Ad35腺病毒载体构建及病毒蛋白免疫原性研究

发布时间：2018-06-03 08:49

  本文选题：35型腺病毒(Ad35) + 病毒载体　； 参考：《北京协和医学院》2009年博士论文

【摘要】：目前以C组5型腺病毒(Ad5)载体为基础构建的腺病毒载体在基因治疗和疫苗研究中得到广泛应用,但是由于其转导作用依赖于细胞表面的柯萨奇-腺病毒受体(CAR),许多重要的细胞如造血干细胞和树突状细胞表面Ad5吸附受体CAR表达很少,一些恶性肿瘤细胞CAR的表达比相应的正常组织细胞要少,Ad5载体难以转导外源基因；同时正常人群Ad35的感染率在90%左右,体内预存的中和抗体可迅速清除腺病毒载体,导致治疗基因消失。这些问题限制了5型腺病毒(Ad5)载体的广泛应用。为此人们在试图利用其他型别的腺病毒开发新型载体,以克服这些不足。 35型腺病毒(Ad35)以几乎在所有的人类体细胞表面都有表达的CD46作为吸附受体,可以转导不易被Ad5感染造血细胞、树突状细胞和恶性肿瘤细胞等靶细胞。而且世界范围内正常人群中Ad35感染率不到5%,病毒载体被抗体清除的概率小,所以Ad35腺病毒作为颇具吸引力的基因治疗载体近年来受到人们的青睐。但目前Ad35载体尚未商品化,且迄今为止仅有的几例有关成功构建Ad35载体一般采用体外连接法,载体构建过程繁琐,病毒包装成功率低。有鉴于此,本文拟利用体外同源重组方法来构建Ad35载体包装系统,以期建立一种操作简便的Ad35载体系统。 为了评价Ad35载体在实际应用中的可行性,为构建Ad35载体提供可靠的理论依据,本文首先对人6个组10种不同型腺病毒基因组序列同源性进行了比较,发现人群普遍感染的C组Ad5腺病毒与人群感染率很低的B组Ad35腺病毒在进化上亲缘关系比较远,六邻体蛋白氨基酸同源性比较显示Ad5和Ad35六邻体的同源性只有75.3%,这提示Ad5和Ad35六邻体之间存在交叉反应的可能性比较小；之后对中国1157份0--80岁健康人群进行了Ad4、Ad5和Ad35抗体水平的本底筛查,结果显示在中国正常人群感染Ad4和Ad5的比例分别为70%和90%左右,而Ad35的感染要远远低于Ad4和Ad5,只有不到5%。 根据细菌内同源重组的原理,克隆了Ad35的全基因组DNA,构建了病毒包装所需的穿梭质粒、骨架质粒和表达Ad35腺病毒E1B的细胞系293-E1B,并通过同源重组的方法成功在293-E1B细胞系中包装出复制缺陷型的Ad35腺病毒,病毒的包装滴度达到106TCID50/ml,接近于野生型腺病毒。同时还对影响病毒包装效率的pXI蛋白进行了初步研究,发现pXI蛋白是影响重组病毒包装效率的主要因素。 目前对Ad35的基因组编码产物研究的还很少,为了阐明Ad35基因组编码产物中与诱导体液免疫有关的蛋白,为今后改进Ad35载体系统打下基础,利用蛋白质组学的方法对Ad35腺病不同毒蛋白免疫原性进行了初步鉴别,首次发现Ad35腺病毒非结构蛋白E2A (DNA binding protein)能够诱导机体产生抗体。 综上所述,本文首次调查了我国人群中Ad35的感染情况、利用细菌内同源重组机制构建了Ad35载体系统并鉴别出一个以前未报道的体液免疫原,这些结果的取得为Ad35载体的开发、应用打下了基础。
[Abstract]:At present , the adenovirus vector constructed on the basis of C - group 5 adenovirus ( Ad5 ) vector has been widely used in gene therapy and vaccine research , but because of its signal transduction , the expression of Ad5 adsorption receptor CAR in many important cells , such as hematopoietic stem cells and dendritic cells , is very little , and the expression of CAR in some malignant cells is less than that of normal tissue cells .
At the same time , the infection rate of Ad35 in the normal population is about 90 % , and the pre - stored neutralizing antibody in the body can rapidly clear the adenovirus vector , leading to the disappearance of the therapeutic gene . These problems limit the wide application of the 5 - type adenovirus ( Ad5 ) vector . To this end , people are trying to exploit other types of adenovirus to develop new vectors to overcome these deficiencies .


Ad35 adenovirus ( Ad35 ) has not been commercialized in recent years , so far only a few cases have been commercialized . So far only a few cases have been successfully constructed .


In order to evaluate the feasibility of Ad35 vector in practical application , it provides a reliable theoretical basis for constructing Ad35 vector . In this paper , we compared the sequence homology of 10 different types of adenovirus genome sequences in six groups . It is found that there is only 75.3 % homology between Ad5 and Ad35 adenovirus in group B with low infection rate . This suggests that there is a small possibility of cross reaction between Ad5 and Ad35 .
The levels of Ad4 , Ad5 and Ad35 were screened in 1157 healthy Chinese in China . The results showed that the rates of Ad4 and Ad5 were 70 % and 90 % , respectively , while Ad35 infection was much lower than Ad4 and Ad5 , only less than 5 % .


Based on the principle of homologous recombination in bacteria , the complete genomic DNA of Ad35 was cloned , the shuttle plasmid , backbone plasmid and cell line 293 - E1B expressing Ad35 adenovirus E1B were constructed , and the replication deficient Ad35 adenovirus was successfully packaged in 293 - E1B cell line by homologous recombination .


At present , there is little research on the genome coding products of Ad35 . In order to clarify the protein related to humoral immunity in Ad35 genome coding products , the immunogenicity of Ad35 adenosis virus protein is preliminarily identified by Proteomics . It is found that Ad35 adenovirus non - structural protein E2A ( DNA binding protein ) can induce the body to produce antibody .


In conclusion , this article first investigated the infection of Ad35 in our population , constructed Ad35 vector system by using homologous recombination mechanism in bacteria , and identified a previously unreported humoral immunity . These results have provided the foundation for the development and application of Ad35 vector .
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R392.1

【参考文献】

相关期刊论文 前1条

1 韦芳,黄倩;腺病毒载体研究进展与展望[J];肿瘤;2005年05期

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本文编号：1972190