以减毒沙门菌为载体的H5亚型禽流感和SARS口服疫苗的研究
发布时间：2019-06-19 00:21
【摘要】： H5亚型禽流感和SARS两种人兽共患疫病严重的危及着社会经济的发展和人类的生命。疫苗是防治本病的重要手段,已有成功应用于人体的H5亚型禽流感疫苗和SARS疫苗,多采用注射接种方式,成本高,需专业人员操作,不易于免疫的普及。针对突如其来的重大疫情,使疫苗快速普及人群,口服是简捷方便的途径,开发口服疫苗势在必行。近年来以减毒沙门菌为载体的口服疫苗成为研究的热点,减毒伤寒沙门菌携带DNA疫苗进入人体的已有安全性报道,只要了解疾病的抗原特性,可以迅速的开发针对该病的口服疫苗,以它为载体还可以开发多价疫苗,生产周期短,成本低。减毒伤寒沙门菌作为疫苗载体菌显示了诱人的开发前景。本实验旨在应用减毒沙门菌为载体,构建H5亚型禽流感和SARS DNA口服疫苗。 本实验应用PCR方法获得了AIV的HA基因,通过人工合成的方法获得了SARS多表位基因S合。对真核表达载体pVAX1进行改造,以asd基因替换pVAX1载体上的Kan+抗性基因,以EGFP为报告基因,成功转染真核细胞;构建了重组真核表达载体pASD,它与减毒沙门菌X4550构成载体-宿主平衡致死系统,解决了质粒载体应用于人体产生抗生素耐药性的问题。将HA基因和S合基因亚克隆于pVAX1和pASD,脂质体法转染293-T细胞,通过RT-PCR检测目的基因的转录,通过间接ELISA和间接免疫荧光法检测目的基因的表达,结果表明,HA基因和S合基因能够正确表达。将携带抗原基因的真核表达质粒电转化入沙门菌X4550,在动物体内、外的研究表明,质粒能够在细菌中稳定存在。细菌的生长曲线测定结果表明,质粒的存在并不影响细菌的生长状态。免疫小鼠确定安全剂量为≤1010CFU。口服疫苗免疫小鼠,结果表明,口服疫苗能够激发小鼠机体产生特异性体液免疫和细胞免疫反应。
[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，
本文编号：2501910
[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，
本文编号：2501910
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