表达密码子优化犬细小病毒VP2蛋白重组犬瘟热弱毒疫苗株的构建

发布时间：2018-03-25 22:08

本文选题：犬细小病毒　切入点：VP2蛋白　出处：《中国农业科学院》2015年硕士论文

【摘要】：犬细小病毒病是由犬细小病毒(Canine parvovirus, CPV)引起的传染犬科动物的,高度接触性、致死性疾病,以出血性肠炎和亚急性心肌炎为特征。犬细小病毒病主要通过粪-口途径传播,幼犬最易感染。目前,犬细小病毒病已经在我国广泛流行,严重威胁着我国的养犬业和皮毛动物养殖业。疫苗接种是防控犬细小病毒病的最有效方法。犬细小病毒病灭活疫苗只能诱导一个短期的免疫反应；由于新的抗原变异毒株的出现,弱毒疫苗接种有时会出现免疫失败。随着反向遗传技术的发展和成熟,单股负链RNA病毒成为了疫苗载体的研究热点。犬瘟热(Canine distemper, CD)是由犬瘟热病毒(Canine distemper virus, CDV)引发的感染多种动物的急性致死性传染病,给我国的犬养殖业和毛皮动物业造成了严重的损失。目前,全世界应用最为广泛的犬瘟热疫苗是弱毒疫苗。利用犬瘟热弱毒疫苗为载体,研制犬细小病毒病、犬瘟热二联活载体疫苗,可实现一苗两防,降低疫苗生产成本,也可针对新出现的抗原变异亚型毒株快速地进行疫苗更新。按照哺乳动物的密码子偏好性对外源抗原蛋白基因进行密码子优化,能够提高该蛋白在哺乳动物体内或细胞内的表达量,为提高疫苗的免疫效力提供了新的方法。本研究,利用本实验已建立的CD弱毒疫苗株CDV/R-20/8的反向遗传技术平台,构建了表达密码子优化CPV VP2蛋白的重组犬瘟热病毒r20/8-CPVVP2opti。免疫荧光实验和Western blot实验表明,重组病毒r20/8-CPVVP2opti感染的BHK-21细胞中,CPV VP2蛋白得到了正确表达。重组病毒r20/8-CPVVP2opti保持了亲本毒株r20/8在Vero细胞上的生长特性,经实验测定具有生长滴度高的特点,其最高生长滴度可达到106.75 TCID50/ml。Western blot证实,密码子优化可显著提高CPVVP2蛋白在重组犬瘟热病毒r20/8-CPVVP2opti感染细胞中的表达量。以表达密码子优化CPVVP2蛋白重组犬瘟热病毒r20/8-CPVVP2opti表达野生型CPVVP2蛋白重组犬瘟热病毒r20/8-CPVVP2和亲本毒株r20/8分别按104.5TCID50/100μl/只的剂量以肌肉注射的方式,两次免疫接种小鼠。r20/8-CPVVP2opti、r20/8-CPVVP2r20/8免疫小鼠,均诱导低水平的CDV中和抗体反应,且3组免疫小鼠血清中CDV中和抗体滴度无显著性差异；然而,r20/8-CPVVP2opti、r20/8-CPVVP2免疫小鼠,均可诱导显著的CPV血凝抑制(HI)抗体反应,加强免疫后1周,CPV HI抗体反应显著增强,且r20/8-CPVVP2opti免疫小鼠血清中CPV HI抗体滴度显著地高于r20/8-CPVVP2免疫小鼠免疫小鼠血清中CPV HI抗体滴度。结果表明,利用密码子优化CPV VP2蛋白基因构建CPV、CDV活载体疫苗,能增强在免疫动物体内诱导的CPV HI抗体反应,有助于提高该疫苗抗CPV感染的免疫效力。本研究结果表明,表达密码子优化CPV VP2蛋白重组CDVr20/8-CPVVP2opti具有良好的免疫原性,具有作为预防犬细小病毒病和犬瘟热二联活载体疫苗的潜力。
[Abstract]:Canine parvovirus disease (CPV) is a highly contagious and fatal disease of Canine parvovirus (CPV), characterized by hemorrhagic enteritis and subacute myocarditis. Puppies are most susceptible to infection. At present, canine parvovirus disease has been widespread in China. Vaccination is the most effective method to prevent and control canine parvovirus disease. Inactivated canine parvovirus vaccine can only induce a short-term immune response. As a result of the emergence of new antigenic variant strains, attenuated vaccines sometimes fail. As reverse genetic technology develops and matures, Single strand negative strand RNA virus has become a hot topic in vaccine vector. Canine distemper (CDV) is an acute fatal infectious disease caused by canine distemper virus (CDV). At present, the most widely used canine distemper vaccine in the world is attenuated virus vaccine. Using canine distemper attenuated vaccine as carrier, canine parvovirus disease is developed. Canine distemper binomial live vector vaccine can realize one vaccine and two prevention, and reduce the cost of vaccine production. It is also possible to quickly update the vaccine against the emerging antigen variant subtype strain, and to optimize the codon of the foreign antigen protein gene according to the codon preference of the mammalian codon. It can increase the expression of the protein in mammalian or in cells, and provide a new method to improve the immune potency of the vaccine. In this study, the reverse genetic technology platform of CD attenuated vaccine strain CDV/R-20/8 has been established in this study. Recombinant canine distemper virus r20 / 8-CPVVP2opti.Immunofluorescence and Western blot experiments showed that the recombinant canine distemper virus r20 / 8-CPVP2optivirus was constructed to express codon to optimize CPV VP2 protein. The recombinant virus r20/8-CPVVP2opti maintained the growth characteristics of the parent strain R20 / 8 on Vero cells and had the characteristics of high growth titer. The highest growth titer can reach 106.75 TCID50/ml.Western blot. Codon optimization can significantly increase the expression of CPVVP2 protein in recombinant canine distemper virus r20/8-CPVVP2opti infected cells. The expression codon optimizes the expression of CPVVP2 protein recombinant canine distemper virus r20/8-CPVVP2opti expression wild-type CPVVP2 protein recombinant canine distemper virus r20/8-CPVVP2 and parent. Strain R20 / 8 was injected intramuscularly at the dose of 104.5TCID50/100 渭 l / mouse, The mice inoculated twice with. R20 / 8-CPVP2optir 20 / 8-CPVP2P2r20 / 8 induced a low level of CDV neutralizing antibody reaction, and there was no significant difference in the titer of CDV neutralizing antibodies in the serum of the three groups of immunized mice; however, R208-CPVP2optir208-CPVP2optir20 / 8-CPVP2VP2 immunized mice. CPV hemagglutination inhibited the anti-HI antibody response significantly, and the HI antibody response was significantly increased at 1 week after enhanced immunization. The titer of CPV HI antibody in the serum of r20/8-CPVVP2opti immunized mice was significantly higher than that of r20/8-CPVVP2 immunized mice. The results showed that the codon optimized CPV VP2 protein gene was used to construct the live vector vaccine. The results showed that the expression codon optimized CPV VP2 protein recombinant CDVr20/8-CPVVP2opti had good immunogenicity. It has the potential as a bivector vaccine against canine parvovirus and canine distemper.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.292

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