5GPR could express Gag/Pol protein detected by IFA after cotransfection with pCDREV expression vector.The work provides the theoretical basis and technical supports for the construction of EIAV four-plasmid gene transfer vector systems. It also offers a new approach for gene therapy, preparation of transgenic animals and the study of gene function.更多还原