HIV Ⅰ型外膜蛋白/干扰素A-2b的构建与表达

目的 构建艾滋病病毒外膜蛋白（env）与干扰素（IFNα-2b）融合基因表达质粒，观察其在痘苗病毒中共表达结果。方法 利用基因重组技术，将IFNα-2b基因片段插入到env基因的nt8383位点，经脂质体转染与血凝素阴性蚀斑筛选，挑出重组痘苗病毒。经免疫荧光、免疫印迹（Western blot）和斑点酶联免疫吸附试验（Dot-ELISA）鉴定表达产物。结果 间接免疫荧光实验结果显示，转染重组质粒的细胞表面有绿色荧光。Westem-blot与Dot-ELISA结果均显示，重组质粒转染细胞的裂解物中存在表达的env／IFNα-2b蛋白。结论 成功地构建了重组真核细胞表达质粒，表达的蛋白具有良好的免疫原性与免疫反应性。     

HIV-1 gag/IFNα-2b表达产物的免疫原性研究

目的构建艾滋病病毒核心蛋白（gag）与干扰素（IFNα-2b）融合基因表达质粒,观察其与痘苗病毒共表达的结果,研究其意义。方法利用基因重组技术,将IFNα-2b基因片段插入到gag基因的nt 531位点,经脂质体转染与血凝素阴性蚀斑筛选,挑出重组病毒。经免疫荧光、蛋白印迹分析（Western blot）和斑点酶联免疫（Dot ELISA）鉴定表达产物。结果间接免疫荧光实验显示,转染重组质粒的细胞表面有绿色荧光。免疫印迹实验与Dot ELISA结果均显示,转染重组质粒的细胞裂解物中存在表达的gag/IFNα-2b蛋白。结论成功地构建了重组真核细胞表达质粒,表达的蛋白具有良好的免疫原性与免疫反应性。     

基于单表达质粒的T7噬菌体病毒样颗粒制备、纯化及形态鉴定

目的 提供一种基于单表达质粒制备T7噬菌体病毒样颗粒（Virus-like particles, VLPs）的新策略,为后续构建基于T7噬菌体病毒样颗粒的载体疫苗或靶向药物奠定基础。方法 将T7噬菌体衣壳蛋白基因gp10A及支架蛋白基因gp9分别克隆至质粒pRSFDuet-1的MCS1及MCS2位点,转入大肠杆菌BL21(DE3)后进行诱导、表达及自主装,采用超高速离心及尺寸排除色谱对T7 VLPs进行纯化,利用 SDS-PAGE、透射电镜和动态光散射技术,对纯化后的T7 VLPs进行形态、结构、表型及粒径分析。结果 琼脂糖凝胶电泳和测序结果表明成功构建pRSFDuet-1-gp9-gp10A质粒,SDS-PAGE电泳表明该质粒能在大肠杆菌BL21(DE3)中大量表达T7噬菌体衣壳蛋白Gp10A及支架蛋白Gp9,经尺寸排除色谱纯化后的纯度较高;通过透射电镜观察其自主装形成大小均一的VLPs结构,该VLPs平均粒径为70 nm,形态似T7噬菌体原衣壳结构。结论 利用pRSFDuet-1质粒同时表达T7噬菌体衣壳蛋白Gp10A和支架蛋白Gp9,能在大肠杆菌BL21(DE3)中自主装形成VLPs。     

登革-日本脑炎嵌合病毒样颗粒的设计与构建

目的设计构建登革-日本脑炎嵌合蛋白表达载体,制备嵌合病毒样颗粒。方法 根据登革病毒样颗粒的形成机制及日本脑炎病毒中和性抗原表位的分布,设计并构建登革-日本脑炎嵌合蛋白表达载体,转染BHK-21细胞,筛选能表达目标蛋白的G418抗性克隆,收集细胞培养上清,纯化病毒样颗粒,用Western-blot和电镜检测病毒样颗粒的形成。结果用RT-PCR扩增、酶切和连接等分子生物学方法成功构建了序列及阅读框均正确的重组表达质粒pCI-SMEJ-14#;将该质粒转染BHK-21细胞,经0.6 g/LG418筛选获得4个能表达目标嵌合蛋白的克隆,从培养的细胞上清中能纯化出直径30~100 nm的病毒样颗粒。结论 所设计的登革-日本脑炎病毒嵌合蛋白能在BHK-21细胞中产生病毒样颗粒,为制备新一代登革-日本脑炎嵌合型疫苗奠定了良好基础。     

人乳头瘤病毒16型晚期蛋白在昆虫细胞中的表达

目的 构建HPV16型晚期蛋白重组杆状病毒，并使其在昆虫细胞中获得高效表达。方法 首先用线性化的杆关病毒DNA与重组杆关病毒转移共杂sf9昆虫细胞进行同源重组，获得2株重组杆状病毒。结果 经鉴定该重组病毒中有目的DNA存在且可表达晚期蛋白；电镜观察发现重组杆关病毒感染的sf9细胞核内有乳头瘤病毒VLPs。结论 HPV16型晚期蛋白在昆虫细胞中的成功表达为HPV16型预防性基因工程亚单位疫苗的研制和诊断试剂的研究开发奠定了基础。     

H7N9禽流感病毒HA2基因真核表达与免疫原性初步研究

目的利用真核表达载体构建H7N9禽流感病毒血凝素刺突茎部(HA2)的真核表达质粒,在293F细胞中表达HA2蛋白,并初步探讨其免疫原性。方法根据pMD18-T-HA中的序列设计H7N9禽流感病毒HA2扩增引物,在下游引物中引入胰酶酶切位点;目的基因经特异性酶切位点克隆入自身带有Fc标签的pFUSE-IgG1-Fc1载体,构建重组质粒HA2-Fc;将重组质粒转染293F细胞,通过间接免疫荧荧光(IFA)和免疫印迹法(WB)鉴定HA2蛋白的表达和免疫原性。结果成功构建H7N9禽流感病毒HA2基因真核表达质粒HA2-Fc,并在293F细胞中表达出分子量大约为50kDa的重组蛋白。IFA和WB显示该蛋白与抗H7N9病毒鼠多抗具有良好的免疫反应。结论成功构建表达HA2亚单位的真核表达系统,重组蛋白具有较高的免疫原性,为筛选H7N9广谱疫苗候选分子、广谱中和抗体,及深入研究其致病机理和免疫机制奠定基础。     

单周期复制的马传染性贫血病毒重组疫苗的构建

目的 构建单周期复制的马传染性贫血病毒（EIAV）重组疫苗。方法 将感染性EIAV分子克隆WU57囊膜（env）基因敲除，在pGPT中克隆4个拷贝的Mason Pfizer猴病毒组成性RNA转运因子（CTE），构建重组质粒pGPTC；构建另一表达Env蛋白的重组质粒pTEB，并分别与重组质粒pGPT、pGPTC共转染进入293细胞；以Western blot对转染细胞外培养液进行检测以鉴定病毒粒子的产生；通过免疫荧光分析鉴定感染细胞内病毒蛋白的表达。结果 pGPTC/pTEB共转染的细胞外培养液中检测到特异性EIAV病毒蛋白，而pGPT／pTEB共转染的细胞外培养液中没有检测到特异性EIAV病毒蛋白。转染细胞产生的病毒粒子EIAVGPTC。感染的原代马肾细胞（EK）内检测到病毒蛋白的表达，而用感染细胞产生的病毒粒子再感染EK细胞，则无病毒蛋白表达。结论 Rev/RRE对于病毒结构蛋白的表达是必需的；CTE可以替代Rev蛋白功能辅助EIAV结构蛋白的表达；转染的293细胞产生了EIAV病毒粒子并分泌到胞外培养液中；产生的病毒粒子EIAVGPTC是一复制缺陷的活重组病毒。     

水痘-带状疱疹病毒糖蛋白I基因在昆虫细胞中的表达及纯化

目的 将北京水痘-带状疱疹病毒(VZV)84-7株克隆糖蛋白I(gpI)基因在杆状病毒-昆虫细胞表达系统中表达,并对其表达产物进行纯化。方法 采用PCR方法从VZVDNA中扩增gpI全基因序列,并将其插入杆状病毒转移质粒pBacPAK9中,获得重组转移质粒pBacVZVgpI,对pBacVZVgpI中的插入基因进行测序。重组转移质粒与线性杆状病毒BacPAK6DNA(Bsu36Idigested)共转染Sf9昆虫细胞,获得重组病毒BacPAK-gpI。通过亲和层析纯化重组蛋白,并检测其抗原性。结果 PCR扩增得到gpI基因,测序结果表明克隆的外源基因正确。经SDS-聚丙烯酰胺凝胶电泳(SDS—PAGE)、免疫印迹(weotem-blot)方法证明gpI基因在昆虫细胞中获得表达,表达产物在培养72h达到高峰,重组蛋白的相对分子质量约为58000和70000,与理论值相符,蛋白质加工与天然蛋白类似。动物实验结果表明,重组蛋白具有较好的免疫原性,可刺激小鼠产生中和抗体。SDS—PAGE检测纯化的重组蛋白,纯度达80％。纯化蛋白经western-blot和ELISA检测后显示,具有特异的抗体结合活性。结论 应用昆虫细胞表达水痘-带状疱疹病毒gpI基因,可为水痘-带状疱疹病毒抗原定量分析、糖蛋白ELISA的研制和制备亚单位疫苗提供基础。     

含肠道病毒71型VP1基因片段RNA病毒样颗粒的构建

[目的]构建含肠道病毒71型(EV71)VP1基因片段的抗核糖核酸酶的病毒样颗粒。[方法]利用PCR技术扩增大肠杆菌MS2噬菌体的外壳蛋白和成熟酶蛋白基因,将其克隆到pET32a中构建中间载体pET32a-MS2。将EV71VP1基因片段连接到中间载体噬菌体基因的下游,构建原核表达载体pET32a-MS2-EV71。将重组质粒pET32a-MS2-EV71转化宿主菌,经IPTG诱导表达获得病毒样颗粒,对病毒样颗粒进行透射电镜观察、RT-PCR检测和稳定性试验。[结果]重组质粒pET32a-MS2-EV71经PCR、双酶切鉴定和测序分析后证实构建成功;透射电镜观察到直径大约23nm的圆形病毒样颗粒;RT-PCR和稳定性试验结果表明,诱导产生的病毒样颗粒含EV71VP1基因片段,并且稳定性良好。[结论]成功构建了含EV71VP1基因片段的病毒样颗粒,稳定性良好,可作为病毒检测时标准品和质控品使用。     

北海道型汉坦病毒核蛋白基因的克隆表达及其免疫原性

目的 构建北海道型汉坦病毒(HOKV)核蛋白(NP)基因的重组杆状病毒表达载体,在昆虫细胞中表达出具有免疫原性的抗原,用于HOKV的检测及诊断.方法 应用RT-PCR方法扩增HOKVFusong-Cr-247株的NP基因,并将该基因片段克隆到PCR[R]2.1TA载体,转化到One ShortTM TOP10感受态细胞构建TA克隆载体,并与pFastBacTM1转座质粒载体分别用Kpn I和Not I双酶切,用T4连接酶连接,构建pFast PUUV-S重组转座质粒,经双酶切、PCR扩增及插入序列方向鉴定后.转化到DH10BacTM E.coli 感受态细胞,经三抗培养基筛选和PCR扩增鉴定后获得重组Bacmid质粒,将重组Baemid质粒转染Sf9昆虫细胞制备重组杆状病毒毒液,并继续扩增重组杆状病毒,以第三代毒液感染Sf9昆虫细胞,96h后收获细胞.用间接免疫荧光(IFA)、SDS-PAGE和Western blot对表达产物进行鉴定和分析.结果 实验应用Bac-to-BacTM杆状病毒表达系统,成功构建了含普马拉类汉坦病毒核蛋白基因的重组杆状病毒表达载体.并在昆虫细胞中获得表达.IFA结果显示,感染细胞的胞浆中有亮绿色荧光颗粒,SDS-PAGE和Western blot检测细胞表达产物的50×103(M)的蛋白条带,证实重组病毒感染昆虫细胞后表达了相应的重组核蛋白,与预计的结果相符.并能与抗汉坦病毒抗体结合.结论 成功表达具有HOKV免疫原性及反应性的重组核蛋白,为研制HOKV的诊断试剂奠定了基础.     

西尼罗病毒特异性抗原片段筛选及ELISA方法的研究

目的筛选西尼罗病毒特异抗原片段,并建立其ELISA快检方法。方法参考亲水性、抗原性、可塑性、表面可及性及二级结构信息,对西尼罗病毒抗原表位进行系统分析,预测得分值较高的抗原区域经原核表达、Western blot筛选及亲和层析纯化后,包被ELISA微孔板,对ELISA反应条件进行系统优化,建立其ELISA快检方法。结果预测获病毒特异性抗原表位29个,对其中11段进行了表达,经筛选,获得西尼罗病毒特异抗原片段及西尼罗与乙型脑炎病毒共同抗原片段各一段。利用西尼罗病毒特异抗原片段WnAg16建立了检测西尼罗病毒抗体的ELISA方法,与所试相近病毒无交叉反应,S/N比值均在23以上,对鼠抗西尼罗病毒多抗的检出下限达1∶204 800。结论利用特异性抗原初步建立了检测西尼罗病毒抗体的ELISA方法。     

Antigenic relationships among Nigerian strains of West Nile virus by complement fixation and agar gel precipitation techniques.

Using two serological techniques, eight Nigerian West Nile virus isolates were investigated to determine antigenic relationships among them, and to find out if these virus isolates were related to West Nile virus strains from the different zoogeographic areas of the world. One virus differed significantly from the seven other strains and was later found to be a strain of Usutu virus. The remaining strains were differentiated into two serological intratypic groups depending on their cross reactions with two strains which served as prototypes for each group. Five virus isolates which constitute one of the antigenic groups were found to be related to the Egypt 101 strain of West Nile virus originating from general Palearctic zone (European and Middle East). The other two virus isolates did not show any relationship to the strains from any of the different zoogeographic zones.     

Infection of the mosquito Aedes aegypti with infectious west Nile virus-antibody complexes

Aedes aegypti fed through chick skin membranes on West Nile virus-homologous antiserum mixtures shown by an anti-globulin neutralization test to be highly infectious complexes (in terms of plaque formation in tissue culture) failed to become infected. Control mosquitoes fed on West Nile virus—normal rabbit serum mixtures containing similar or smaller amounts of infectious virus were shown to become infected.Mosquitoes ingesting suspensions of West Nile virus previously incubated with Murray Valley encephalitis or Ntaya antiserum became infected at significantly lower rates (P = < 0.05) than controls ingesting West Nile virus—normal rabbit serum mixtures.West Nile virus—17D yellow fever or dengue—2 antiserum mixtures did not produce significantly reduced infection rates in Ae. aegypti when compared to controls.     

Isolation of two strains of West Nile virus during an outbreak in southern Russia, 1999

From July to September 1999, a widespread outbreak of meningoencephalitis associated with West Nile virus (Flavivirus, Flaviviridae) occurred in southern Russia, with hundreds of cases and dozens of deaths. Two strains of West Nile virus isolated from patient serum and brain-tissue samples reacted in hemagglutination-inhibition and neutralization tests with patients' convalescent-phase sera and immune ascites fluid from other strains of West Nile virus.     

西尼罗病毒E蛋白的结构和应用进展

西尼罗病毒是一种单股正链RNA病毒,属于黄病毒属黄病毒科,主要通过库蚊传播,能引起西尼罗热及神经侵袭性疾病如脑炎、脑膜炎等。近年来,该病毒在多个国家和地区间广泛流行,给发病国家带来了极大的危害。目前对于西尼罗病毒尚未有可用于人类的疫苗上市,也没有特定的抗病毒药物可用,因此加强病毒监测、研发新的疫苗及掌握实验室诊断方法变得尤为重要。本文主要通过对西尼罗病毒E蛋白的结构、疫苗及检测方法的最新进展加以探讨,为研发西尼罗病毒疫苗及诊断试剂盒提供理论依据。     

Mental status after West Nile virus infection

Mental status after acute West Nile virus infection has not been examined objectively. We compared Telephone Interview for Cognitive Status scores of 116 patients with West Nile fever or West Nile neuroinvasive disease. Mental status was poorer and cognitive complaints more frequent with West Nile neuroinvasive disease (p = 0.005).     

Preclinical and clinical development of YFV 17D-based chimeric vaccines against dengue,West Nile and Japanese encephalitis viruses

Dengue viruses (DENV), West Nile virus (WNV) and Japanese encephalitis virus (JEV) are major global health and growing medical problems. While a live-attenuated vaccine exists since decades against the prototype flavivirus, yellow fever virus (YFV), there is an urgent need for vaccines against dengue or West Nile diseases, and for improved vaccines against Japanese encephalitis. Live-attenuated chimeric viruses were constructed by replacing the genes coding for Premembrane (prM) and Envelope (E) proteins from YFV 17D vaccine strain with those of heterologous flaviviruses (ChimeriVax™ technology). This technology has been used to produce vaccine candidates for humans, for construction of a horse vaccine for West Nile fever, and as diagnostic reagents for dengue, Japanese encephalitis, West Nile and St. Louis encephalitis infections. This review focuses on human vaccines and their characterization from the early stages of research through to clinical development. Phenotypic and genetic properties and stability were examined, preclinical evaluation through in vitro or animal models, and clinical testing were carried out. Theoretical environmental concerns linked to the live and genetically modified nature of these vaccines have been carefully addressed. Results of the extensive characterizations are in accordance with the immunogenicity and excellent safety profile of the ChimeriVax™-based vaccine candidates, and support their development towards large-scale efficacy trials and registration.     

Antigenic relationship of West Nile strains by titre ratios calculated from cross-neutralization test results

The antigenic relationship of ten South African West Nile isolates, the South African prototype virus H442, the Egyptian strain EG101 and the Indian strain G2266 were compared using titre ratios. The titre ratios or 'R' values were calculated from heterologous and homologous neutralization titres and expressed as a percentage. Substantial antigenic differences were demonstrated between the South African prototype strain and the majority of the recently obtained South African isolates, seven of which were fairly closely related and possibly form a distinct antigenic sub-set. The recent isolates also differed from the Egyptian and Indian West Nile isolates. The heterologous results between the South African West Nile strains and the Indian strain G2266 suggest that prior infection with an Indian West Nile virus would give poor protection against the South African viruses, whereas the reverse would not be so.     

Entomological studies along the Colorado Front Range during a period of intense West Nile virus activity

To better understand the ecology of West Nile virus transmission in Northern Colorado, field studies were conducted in Larimer and Weld counties from September 2003 through March 2005. During summer studies, 18,540 adult mosquitoes were collected using light traps and gravid traps. West Nile virus RNA was detected in 24 of the 2,140 mosquito pools tested throughout the study area in 2003 and 2004. Culex tarsalis had the highest minimum infection rate (MIR) in both 2003 (MIR = 34.48) and in 2004 (MIR = 8.74). During winter studies, 9,391 adult mosquitoes were collected by aspirator from various overwintering sites including bridges and storm drains. The most frequently collected species was Culex pipiens. West Nile virus was not detected in our overwintering collections. The relationship between spring adult emergence and temperature inside and outside overwintering sites is described. Species composition of collections as well as the spatial and temporal distribution of West Nile virus detections are presented.     

Migratory birds and spread of West Nile virus in the Western Hemisphere

West Nile virus, an Old World flavivirus related to St. Louis encephalitis virus, was first recorded in the New World during August 1999 in the borough of Queens, New York City. Through October 1999, 62 patients, 7 of whom died, had confirmed infections with the virus. Ornithophilic mosquitoes are the principal vectors of West Nile virus in the Old World, and birds of several species, chiefly migrants, appear to be the major introductory or amplifying hosts. If transovarial transmission or survival in overwintering mosquitoes were the principal means for its persistence, West Nile virus might not become established in the New World because of aggressive mosquito suppression campaigns conducted in the New York area. However, the pattern of outbreaks in southern Europe suggests that viremic migratory birds may also contribute to movement of the virus. If so, West Nile virus has the potential to cause outbreaks throughout both temperate and tropical regions of the Western Hemisphere.