【作者】 陈伟；

【导师】 王明丽；

【作者基本信息】 安徽医科大学 ， 微生物学， 2009， 硕士

【摘要】 目的肠道病毒71型(Enterovirus 71, EV71)是手足口病(Hand, foot and mouth disease, HFMD)的主要病原体之一,且常并发多种神经系统症状,甚至导致患者死亡。目前对EV71重症感染尚无有效的预防与治疗手段。本研究采用从六安地区手足口病患儿标本中分离病毒的方法得到EV71地方流行株;再采用分子生物学技术以该地方株的基因组为模板,扩增EV71衣壳蛋白VP1基因全长,构建VP1基因的重组真核表达载体pcDNA3.1(+)∕VP1,转染COS-7细胞并表达;通过肌肉注射免疫BALB∕c小鼠,观察pcDNA3.1(+)∕VP1诱导小鼠产生的体液免疫和细胞免疫应答水平,为预防EV71感染寻找新的有效途径。方法本研究主要分为三部分内容:⑴EV71地方株的分离与鉴定:首先将采自33例临床诊断为手足口病的患儿的51份标本(其中咽拭子33份,疱疹液12份,脑脊液6份)接种Vero细胞进行病毒分离。将分离得到的可疑病毒株利用透射电镜进行形态学观察,用EV71型特异性免疫血清及型特异性引物分别进行中和试验及RT-PCR检测,以对分离物进行EV71型别鉴定;⑵VP1基因真核表达载体的构建:提取分离并经鉴定证实为EV71的1株地方株19-Luan-CHN-08的基因组RNA,以其为模板采用RT-PCR法扩增VP1基因片段全长,将其插入至pMD18-T simple克隆载体,转化大肠杆菌感受态细胞,筛选阳性克隆,进行PCR鉴定、酶切鉴定及序列测定。并将序列与EV71 BrCr株VP1基因序列比较其同源性。将测序正确的VP1基因克隆至真核表达载体pcDNA3.1(+),命名为pcDNA3.1(+)∕VP1,转化大肠杆菌感受态细胞,筛选阳性克隆,进行PCR鉴定及酶切鉴定。将pcDNA3.1(+)∕VP1转染COS-7细胞,用Western blot法和RT-PCR法检测VP1基因在COS-7细胞中的表达;⑶重组真核表达载体pcDNA3.1(+)∕VP1的免疫活性初步研究:将pcDNA3.1(+)∕VP1免疫18只6-8周龄BALB∕c小鼠,共免疫三次,小鼠随机分为A, B和C 3组,每组6只,其中A组为实验组,每只小鼠分别在第0、2和4周经双后肢股四头肌注射pcDNA3.1(+)∕VP1重组质粒DNA 100μg,B组为空载体阴性对照组,每只小鼠分别在第0、2和4周经双后肢股四头肌注射pcDNA3.1(+)空质粒100μg,C组为PBS空白对照组,每只小鼠分别在第0、2和4周经双后肢股四头肌注射无菌PBS 100μl。每次免疫前24h,于每只小鼠双后肢股四头肌注射0.75%盐酸布比卡因30μl。分别于每次免疫前经鼠尾静脉采血收集血清,末次免疫后的第2周眼眶取血收集血清并处死小鼠,取各组小鼠脾细胞。间接ELISA法检测各组小鼠血清中的特异性EV71 VP1抗体水平,中和试验检测其抗EV71的中和效应,MTT比色法测定T淋巴细胞的增殖反应以检测细胞免疫水平。结果⑴本次研究中,14例患儿咽拭子标本分离阳性,阳性率为42.4%(14/33),其中2例患儿的疱疹液标本同时阳性,阳性率16.7%(2/12),共分离培养得到14株病毒分离株。透射电镜观察,在Vero细胞胞浆内可见有直径约30nm的球形无包膜肠道病毒样颗粒;而中和试验及型特异性引物RT-PCR检测结果证实其中6株为EV71,分别命名为01-Luan-CHN-08、06-Luan-CHN-08、11-Luan- CHN-08、13- Luan-CHN-08、14-Luan-CHN-08和19-Luan-CHN-08;⑵成功克隆了VP1基因片段全长,测序结果表明与EV71 BrCr株RNA同源性达98.4%,氨基酸同源性达96.3%。将此VP1基因片段与真核表达载体pcDNA3.1(+)连接,构建了重组真核表达载体pcDNA3.1(+)∕VP1,RT-PCR检测到VP1 mRNA表达,Western blot检测表明pcDNA3.1(+)∕VP1转染的COS-7细胞裂解液中出现一条约34KD的蛋白条带,与EV71 VP1蛋白分子量一致,其可被EV71 VP1多克隆抗体特异性识别;⑶在用pcDNA3.1(+)∕VP1免疫的BALB∕c小鼠血清中,初次免疫后2周特异性IgG抗体水平即有明显增高,OD450值为0.218,以后随免疫次数增加而逐渐增高,末次免疫后2周OD450值为0.559,经统计学分析,与B组及C组比较差异有显著性(p < 0.01);中和试验结果显示pcDNA3.1(+)∕VP1免疫的BALB∕c小鼠血清可在体外对EV71产生中和效应;三次免疫后,脾淋巴细胞增殖反应测定,A组小鼠T淋巴细胞增殖活性升高,刺激指数分别为:A组(2.410±0.12)、B组(1.400±0.21)、C组(1.241±0.19),A组与B组及C组比较差异有显著性(p<0.01)。结论⑴EV71是此次六安地区HFMD主要病原体之一,采用Vero细胞株培养可对其进行有效分离,其中咽拭子标本阳性分离率最高,其次为疱疹液标本,脑脊液标本阳性分离率最低;⑵成功构建了EV71地方分离株VP1重组真核表达载体pcDNA3.1(+)∕VP1,并证实该重组载体在COS-7细胞中获得表达,且表达产物具有抗原性;⑶小鼠注射重组真核表达载体pcDNA3.1(+)∕VP1后可获得一定程度的特异性体液免疫和细胞免疫应答,免疫小鼠血清可中和EV71病毒,抑制其致细胞病变效应。本研究为进一步研制EV71核酸疫苗奠定了一定的实验基础,将有助于对EV71感染的预防与控制。更多还原

【Abstract】 Objective Enterovirus 71 (EV71) is a major causative viral agent responsible for hand,foot and mouth disease (HFMD) and often causes a variety of nervous system complications. There is no effective antiviral treatment for severe EV71 infection and no vaccine is available. In this study, we isolated EV71 from specimens of patients with HFMD in Luan. Full-length VP1 gene was amplified and cloned into eukaryotic expression vector pcDNA3.1(+).To observe the humoral immune response and cellular immune response in mice induced by intramuscular injection in BALB/c mice. Find a new and effective way to prevent EV71 infection .Methods⑴First, in this study,we collected 51 specimens from 33 cases of children with HFMD (33 throat swab, 12 herpes fluid, 6 cerebrospinal fluid) and isolated virus using of Vero cell culture. All positive specimens were observed using transmission electron microscope and identified for EV71 by neutralization test using EV71 standard serum and by RT-PCR using EV71 specific primers;⑵Subsequently, the RNA were extracted from one EV71 local strains.And then the VP1 gene were amplified using RT-PCR and cloned into the pMD18-T simple vector. The constructs were transformed into competent E.coli cells and the positive clones were selected using restriction digestion, RT-PCR and DNA sequencing analysis. The correct sequencing of the VP1 gene was inserted into eukaryotic expression vector, pcDNA3.1(+). The recombinant plasmid were test for protein expression in COS-7 cells;⑶Eventually, in the in vivo studies, 18 female BALB/c mice aged between 6-8 weeks were randomly divided in 3 groups of 6 and were immunised by intramuscular injection with the recombinant plasmid., in which group A, the mice were injected with 100μg/100μl the recombinant plasmid pcDNA3.1(+)/VP1 per mouse. The two negative control groups were injected with the backbone vector pcDNA3.1(+) (100μg/100μl per mouse) and PBS (100μl per mouse). The mice were given booster injections at 2 and 4 weeks post-immunisation. Blood samples were collected from the mice via the tail vein before immunization and via the orbital artery and vein after the last immunization 2 weeks, meanwhiles, prepared T lymphocytes from the mouse. The indirect Enzyme Linked Immunosorbent Assay(ELISA) were performed to detect the presence of anti-VP1 IgG in the sera of immunised mice and the virus neutralizing assay were performed to assess the ability of the mice serum to neutralise the live EV71. The proliferation of T lymphocytes by MTT assay in order to observe the cell immunity.Results⑴In this study, 14 suspicimens enterovirus positive throat swab specimens (42.4%) were found from 14 patients with HFMD, in which 2 herpes fluid specimens (16.7%) at the same time.We confirmed they were enterovirus virus by transmission electron microscope.In the end,6 EV71 positive specimens(42.9%) were found by micro-neutralization test using EV71 standard serum and by RT-PCR using EV71 specific primers;⑵Sequencing results showed that the homology of nucleotide and amino acid between the VP1 of local strain and EV71 BrCr strain were 98.4% and 96.3%. Identified by restriction digestion analysis and RT-PCR analysis, the recombinant eukaryotic expression plasmid pcDNA3.1(+)/VP1 was successfully constructed. By RT-PCR,mRNA of VP1 gene fragment could be detected in transfected COS-7 cells.The result of Western blot showed that there was a protein band about 34KD in cell lysis of transfected COS-7 cells,and it could be specifically recognized by EV71 polyclonal antibody;⑶The recombinant eukaryotic expression plasmid pcDNA3.1 (+)/VP1 was used to immune BALB/c mice.The serum-specific IgG antibody levels significantly increased after the initial immunization, OD450 was 0.218, and then increased along with the number of immune increased. OD450 was 0.559 in week 2 after the last immunization. A significantly difference was test between A group and B group or C group.(p <0.01). The result of neutralizing assay showed that the anti-VP1 IgG had neutralising activity against EV71. The activity of T lymphocytes proliferation increases and the stimulation index (SI) were: A group 2.410±0.12, B group 1.400±0.21, C group 1.241±0.19, The SI of A group were significantly higher than B group and C group (p <0.01).Conclusions⑴EV71 is one of the main pathogens for the hand, foot and mouth disease occored in Liuan region.⑵The recombinant eukaryotic expression plasmid pcDNA3.1(+)/VP1 was successfully constructed, and VP1 protein express in COS-7 cells.⑶The female mice injected recombinant eukaryotic expression plasmid pcDNA3.1(+)/VP1 were available for a certain degree of humoral immunity and cellular immune responses.This study has given to a solid foundation for the nucleic acid vaccine against EV71.更多还原