【摘要】：目的:呼吸道合胞病毒(Respiratory Syncytial Virus,RSV)廣泛分布于世界各地,是導(dǎo)致嬰幼兒嚴(yán)重下呼吸道感染的最重要的病毒病原。病毒感染機(jī)體后的免疫保護(hù)機(jī)制尚未明確,也無(wú)特異性防治方法。RSV融合糖蛋白(fusion glycoprotein,F)和吸附蛋白(attachment protein,G)是RSV僅有的兩種中和抗原。由于G蛋白的差異,RSV有A和B兩種抗原亞型。我國(guó)流行的RSV主要是以A亞型為主。考慮到RSV病毒結(jié)構(gòu)和感染的特點(diǎn),研究以共表達(dá)RSV A亞型G、F糖蛋白的非復(fù)制型重組腺病毒和DNA疫苗進(jìn)行聯(lián)合免疫預(yù)防RSV感染。采取這種抗原組合的優(yōu)點(diǎn)在于:結(jié)構(gòu)上類(lèi)似減毒活疫苗,有助于誘導(dǎo)平衡的免疫反應(yīng);提高外源基因轉(zhuǎn)移及表達(dá)效率;一次接種產(chǎn)生兩種中和抗原,可改善接種效率,減少嬰幼兒接種疫苗的創(chuàng)痛,降低疫苗成本。 共表達(dá)策略目前主要用于以疾病治療為目的基因治療領(lǐng)域,或用于表達(dá)異源多聚體蛋白的亞單位。用于疫苗研究的報(bào)道較少。美國(guó)BD Bioscience Clontech公司已經(jīng)有商品化的真核雙表達(dá)載體pIRES,但尚缺乏可方便地用于構(gòu)建雙順?lè)醋庸脖磉_(dá)的腺病毒載體系統(tǒng)。為此,本研究利用內(nèi)部核糖體進(jìn)入位點(diǎn)(internal ribosome entry site,IRES)構(gòu)建可用于雙順?lè)醋庸脖磉_(dá)的腺病毒重組體。 方法:本研究依據(jù)IRES的堿基序列,設(shè)計(jì)并合成一對(duì)特異性引物,以商品化的真核雙表達(dá)載體pIRES為模板,擴(kuò)增IRES及其調(diào)控序列IVS,克隆入T載體,獲得pGEMT-IRES克隆載體(MCSA、MCVB分別位于IRES序列的上、下游)。根據(jù)IRES上、下游多克隆位點(diǎn),設(shè)計(jì)并合成兩對(duì)用于擴(kuò)增目的基因的PCR引物,分別擴(kuò)增出F基因和增強(qiáng)型綠色熒光蛋白基因(Enhanced Green Fluorescent Protein,EGFP),分別克隆至pGEMT-IRES,獲得重組質(zhì)粒pGEMT-F/EGFP。為構(gòu)建共表達(dá)RSV F和EGFP的非復(fù)制型腺病毒重組體,本研究采用了AdEasy系統(tǒng)。該系統(tǒng)是利用生長(zhǎng)周期短,便于操作的E. coli BJ5183細(xì)胞完成腺病毒骨架質(zhì)粒與克隆有外源基因的穿梭載體同源重組。在這一過(guò)程中,包括三個(gè)依次遞進(jìn)的步驟:將外源性基因(F-IRES-EGFP)亞克隆于穿梭載體pShuttle-CMV;重組穿梭載體與腺病毒骨架載體pAdEasy-1在E. coli BJ5183細(xì)胞內(nèi)同源
[Abstract]:Objective: respiratory syncytial virus (Respiratory Syncytial) is widely distributed all over the world, and it is the most important viral pathogen leading to severe lower respiratory tract infection in infants. The mechanism of immune protection after virus infection is not clear, and there is no specific prevention and treatment method. Fusion glycoprotein F and attachment protein G are the only two neutralizing antigens of RSV. Because of the difference of G protein, RSV has two antigenic subtypes, A and B. The prevalent RSV in China is mainly subtype A. Considering the structure and infection characteristics of RSV virus, the non-replicating recombinant adenovirus and DNA vaccine co-expressing RSV A subtype GnF glycoprotein were studied to prevent RSV infection. The advantages of adopting this antigen combination are that it is structurally similar to a live attenuated vaccine and contributes to the induction of a balanced immune response; increases the efficiency of foreign gene transfer and expression; and produces two neutralizing antigens at one time, which can improve the inoculation efficiency. Reduce the pain of infant vaccination and reduce the cost of vaccine. Coexpression strategy is mainly used in the field of gene therapy for disease treatment or for the expression of heteropolymer protein subunits. There have been few reports of vaccine research. BD Bioscience Clontech company in the United States already has the commercial eukaryotic double expression vector pIRESbut still lacks of the adenovirus vector system which can be used to construct the double cistronic coexpression vector conveniently. In this study, we constructed an adenovirus recombinant which could be used for the co-expression of double cistrons using internal ribosomal entry site (internal ribosome entry sitesires). Methods: according to the base sequence of IRES, a pair of specific primers were designed and synthesized. Using commercial eukaryotic double expression vector pIRES as template, IRES and its regulatory sequence were amplified and cloned into T vector. The pGEMT-IRES clone vector was obtained (MCSA-MCVB was located in the upstream and downstream of the IRES sequence, respectively). According to the polyclonal site downstream of IRES, two pairs of PCR primers were designed and synthesized to amplify the target gene. The F gene and the enhanced green fluorescent protein gene (Enhanced Green Fluorescent protein EGFP) were amplified, respectively, and cloned into pGEMT-IRES. the recombinant plasmid pGEMT-Fr / EGFPwas obtained. In order to construct a non-replicating adenovirus recombinant expressing RSV F and EGFP, AdEasy system was used in this study. In this system, adenovirus skeleton plasmids were homologous recombined with the shuttle vector cloned with foreign genes by using E. coli BJ5183 cells with short growth cycle and easy to operate. In this process, there are three progressive steps: subcloning of exogenous gene (F-IRES-EGFP) into shuttle vector pShuttle-CMV, and homology between recombinant shuttle vector and adenovirus skeleton vector pAdEasy-1 in E. coli BJ5183 cells
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2005
【分類(lèi)號(hào)】：R346

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