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狂犬病病毒G蛋白的原核表达和免疫原性分析

发布时间:2018-07-02 12:43

  本文选题:狂犬病病毒 + G蛋白 ; 参考:《西北农林科技大学》2017年硕士论文


【摘要】:狂犬病(Rabies)是由狂犬病病毒(Rabies Virus,RABV)引起的,是世界上最致命的传染病之一,死亡率接近100%。狂犬病毒是狂犬病毒属(Lyssavirus genus),弹状病毒科(Rhabdoviridae family)的一种单股负链RNA病毒,可引起人类,家畜及野生动物急性脑膜炎而导致死亡,并可在大多数哺乳动物中广泛传播。狂犬病主要是通过被狂犬病毒感染的动物的撕咬、抓挠受伤而传播。在中国,狂犬病的主要传播来源是被RABV感染的狗和猫等。当前,对于狂犬病感染或发病后的治疗方法尚未实现,因而及时接种狂犬病疫苗可在很大程度上有效防制狂犬病。目前我国动物用狂犬病疫苗主要是传统灭活疫苗和弱毒疫苗,弱毒疫苗存在返毒的危险,而灭活疫苗则存在灭活不彻底的风险。基因工程亚单位疫苗由于不含有强毒的感染性组分,所以不必进行灭活措施,是新型疫苗研究的重要发展方向。G(glycoprotein)蛋白是狂犬病毒的五种组成蛋白之一,由524个氨基酸组成,蛋白分子质量约为62KD。G蛋白是狂犬病毒中唯一糖基化的,以及唯一可诱导机体产生中和抗体的结构蛋白。因此,G蛋白既是狂犬病毒中最有效的保护性抗原,同时也是研究基因工程亚单位疫苗的首选抗原,是狂犬病基因工程亚单位疫苗研究的热点。本课题研究中,利用狂犬病病毒CTN-1V10株为模板,以生物工程合成的方法合成编码G蛋白的基因片段G。将该片段插入大肠杆菌pET-28a载体中,构建重组质粒,构建完成后,将重组质粒转化入大肠杆菌感受态细胞BL21(DE3)表达目的蛋白,SDS-PAGE电泳及Western blot结果表明,重组蛋白在大肠杆菌系统中实现了大量可溶性表达,在此基础上,进一步对该蛋白的反应原性和免疫原性进行了研究与分析。首先以狂犬病病毒CTN-1V10株(Genebank:AEV43285)为模板,设计相应的特异性引物,以生物工程合成的方法合成编码G蛋白的基因片段G,片段大小为1884bp。PCR结果表明,合成的目的片段长度正确。随后经转化,连接等步骤将目的片段与大肠杆菌pET-28a载体构建重组质粒。然后对重组蛋白进行了诱导表达及其条件优化。将重组质粒转化入大肠杆菌感受态细胞JM109,经菌液PCR鉴定阳性的菌株命名为pET-G。提取重组成功的质粒,将质粒转化入大肠杆菌感受态细胞BL21(DE3)中,经菌液PCR,酶切,测序鉴定,三种鉴定均成功后,方可用于后续的蛋白表达试验中。分别从温度、诱导时间梯度、诱导剂三个方面入手,对G蛋白的诱导表达的条件进行逐步优化。SDS-PAGE电泳检测,以及Western blot鉴定结果表明,在20°C,IPTG浓度为0.2 mM,诱导时长为12 h时,重组蛋白的可溶性表达量最高。且蛋白可与抗RABV单克隆抗体发生特异性反应,具有良好的反应原性。接下来进行了重组蛋白的纯化条件摸索。利用Ni填料亲和层析、离子交换层析、凝胶过滤层析等方法对重组G蛋白进行纯化,以获得相对纯度较高的目的蛋白,并检测蛋白活性,为后续实验做准备。最后进行了小鼠免疫试验及G蛋白的免疫原性分析。将30只6周龄的BALB/c小鼠按照免疫原的区别分为6组,分别为Tris buffer组(NC),商品灭活苗免疫组(PC),G-BI-Tris组,G-BI-ISA组,G-BB-Tris组,G-BB-ISA组。其中BI,BB为免疫增强剂,ISA为佐剂。按照剪耳编号,剪尾,采血,背部皮下多点注射的顺序免疫小鼠,每只小鼠注射100μL。28天后进行二免,至56天为止,期间每周采血一次,获得血清后-40°C冻存备用。56天后取28天和56天的血清用狂犬病病毒中和抗体ELISA检测试剂盒进行抗体检测。检测结果表明除阴性对照组外,其余各组一免后均产生抗体,且直至二免后28天为止抗体量一直呈上升水平。这说明,本实验中的重组G蛋白不仅具有良好的反应原性,而且具有相应的的免疫原性,为制备狂犬病病毒基因工程亚单位疫苗奠定了实验基础。
[Abstract]:Rabies is caused by the Rabies Virus (RABV) and is one of the most deadly infectious diseases in the world. The death rate is close to the 100%. rabies virus (Lyssavirus genus), a single strand of negative chain RNA virus of the family of Rhabdoviridae family (Rhabdoviridae family), which can cause acute meningitis in humans, livestock and wild animals. It leads to death and is widely spread in most mammals. Rabies is mainly transmitted by the bite of an animal infected by a rabies virus and scratching. In China, the main source of rabies is RABV infected dogs and cats. Currently, treatment for rabies has not been achieved and therefore timely. The vaccination of rabies vaccine can effectively prevent rabies to a great extent. At present, the main animal rabies vaccine in China is the traditional inactivated vaccine and the weak virus vaccine, the weakly toxic vaccine has the risk of returning to poison, and the inactivated vaccine has the risk of inactivation. .G (glycoprotein) protein is one of the five constituent proteins of rabies virus, which consists of 524 amino acids. Protein molecular mass about 62KD.G protein is the only glycosylated protein in rabies virus and the only structural protein that can induce the body to produce neutralizing antibodies. So, G Protein is not only the most effective protective antigen in rabies virus, but also the first antigen to study the subunit vaccine of genetic engineering. It is a hot spot in the study of the subunit vaccine of rabies gene engineering. In this study, the CTN-1V10 strain of rabies virus was used as a template to synthesize the gene fragment G. encoding G protein by bioengineering synthesis. The recombinant plasmid was inserted into the Escherichia coli pET-28a vector to construct the recombinant plasmid. After the construction was completed, the recombinant plasmid was transformed into the BL21 (DE3) expression target protein of the Escherichia coli cell, and the results of SDS-PAGE electrophoresis and Western blot showed that the recombinant protein was expressed in large amount of soluble expression in the Escherichia coli system. On this basis, the recombinant protein was further expressed. The reactivity and immunogenicity of the protein were studied and analyzed. First, specific primers were designed with the CTN-1V10 strain (Genebank:AEV43285) of rabies virus (Genebank:AEV43285), and the gene fragment encoding G protein was synthesized by bioengineering synthesis. The size of the fragment was 1884bp.PCR, and the length of the synthesized target fragment was correct. Then the recombinant plasmid was constructed by transformation, connection and other steps. The recombinant protein was induced and optimized. The recombinant plasmid was transformed into the JM109 of Escherichia coli receptor, and the positive strain was named pET-G. to extract the successful recombinant plasmid by PCR, and the plasmid was transferred to the recombinant plasmid by PCR. Into the BL21 (DE3) of Escherichia coli cells, the three kinds of identification were successfully identified by PCR, enzyme digestion and sequencing, and they could be used in the subsequent protein expression test. From the temperature, the induction time gradient and the inducer in three aspects, the conditions of the induced expression of G protein were gradually optimized by.SDS-PAGE electrophoresis and Western B. The results of lot identification showed that the soluble expression of the recombinant protein was the highest when the concentration of IPTG was 0.2 mM and the length of IPTG was 12 h. And the protein could react with the anti RABV monoclonal antibody and had a good reactivity. Then the purified protein of the recombinant protein was explored. Ni packing affinity chromatography and ion exchange chromatography were used. The recombinant G protein was purified by gel filtration chromatography to obtain the target protein with relatively high purity, and to detect the protein activity and prepare for the follow-up experiment. Finally, the immune test of mice and the immunogenicity of G protein were carried out. The 30 6 week old BALB/c mice were divided into 6 groups according to the difference of immunogen, which were Tris buffer group, respectively. (NC), inactivated vaccine group (PC), group G-BI-Tris, group G-BI-ISA, group G-BB-Tris, group G-BB-Tris, G-BB-ISA group. In which BI, BB were immune enhancers and ISA as adjuvant. Mice were immunized in order of ear numbering, cutting tail, collecting blood and subcutaneous injection on the back. Each mouse was injected with 100 mu and L.28 days after two exempts. To 56 days, blood was collected once a week and obtained once a week, obtained every week, obtained every week, obtained every week, obtained every week, obtained each week, obtained weekly, obtained a weekly blood obtained, obtained once a week, obtained. The serum -40 degree C after.56 days was stored for 28 days and 56 days to detect the antibody test of the serum rabies virus neutralization antibody ELISA detection kit. The results showed that the antibody was produced in all the other groups except the negative control group, and the anti body weight of the other groups was increased until the 28 day after two. This indicated that the recombinant G in this experiment was in this experiment. The protein not only has good reactivity, but also has the corresponding immunogenicity. It lays the experimental foundation for the preparation of rabies virus genetic engineering subunit vaccine.
【学位授予单位】:西北农林科技大学
【学位级别】:硕士
【学位授予年份】:2017
【分类号】:S852.65

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