本文選題：狂犬病病毒 + G蛋白��； 參考：《西北農(nóng)林科技大學(xué)》2017年碩士論文

【摘要】：狂犬病(Rabies)是由狂犬病病毒(Rabies Virus,RABV)引起的,是世界上最致命的傳染病之一,死亡率接近100%。狂犬病毒是狂犬病毒屬(Lyssavirus genus),彈狀病毒科(Rhabdoviridae family)的一種單股負(fù)鏈RNA病毒,可引起人類,家畜及野生動物急性腦膜炎而導(dǎo)致死亡,并可在大多數(shù)哺乳動物中廣泛傳播。狂犬病主要是通過被狂犬病毒感染的動物的撕咬、抓撓受傷而傳播。在中國,狂犬病的主要傳播來源是被RABV感染的狗和貓等。當(dāng)前,對于狂犬病感染或發(fā)病后的治療方法尚未實(shí)現(xiàn),因而及時接種狂犬病疫苗可在很大程度上有效防制狂犬病。目前我國動物用狂犬病疫苗主要是傳統(tǒng)滅活疫苗和弱毒疫苗,弱毒疫苗存在返毒的危險,而滅活疫苗則存在滅活不徹底的風(fēng)險。基因工程亞單位疫苗由于不含有強(qiáng)毒的感染性組分,所以不必進(jìn)行滅活措施,是新型疫苗研究的重要發(fā)展方向。G(glycoprotein)蛋白是狂犬病毒的五種組成蛋白之一,由524個氨基酸組成,蛋白分子質(zhì)量約為62KD。G蛋白是狂犬病毒中唯一糖基化的,以及唯一可誘導(dǎo)機(jī)體產(chǎn)生中和抗體的結(jié)構(gòu)蛋白。因此,G蛋白既是狂犬病毒中最有效的保護(hù)性抗原,同時也是研究基因工程亞單位疫苗的首選抗原,是狂犬病基因工程亞單位疫苗研究的熱點(diǎn)。本課題研究中,利用狂犬病病毒CTN-1V10株為模板,以生物工程合成的方法合成編碼G蛋白的基因片段G。將該片段插入大腸桿菌pET-28a載體中,構(gòu)建重組質(zhì)粒,構(gòu)建完成后,將重組質(zhì)粒轉(zhuǎn)化入大腸桿菌感受態(tài)細(xì)胞BL21(DE3)表達(dá)目的蛋白,SDS-PAGE電泳及Western blot結(jié)果表明,重組蛋白在大腸桿菌系統(tǒng)中實(shí)現(xiàn)了大量可溶性表達(dá),在此基礎(chǔ)上,進(jìn)一步對該蛋白的反應(yīng)原性和免疫原性進(jìn)行了研究與分析。首先以狂犬病病毒CTN-1V10株(Genebank:AEV43285)為模板,設(shè)計相應(yīng)的特異性引物,以生物工程合成的方法合成編碼G蛋白的基因片段G,片段大小為1884bp。PCR結(jié)果表明,合成的目的片段長度正確。隨后經(jīng)轉(zhuǎn)化,連接等步驟將目的片段與大腸桿菌pET-28a載體構(gòu)建重組質(zhì)粒。然后對重組蛋白進(jìn)行了誘導(dǎo)表達(dá)及其條件優(yōu)化。將重組質(zhì)粒轉(zhuǎn)化入大腸桿菌感受態(tài)細(xì)胞JM109,經(jīng)菌液PCR鑒定陽性的菌株命名為pET-G。提取重組成功的質(zhì)粒,將質(zhì)粒轉(zhuǎn)化入大腸桿菌感受態(tài)細(xì)胞BL21(DE3)中,經(jīng)菌液PCR,酶切,測序鑒定,三種鑒定均成功后,方可用于后續(xù)的蛋白表達(dá)試驗(yàn)中。分別從溫度、誘導(dǎo)時間梯度、誘導(dǎo)劑三個方面入手,對G蛋白的誘導(dǎo)表達(dá)的條件進(jìn)行逐步優(yōu)化。SDS-PAGE電泳檢測,以及Western blot鑒定結(jié)果表明,在20°C,IPTG濃度為0.2 mM,誘導(dǎo)時長為12 h時,重組蛋白的可溶性表達(dá)量最高。且蛋白可與抗RABV單克隆抗體發(fā)生特異性反應(yīng),具有良好的反應(yīng)原性。接下來進(jìn)行了重組蛋白的純化條件摸索。利用Ni填料親和層析、離子交換層析、凝膠過濾層析等方法對重組G蛋白進(jìn)行純化,以獲得相對純度較高的目的蛋白,并檢測蛋白活性,為后續(xù)實(shí)驗(yàn)做準(zhǔn)備。最后進(jìn)行了小鼠免疫試驗(yàn)及G蛋白的免疫原性分析。將30只6周齡的BALB/c小鼠按照免疫原的區(qū)別分為6組,分別為Tris buffer組(NC),商品滅活苗免疫組(PC),G-BI-Tris組,G-BI-ISA組,G-BB-Tris組,G-BB-ISA組。其中BI,BB為免疫增強(qiáng)劑,ISA為佐劑。按照剪耳編號,剪尾,采血,背部皮下多點(diǎn)注射的順序免疫小鼠,每只小鼠注射100μL。28天后進(jìn)行二免,至56天為止,期間每周采血一次,獲得血清后-40°C凍存?zhèn)溆谩?6天后取28天和56天的血清用狂犬病病毒中和抗體ELISA檢測試劑盒進(jìn)行抗體檢測。檢測結(jié)果表明除陰性對照組外,其余各組一免后均產(chǎn)生抗體,且直至二免后28天為止抗體量一直呈上升水平。這說明,本實(shí)驗(yàn)中的重組G蛋白不僅具有良好的反應(yīng)原性,而且具有相應(yīng)的的免疫原性,為制備狂犬病病毒基因工程亞單位疫苗奠定了實(shí)驗(yàn)基礎(chǔ)。
[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.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S852.65

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