本文選題：35型腺病毒(Ad35) + 病毒載體　； 參考：《北京協(xié)和醫(yī)學(xué)院》2009年博士論文

【摘要】：目前以C組5型腺病毒(Ad5)載體為基礎(chǔ)構(gòu)建的腺病毒載體在基因治療和疫苗研究中得到廣泛應(yīng)用,但是由于其轉(zhuǎn)導(dǎo)作用依賴于細(xì)胞表面的柯薩奇-腺病毒受體(CAR),許多重要的細(xì)胞如造血干細(xì)胞和樹(shù)突狀細(xì)胞表面Ad5吸附受體CAR表達(dá)很少,一些惡性腫瘤細(xì)胞CAR的表達(dá)比相應(yīng)的正常組織細(xì)胞要少,Ad5載體難以轉(zhuǎn)導(dǎo)外源基因；同時(shí)正常人群Ad35的感染率在90%左右,體內(nèi)預(yù)存的中和抗體可迅速清除腺病毒載體,導(dǎo)致治療基因消失。這些問(wèn)題限制了5型腺病毒(Ad5)載體的廣泛應(yīng)用。為此人們?cè)谠噲D利用其他型別的腺病毒開(kāi)發(fā)新型載體,以克服這些不足。 35型腺病毒(Ad35)以幾乎在所有的人類體細(xì)胞表面都有表達(dá)的CD46作為吸附受體,可以轉(zhuǎn)導(dǎo)不易被Ad5感染造血細(xì)胞、樹(shù)突狀細(xì)胞和惡性腫瘤細(xì)胞等靶細(xì)胞。而且世界范圍內(nèi)正常人群中Ad35感染率不到5%,病毒載體被抗體清除的概率小,所以Ad35腺病毒作為頗具吸引力的基因治療載體近年來(lái)受到人們的青睞。但目前Ad35載體尚未商品化,且迄今為止僅有的幾例有關(guān)成功構(gòu)建Ad35載體一般采用體外連接法,載體構(gòu)建過(guò)程繁瑣,病毒包裝成功率低。有鑒于此,本文擬利用體外同源重組方法來(lái)構(gòu)建Ad35載體包裝系統(tǒng),以期建立一種操作簡(jiǎn)便的Ad35載體系統(tǒng)。 為了評(píng)價(jià)Ad35載體在實(shí)際應(yīng)用中的可行性,為構(gòu)建Ad35載體提供可靠的理論依據(jù),本文首先對(duì)人6個(gè)組10種不同型腺病毒基因組序列同源性進(jìn)行了比較,發(fā)現(xiàn)人群普遍感染的C組Ad5腺病毒與人群感染率很低的B組Ad35腺病毒在進(jìn)化上親緣關(guān)系比較遠(yuǎn),六鄰體蛋白氨基酸同源性比較顯示Ad5和Ad35六鄰體的同源性只有75.3%,這提示Ad5和Ad35六鄰體之間存在交叉反應(yīng)的可能性比較�。恢髮�(duì)中國(guó)1157份0--80歲健康人群進(jìn)行了Ad4、Ad5和Ad35抗體水平的本底篩查,結(jié)果顯示在中國(guó)正常人群感染Ad4和Ad5的比例分別為70%和90%左右,而Ad35的感染要遠(yuǎn)遠(yuǎn)低于Ad4和Ad5,只有不到5%。 根據(jù)細(xì)菌內(nèi)同源重組的原理,克隆了Ad35的全基因組DNA,構(gòu)建了病毒包裝所需的穿梭質(zhì)粒、骨架質(zhì)粒和表達(dá)Ad35腺病毒E1B的細(xì)胞系293-E1B,并通過(guò)同源重組的方法成功在293-E1B細(xì)胞系中包裝出復(fù)制缺陷型的Ad35腺病毒,病毒的包裝滴度達(dá)到106TCID50/ml,接近于野生型腺病毒。同時(shí)還對(duì)影響病毒包裝效率的pXI蛋白進(jìn)行了初步研究,發(fā)現(xiàn)pXI蛋白是影響重組病毒包裝效率的主要因素。 目前對(duì)Ad35的基因組編碼產(chǎn)物研究的還很少,為了闡明Ad35基因組編碼產(chǎn)物中與誘導(dǎo)體液免疫有關(guān)的蛋白,為今后改進(jìn)Ad35載體系統(tǒng)打下基礎(chǔ),利用蛋白質(zhì)組學(xué)的方法對(duì)Ad35腺病不同毒蛋白免疫原性進(jìn)行了初步鑒別,首次發(fā)現(xiàn)Ad35腺病毒非結(jié)構(gòu)蛋白E2A (DNA binding protein)能夠誘導(dǎo)機(jī)體產(chǎn)生抗體。 綜上所述,本文首次調(diào)查了我國(guó)人群中Ad35的感染情況、利用細(xì)菌內(nèi)同源重組機(jī)制構(gòu)建了Ad35載體系統(tǒng)并鑒別出一個(gè)以前未報(bào)道的體液免疫原,這些結(jié)果的取得為Ad35載體的開(kāi)發(fā)、應(yīng)用打下了基礎(chǔ)。
[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 .
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2009
【分類號(hào)】：R392.1

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 韋芳,黃倩;腺病毒載體研究進(jìn)展與展望[J];腫瘤;2005年05期

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本文編號(hào)：1972190