汉坦病毒G1S0.7嵌合基因的真核表达及其经基因枪免疫的初步研究

发布时间：2018-04-16 19:11

本文选题：汉坦病毒 + M基因　；参考：《第四军医大学》2010年硕士论文

【摘要】： 汉坦病毒(Hantavirus,HV)属于世界范围内分布的、尚不能控制的对人类有致死性威胁疾病的病原体之一,可以引起肾综合征出血热(hemorrhagic fever with renal syndrome,HFRS)和汉坦病毒肺综合征(Hantavirus pulmonary syndrome,HPS)。HFRS是一种全球性的急性传染病,疫区分布广泛、发病机制复杂、病情危重易变,并发症多而难治,病死率居高不下,严重危害人类的生命健康。我国是世界上受HFRS危害最重的国家,HFRS发病人数占世界总发病人数的90%以上,在我国是除病毒性肝炎之外危害最大的病毒性传染病,每年约发生5万～10万例,疫情仍得不到有效控制。目前尚无特效治疗药物。“预防为主”是控制传染病的基本原则,因此研制安全、有效的疫苗是控制本病流行和发生的关键。 HV属于布尼亚病毒科汉坦病毒属,由核衣壳和囊膜组成,其核酸类型为单负链RNA,分为小(S)、中(M)和大(L)三个基因片段,分别编码病毒的核衣壳蛋白(nucleocapsid protein,NP)、囊膜糖蛋白(glycoprotein,GP)G1和G2以及RNA聚合酶(RNA dependent RNA polymerase,RDRP)。研究表明,HV的囊膜糖蛋白GP及核蛋白NP均与其保护性免疫有关。一般认为GP主要诱发保护性体液免疫应答,其可刺激机体产生中和抗体,对感染动物和人体均具有保护作用。GP上具有病毒毒力位点、中和抗原及型特异性抗原位点等多种抗原决定簇,还具有血凝活性位点,是重要的保护性抗原。但GP的免疫原性较弱,刺激机体产生的抗体出现晚,滴度不高。而核蛋白NP是HV另一重要的结构蛋白,其在宿主体内的表达量最高,有很强的抗原性和免疫原性,其刺激机体产生的特异性抗体出现早、滴度高、维持时间长。国内外研究结果证实NP具有B细胞和T细胞决定簇,可诱导机体同时产生保护性体液免疫和细胞免疫。本室前期研究表明,NP上也存在中和抗原位点,并且抗原位点大多位于S基因5′端约0.7kb片段编码的蛋白上。本实验采用HV国际标准株——汉滩病毒76-118株,将其M基因G1片段和S基因5′端约0.7 kb片段进行了融合表达,以期达到不同结构蛋白在刺激机体免疫应答中的优势互补。我们利用本室前期构建并经测序的重组质粒pShuttle-G1S0.7,经双酶切回收得到片段G1S0.7后,克隆入真核表达载体pVAX1,得到重组质粒pVAX-G1S0.7,再以脂质体介导转染人胚肾(HEK)293细胞,并采用ELISA和Western-blot对表达产物进行鉴定。ELISA检测结果和Western-blot结果显示,汉坦病毒G1S0.7嵌合基因在HEK293细胞中得到了表达,所表达的融合蛋白分子量约90kD,与预期大小一致,并且表达产物可与抗汉坦病毒NP mAb特异性结合。在此基础上,我们对该重组质粒的基因免疫效果进行了初步研究。采用基因枪免疫途径,将上述重组质粒DNA包被于金颗粒上制成基因枪子弹轰击C57小鼠腹部皮肤进行接种。每隔两周免疫一次,共免疫四次。用ELISA法和ELISpot法分别检测免疫鼠的体液免疫应答和细胞免疫应答效果。对各组小鼠不同免疫时间血清中特异性抗体的ELISA检测结果表明,重组质粒经基因枪免疫后可诱导小鼠产生抗汉坦病毒GP的抗体,但抗体出现晚,滴度低。ELISpot试验检测免疫鼠脾脏中特异性分泌IFN-γ的T淋巴细胞(SFC,spot forming cells)数目,结果表明,重组质粒刺激小鼠产生的SFC数量明显高于空载体对照组及空白对照组,说明其可诱导机体产生特异性的细胞免疫应答。以上实验结果表明,构建的重组质粒通过基因枪接种方式,不仅可刺激机体产生特异性的低滴度的体液免疫应答,还可刺激机体产生特异性的细胞免疫应答,不足之处在于基因枪免疫途径未能显著提高免疫小鼠的体液应答水平。在以后的研究中,需要进一步摸索基因枪技术使用的最佳条件,还需在载体选择、注射时间、注射剂量等方面作更深入的研究,进一步探索如何提高基因免疫的效应,为研制出汉坦病毒基因疫苗奠定基础。
[Abstract]:Hantaviruses ( HV ) belong to one of the pathogen of worldwide distribution , which is not yet controlled by lethal threat to humans , and can cause hemorrhagic fever with renal syndrome , hemorrhagic fever with renal syndrome and Hantan pulmonary syndrome . Hemorrhagic Fever is a global acute infectious disease , the epidemic area is widespread , the pathogenesis is complex , the disease is critical , the complications are many , the morbidity is high , the mortality rate is high , serious harm to the human life health . China is the world of the most dangerous virus infectious disease , the epidemic is still not effectively controlled . At present , there is no special effect treatment medicine . " Prevention is the main " is the basic principle of controlling infectious diseases , so the development of safe and effective vaccine is the key to control the epidemic and the occurrence of this disease .

HV belongs to the genus Hantaviruses of Virginia , consisting of a nuclear capsid and a capsule . The nucleic acid type is single negative strand RNA . It is divided into three gene segments of small ( S ) , medium ( M ) and large ( L ) , respectively , encoding the virus ' s nuclear capsid protein ( NP ) , glycoprotein ( GP ) G1 and G2 , and RNA polymerase ( RNA dependent RNA polymerase , Rdrp ) . The results of this study show that the NP is a major structural protein , which has the highest expression level in the host , and has high antigenicity and immunogenicity . The results of the study show that NP has the highest expression level in host , and has high antigenicity and immunogenicity .

The recombinant plasmid pShuttle - G1S0.7 was constructed and sequenced . The recombinant plasmid pShuttle - G1S0.7 was cloned into eukaryotic expression vector pShuttle - G1S0.7 . The recombinant plasmid pShuttle - G1S0.7 was cloned into eukaryotic expression vector pShuttle - G1S0.7 . The recombinant plasmid pShuttle - G1S0.7 was cloned into eukaryotic expression vector pShuttle - G1S0.7 . The recombinant plasmid pShuttle - G1S0.7 was cloned into eukaryotic expression vector pShuttle - G1S0.7 . The molecular weight of the expressed fusion protein was about 90kD , which was consistent with the expected size , and the expression product could be specifically bound to the anti - Hantanvirus NP mAb .

On the basis of this , we have carried out a preliminary study on the gene immunization effect of the recombinant plasmid . The recombinant plasmid DNA was coated on the gold particles by gene gun immunization . The humoral immune responses and cellular immune responses of the immunized mice were detected by ELISA . The results showed that the number of specific antibodies produced by the recombinant plasmid was higher than that of the empty vector control group and the blank control group . The results showed that the recombinant plasmid could induce the specific cellular immune response .

The results show that the recombinant plasmid constructed can not only stimulate the body fluid immune response with low titer , but also stimulate the organism to produce specific cellular immune response , and the deficiency is that the immune pathway of gene gun does not significantly improve the humoral response level of immune mice . In the future research , it is necessary to further study the optimal conditions for the use of gene gun technology , and further explore how to improve the effect of gene immunization and lay the foundation for the development of the Hantaviruses gene vaccine .

【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

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