【摘要】： H5亞型禽流感和SARS兩種人獸共患疫病嚴(yán)重的危及著社會經(jīng)濟(jì)的發(fā)展和人類的生命。疫苗是防治本病的重要手段,已有成功應(yīng)用于人體的H5亞型禽流感疫苗和SARS疫苗,多采用注射接種方式,成本高,需專業(yè)人員操作,不易于免疫的普及。針對突如其來的重大疫情,使疫苗快速普及人群,口服是簡捷方便的途徑,開發(fā)口服疫苗勢在必行。近年來以減毒沙門菌為載體的口服疫苗成為研究的熱點(diǎn),減毒傷寒沙門菌攜帶DNA疫苗進(jìn)入人體的已有安全性報(bào)道,只要了解疾病的抗原特性,可以迅速的開發(fā)針對該病的口服疫苗,以它為載體還可以開發(fā)多價(jià)疫苗,生產(chǎn)周期短,成本低。減毒傷寒沙門菌作為疫苗載體菌顯示了誘人的開發(fā)前景。本實(shí)驗(yàn)旨在應(yīng)用減毒沙門菌為載體,構(gòu)建H5亞型禽流感和SARS DNA口服疫苗。 本實(shí)驗(yàn)應(yīng)用PCR方法獲得了AIV的HA基因,通過人工合成的方法獲得了SARS多表位基因S合。對真核表達(dá)載體pVAX1進(jìn)行改造,以asd基因替換pVAX1載體上的Kan+抗性基因,以EGFP為報(bào)告基因,成功轉(zhuǎn)染真核細(xì)胞;構(gòu)建了重組真核表達(dá)載體pASD,它與減毒沙門菌X4550構(gòu)成載體-宿主平衡致死系統(tǒng),解決了質(zhì)粒載體應(yīng)用于人體產(chǎn)生抗生素耐藥性的問題。將HA基因和S合基因亞克隆于pVAX1和pASD,脂質(zhì)體法轉(zhuǎn)染293-T細(xì)胞,通過RT-PCR檢測目的基因的轉(zhuǎn)錄,通過間接ELISA和間接免疫熒光法檢測目的基因的表達(dá),結(jié)果表明,HA基因和S合基因能夠正確表達(dá)。將攜帶抗原基因的真核表達(dá)質(zhì)粒電轉(zhuǎn)化入沙門菌X4550,在動物體內(nèi)、外的研究表明,質(zhì)粒能夠在細(xì)菌中穩(wěn)定存在。細(xì)菌的生長曲線測定結(jié)果表明,質(zhì)粒的存在并不影響細(xì)菌的生長狀態(tài)。免疫小鼠確定安全劑量為≤1010CFU�？诜呙缑庖咝∈�,結(jié)果表明,口服疫苗能夠激發(fā)小鼠機(jī)體產(chǎn)生特異性體液免疫和細(xì)胞免疫反應(yīng)。
[Abstract]:H5 avian influenza and SARS are two kinds of zoonotic diseases, which seriously endanger the development of social economy and human life. Vaccine is an important means to prevent and cure this disease. H5 avian influenza vaccine and SARS vaccine have been successfully used in human body. Most of them are injected and vaccinated, which has high cost and needs to be operated by professionals, so it is not easy to popularize immunization. In view of the sudden major epidemic situation, oral administration is a simple and convenient way to popularize the vaccine rapidly, so it is imperative to develop oral vaccine. In recent years, attenuated Salmonella typhimurium oral vaccine with attenuated Salmonella typhimurium as carrier has become a hot research topic. Attenuated Salmonella typhimurium vaccine carrying DNA vaccine into human body has been reported. As long as we understand the antigen characteristics of the disease, we can quickly develop an oral vaccine for the disease, and we can also develop a multivalent vaccine with short production cycle and low cost. Attenuated Salmonella typhimurium as vaccine carrier bacteria has shown attractive development prospects. The purpose of this study was to construct H5 avian influenza and SARS DNA oral vaccines with attenuated Salmonella as vector. In this experiment, the HA gene of AIV was obtained by PCR, and the multiple epitope gene S of SARS was obtained by synthetic method. The eukaryotic expression vector pVAX1 was modified to replace the Kan resistant gene on pVAX1 vector with asd gene and EGFP as reporter gene, and the recombinant eukaryotic expression vector pASD, was constructed to form a vector-host balanced lethal system with attenuated Salmonella X4550, which solved the problem that plasmid vector was used in human body to produce antibiotic resistance. HA gene and S gene were subcloned into 293T cells by pVAX1 and pASD, liposomes. The transcription of the target gene was detected by RT-PCR, and the expression of the target gene was detected by indirect ELISA and indirect immunofluorescence. The results showed that HA gene and S gene could be expressed correctly. The eukaryotic expression plasmid carrying antigen gene was electrotransformed into Salmonella X4550. Studies in vivo and in vitro showed that the plasmid could exist stably in the bacteria. The results of bacterial growth curve showed that the existence of plasmid did not affect the growth state of bacteria. The safe dose of immunized mice was 鈮，

本文編號：2501910