人乳头瘤病毒16型L1的表达、病毒样颗粒组装及其免疫原性研究

目的 在原核表达系统中表达HPV16 L1蛋白,纯化后在体外自组装成VLPs并鉴定。方法 优化GenBank中HPV16 L1基因序列并截短C末端25个氨基酸,构建至原核表达载体pET-28a上,获得重组表达载体pET28a-16L1△C25。采用镍亲和层析法纯化超声上清,于体外解组装-重组装HPV16 VLPs,采用动态光散射和透射电镜进行形貌分析,纯化后于第0、2和4周免疫小鼠,假病毒中和试验检测HPV16 VLPs免疫后血清中和抗体。结果 双酶切和测序结果表明成功构建pET28a-16L1△C25重组质粒,诱导表达后,经SDS-PAGE和Western blotting分析显示表达的L1蛋白大部分以可溶性形式存在,纯化后的蛋白样品于体外重新组装,动态光散射和透射电镜能够观察到形态与天然病毒颗粒相似的VLPs,第6周小鼠血清中和抗体滴度Log10平均值达到4.43。结论 利用原核表达系统成功表达了截短型HPV16 L1蛋白,并于体外组装成结构完整的VLPs,且具有较好的免疫原性,为低成本HPV预防性疫苗的研发奠定基础。     

诺如病毒病毒样颗粒的表达及其与Caco-2细胞的结合活性研究

目的在杆状病毒表达系统转染的昆虫SF-9细胞中表达诺如病毒（NorovirUSes,NoV）的衣壳蛋白VPl,并芎令证其与Caco-2细胞的结合活性。方法利用杆状病毒表达系统转染的昆虫SF-9细胞进行诺如病毒VPl蛋白表达,用蔗糖密度梯瞍超速离心方法纯化,采用Westernblot鉴定表达产物,采用流式细胞术检测NoV病毒样颗粒（Virus-likeparticles,VLPs）与Caco-2细胞的结合活性。结果重组质粒转染细胞表达产物经蔗糖密度梯度超速离心后进行SDS~PAGE,在分子质量单位55~70ku之间有3条蛋白带,与预期重组蛋白NoV—VI-Ps大小相符;Westernblot分析各蛋白组分均能被兔抗NoV多抗血清识别;流式细胞仪分析届示,加入重组蛋白NoV—VI—Ps后荧光信号较阴性对照组显著增强,表明表达的重组蛋自NoV—VI．Ps能够与Caco-2结合,且3个组分的结合能力不同（P〈i0·05）,以组分5与Caco-2细胞结合的能力最强（P〈O．05）。结论NoVVI．Ps表达成功,并证明NoV-VI—Ps具有与Caco2细胞结合活性,为N。V疫苗的开发和防治药物的研制奠定了基础。     

甲型流感病毒M2基因真核表达载体的构建及序列分析

目的 构建含有甲型流感病毒M2基因的真核表达载体并分析插入的M2基因序列。方法从接种人流感病毒株A／PR／8／34（H1N1）的鸡胚尿囊液中提取病毒RNA，用特异引物进行RT-PCR，扩增M2基因。通过分子克隆技术将所扩增片段克隆入真核表达质粒载体pcDNA3．1（＋）。经双酶切、PCR鉴定后挑选阳性克隆测序鉴定并对插入的M2基因序列进行分析。结果经双酶切、PCR及测序鉴定证实M2基因的真核表达载体构建成功。序列分析有4个氨基酸出现变异，提交Genbank进行Blast比对显示同源性97％（Genbank／NCBI AY768951）。结论M2四聚体蛋白具有H^＋通道功能，能够协助病毒与宿主细胞膜融合后释放RNP复合体，还能稳定HA蛋白的结构。M2蛋白的高度保守性和具有交叉保护能力特性使之成为新型的通用流感疫苗的突破口。该结果将为甲型流感病毒基因工程疫苗，通用疫苗和核酸疫苗的研究打下基础。     

亚洲1型口蹄疫病毒衣壳蛋白前体P1-2A基因和蛋白酶3C基因在昆虫细胞中的共表达

目的在昆虫细胞中表达亚洲1型口蹄疫病毒(Foot-and-mouth disease virus,FMDV)衣壳蛋白前体P1-2A基因和蛋白酶3C基因,为进一步研究FMDV空衣壳抗原和疫苗奠定基础。方法从质粒pTOP-P1-2A和pTOP-3C中分别扩增出编码亚洲1型口蹄疫病毒衣壳蛋白前体P1-2A基因和3C蛋白酶基因,构建转移载体pDual-P1-2A-3C并与大肠杆菌(Escherichia coli)DH10Bac中的Bacmid质粒重组,构建重组转座质粒rBacmid-P1-3C。在脂质体介导下将rBacmid-P1-3C转染Sf9昆虫细胞获得重组杆状病毒。在Sf9昆虫细胞中共表达P1-2A和3C蛋白,通过SDS-PAGE、Western blot和Dot-ELISA试验鉴定重组蛋白。结果目的基因在昆虫细胞中得到了正确的表达,表达蛋白与抗亚洲1型FMDV血清发生特异性反应,有良好的抗原性。结论成功在昆虫细胞中表达亚洲1型口蹄疫病毒衣壳蛋白前体P1-2A基因和蛋白酶3C基因。     

人乳头瘤病毒16型晚期基因L1的原核表达质粒的构建和鉴定

目的 构建人类乳头瘤病毒（ＨＰＶ）１６型晚期基因Ｌ１的原核表达质粒,以期从中选出能够高效表达ＨＰＶ１６Ｌ１的重组子,为进一步表达Ｌ１蛋白奠定基础。方法 应用定向克隆策略,将ＨＰＶ１６Ｌ１基因分别克隆到原核表达载体ｐＴｒａｃ９９Ａ和ｐＥＴ－２１ｃ中。结果 通过酶切鉴定选出了重组子ｐＴｒｃ９９Ａ－ＨＰＶ１６Ｌ１和ｐＥＴ－２１ｃ－ＨＰＶ１６Ｌ１。结论 ＨＰＶ１６Ｌ１的原核表达质粒构建成功。     

人乳头瘤病毒16型表位嵌合病毒样颗粒的构建及免疫原性研究

目的 制备E7_49-57表位嵌合病毒样颗粒,并进行免疫原性分析。方法 利用分子模拟软件Discovery Studio预测HPV16 L1的E7_49-57最佳嵌合位点,将E7_49-57表位嵌入预测位点。构建pET28a-16L1-E7_49-57重组质粒,于大肠杆菌中诱导表达HPV16L1-E7_49-57蛋白并利用镍柱进行亲和层析纯化。于体外重组VLPs后,进行动态光散射粒径和透射电镜分析。用E7_49-57嵌合VLPs免疫小鼠,假病毒中和试验检测免疫血清中和抗体滴度。流式多因子法检测Th1和Th2型细胞因子水平。结果 HI loop区355/356为最佳表位嵌合位点。正确表达了HPV16 L1-E7_49-57蛋白并组装E7_49-57嵌合VLPs。E7_49-57嵌合VLPs免疫小鼠3次后,小鼠血清中和抗体滴度log10平均值达到4.23,略低于野生型VLPs(log10平均值为4.45)。但是,相对于野生型VLPs,E7_49-57嵌合VLPs诱导产生Th1型细胞因子(INF-γ, IL-2, TNF-α)的水平显著提高。结论 本研究制备的E7_49-57嵌合VLPs 能刺激机体同时产生较强的体液和细胞免疫应答,可能兼具预防和治疗双重功效,为宫颈癌治疗性疫苗的研制奠定基础。     

HPV16 L1在整合型重组毕赤酵母中的表达及病毒样颗粒的纯化

目的 构建可稳定表达HPV16 L1的整合型重组毕赤酵母,并纯化自主组装成的HPV16 L1病毒样颗粒（VLPs）.方法 根据酵母密码子偏爱性优化HPV16 L1基因并克隆到pPIC3.5K表达载体,构建pPIC3.5K/HPV16 L1重组质粒;重组质粒经Bgl Ⅱ酶切线性化后,电转化至GS115菌株中,筛选HPV16 L1重组毕赤酵母.阳性整合菌株甲醇诱导后,以HPV16 L1单克隆抗体检测目的蛋白表达;采用肝素亲和层析法纯化HPV16 L1VLPs并进行透射电镜观察.结果 PCR、酶切和测序分析表明成功构建了pPIC3.5K/HPV16 L1重组质粒.成功构建的HPV16 L1重组毕赤酵母甲醇诱导后,Western blot证实重组酵母菌裂解产物存在HPV16 L1目的蛋白.肝素亲和纯化后,透射电镜观察到了直径大约55 nm的VLPs,其形态与HPV16天然病毒颗粒相似.结论 利用整合型重组毕赤酵母表达系统成功表达了HPV16L1蛋白,并用肝素亲和纯化可快速获得结构完整的HPV16 L1VLPs,为HPV16预防性疫苗的研制奠定基础.     

昆虫细胞偏爱密码子优化的HPV16L1基因在昆虫和哺乳动物细胞中的表达

目的获得有效表达人乳头瘤病毒16型（HPV16）L1基因的重组杆状病毒和腺病毒，为研究HPV的免疫保护机制提供材料。方法按照昆虫细胞密码子偏爱优化并合成HPV16LI基因，利用Bac—to-Bac昆虫表达系统获得表达HPV16L1基因的重组杆状病毒，利用AdEasy腺病毒载体系统获得表达HPV16L1基因的重组腺病毒载体。通过间接免疫荧光和Westernblot对HPV16L1基因表达进行鉴定，利用负染电子显微镜观察病毒样颗粒（VLP）的形成。结果获得了稳定表达HPV16L1蛋白的重组杆状病毒和重组腺病毒载体，在Sf9细胞和293细胞中可有效表达能被抗HPV16L1单克隆抗体识别的L1蛋白，分子质量单位为56ku，在Sf9细胞中可观察到VLP的形成。结论按照昆虫细胞密码子偏爱进行优化的HPV16L1基因，在昆虫细胞和哺乳动物细胞内均可有效表达。     

人乳头瘤病毒16与胃癌相关性的研究

目的探讨人乳头瘤病毒１６（ＨＰＶ１６）与胃癌发生的关系。方法用聚合酶链式反应（ＰＣＲ）扩增出ＨＰＶ１６早期区Ｅ６的１２０ｂｐＤＮＡ片段，扩增产物与５＇末端３２Ｐ标记的特异性寡核苷酸探针进行斑点杂交及放射性自显影，用此方法对３４６份新鲜的及福尔马林固定石蜡包埋的胃组织标本中ＨＰＶ１６ＤＮＡ进行检测。结果胃腺癌、癌旁粘膜、胃局部淋巴结及正常胃粘膜组织中ＨＰＶ１６的检出率分别为：２２．７２％（３０／１３２）、５．８８％（４／６８）、０％（０／４２）、２．８８％（３／１０４），胃癌组ＨＰＶ１６的检出率高于其他各组，统计学上差异均有显著性（Ｐ＜０．０１）。结论ＨＰＶ１６可感染胃粘膜上皮细胞，可能是胃癌的致癌因素之一。     

流感通用疫苗的研究进展

流感是一种呼吸道传染性疾病.目前制备流感疫苗主要利用流感病毒的HA和NA诱导相应的抗体,但存在抗原漂移和抗原转换等问题.解决办法之一就是研制流感通用疫苗.M2e是流感病毒M2蛋白的胞外区,它的序列相对保守.利用HBc作为病毒样颗粒包装M2e是至今为止制备流感通用疫苗最可行的方法.实验证明,M2e-HBc融合蛋白不仅能诱导出足够的抗体,还能增强T细胞反应,产生交叉性保护效应,大大降低流感病毒的致病率、致死率,促进疾病的恢复.此外,还有流感病毒的NP和HA等保守序列,结合佐剂及黏膜给药方式都不失为通用疫苗的可行选择.     

甲型流感病毒M2蛋白胞外功能区真核表达质粒的构建和表达分析

目的 构建流感病毒M2蛋白胞外功能区（M2e）真核表达质粒，并研究其在真核细胞中的表达。方法 根据Genbank（NCBI ISDN13425）中查得的M2e编码序列，设计并合成两条DNA单链;在两条链两端添加碱基构成酶切位点的粘性末端，退火后使之互补结合成为M2e编码序列；然后将其插入到经双酶切的真核表达载体pcDNA3．1（＋）中，构建重组真核表达质粒pcDNA3．1（＋）-M2ef经酶切和DNA测序鉴定后，转染HEK293细胞。用免疫荧光技术检测pcD—NA3．1（＋）-M2e在HEK293细胞的表达。并通过MTT检测刺激淋巴细胞增殖及M2e蛋白的分泌情况。结果 合成的寡核苷酸链经退火形成双链，插入酶切的真核表达载体后构建成pcDNA3．1（＋）-M2e。免疫荧光技术证实该质粒表达的M2e蛋白定位于细胞膜上，转染细胞的培养上清经MTT实验证实能刺激淋巴细胞增殖，表明表达的M2e蛋白也可分泌至细胞外。结论成功构建了流感病毒M2e的真核表达质粒pcDNA3．1（＋）-M2e，表达的M2e蛋白不仅存在于绳胞膜上，也可分泌到细胞外。流感病毒M2e基因真核表达质粒的构建及成功表达为流感病毒基因工程疫苗、通用疫苗和核酸疫苗的研究奠定了基础。     

Live attenuated influenza virus vaccine reduces virus shedding of newborn piglets in the presence of maternal antibody

Background

Influenza A virus in swine (IAV‐S) causes an acute respiratory disease of swine which results in great economic losses in pig production. Major control strategies include the use of killed vaccines (KV) in breeding females to confer passive immunity to their offspring. A bivalent H1N1 and H3N2 NS1‐truncated live attenuated IAV‐S vaccine have recently become available, which showed promising results in young pigs.

Objective

The aim of this study was to investigate the effect of an intranasal vaccination of newborn pigs with or without maternally derived antibodies (MDA) on virus shedding (via nasal swabs tested by virus isolation).

Methods

The study was performed as intratracheal challenge experiments with either a heterologous H1N2 or H3N2 viruses.

Results and conclusion

The results of this study showed a significant decrease in the incidence and duration of shedding viable virus for vaccinated newborn piglets with or without MDA, providing strong evidence that intranasal vaccination is overcoming passively acquired maternal immunity. This study indicates that intranasal vaccination with a truncated NS1 live attenuated IAV‐S vaccine of newborn piglets with maternal antibodies can be a valuable tool for reducing the prevalence of heterologous H1N2 and H3N2 IAV‐S in pig herds.     

流感病毒蛋白糖基化及其功能研究进展

甲型流感病毒容易发生变异,可以引发局部或全球范围的流行。每次流感病毒新型别的出现都对人类健康和社会经济产生极大的危害,其一直是研究的热点。本文对甲型流感病毒血凝素(hemagglutinin,HA)和神经氨酸酶(neuraminidase,NA)两种蛋白糖基化及其功能、研究方法等方面进行综述。     

重组蛋白ASP-1对高致病性禽流感M2e疫苗分子的佐剂活性

目的探讨重组蛋白ASP-1对高致病性禽流感M2e疫苗分子的免疫佐剂作用。方法在大肠杆菌原核表达系统中获得融合了pET32（＋）表达载体蛋白trxA和M2e三联体的重组蛋白TrxA-M2e3作为高致病性禽流感M2e疫苗分子;在大肠杆菌中截短表达盘尾丝虫活化相关蛋白ASP,获得截短片段ASP-1作为佐剂蛋白。将纯化后的重组蛋白TrxA-M2e3和ASP-1混合或者单独使用,免疫BALB/c小鼠后检测血清中M2e抗体水平。使用不同进化分支H5N1病毒对免疫小鼠进行致死性攻击,检测交叉免疫保护效果。结果获得了纯化的重组蛋白TrxA-M2e3和ASP-1;与TrxA-M2e3单独免疫组相比,应用佐剂蛋白ASP-1可显著提高免疫小鼠血清中M2e特异性IgG抗体滴度（P〈0.05）。在不同进化分支H5N1病毒的致死性攻击后,应用ASP-1与TrxA-M2e3联合免疫小鼠的肺组织病毒复制得到有效抑制,免疫小鼠全部存活。而其它免疫组小鼠全部死亡。结论 M2e是发展新型禽流感疫苗应对病毒变异的理想靶抗原分子,而重组蛋白ASP-1可以为一种新型佐剂在M2e疫苗分子诱导交叉免疫保护中发挥关键作用。     

鹧鸪禽流感病毒和人流感病毒唾液酸受体分布特征

目的 探讨鹧鸪禽流感病毒和人流感病毒唾液酸受体分布特点及其作用。方法 用亲和组化法检测鹧鸪呼吸道与消化道流感病毒唾液酸受体的分布,荧光素Alexa488标记禽流感病毒H9N1和人流感病毒H1N1,分别与鹧鸪呼吸道、消化道各解剖部位组织细胞结合。结果 鹧鸪呼吸道同时表达SAα-2,3Gal和SAα-2,6Gal两种受体,但其消化道仅表达SAα-2,3Gal受体。SAα-2,3Gal受体在鹧鸪呼吸道各部位和消化道结肠上皮细胞呈强阳性弥漫分布;SAα-2,6Gal受体在呼吸道气管、支气管和次级支气管呈强阳性弥漫分布,而副支气管、消化道结肠均为缺乏或仅表达极少量。H9N1禽流感病毒与鹧鸪呼吸道各部位和消化道结肠均结合;人流感病毒H1N1与鹧鸪的气管、支气管、次级支气管结合,但未见与副支气管和结肠结合,此结果与鹧鸪呼吸道和消化道SAα-2,3Gal和SAα-2,6Gal受体分布总体一致。结论 鹧鸪同时表达SAα-2,3Gal和SAα-2,6Gal受体,能与禽流感病毒和人流感病毒结合,有助于禽流感病毒与人流感病毒基因重配,提示其可充当流感病毒潜在的中间宿主。     

Improving influenza vaccine virus selection: report of a WHO informal consultation held at WHO headquarters, Geneva, Switzerland, 14-16 June 2010

? For almost 60 years, the WHO Global Influenza Surveillance and Response System (GISRS) has been the key player in monitoring the evolution and spread of influenza viruses and recommending the strains to be used in human influenza vaccines. The GISRS has also worked to continually monitor and assess the risk posed by potential pandemic viruses and to guide appropriate public health responses. ? The expanded and enhanced role of the GISRS following the adoption of the International Health Regulations (2005), recognition of the continuing threat posed by avian H5N1 and the aftermath of the 2009 H1N1 pandemic provide an opportune time to critically review the process by which influenza vaccine viruses are selected. In addition to identifying potential areas for improvement, such a review will also help to promote greater appreciation by the wider influenza and policy-making community of the complexity of influenza vaccine virus selection. ? The selection process is highly coordinated and involves continual year-round integration of virological data and epidemiological information by National Influenza Centres (NICs), thorough antigenic and genetic characterization of viruses by WHO Collaborating Centres (WHOCCs) as part of selecting suitable candidate vaccine viruses, and the preparation of suitable reassortants and corresponding reagents for vaccine standardization by WHO Essential Regulatory Laboratories (ERLs). ? Ensuring the optimal effectiveness of vaccines has been assisted in recent years by advances in molecular diagnosis and the availability of more extensive genetic sequence data. However, there remain a number of challenging constraints including variations in the assays used, the possibility of complications resulting from non-antigenic changes, the limited availability of suitable vaccine viruses and the requirement for recommendations to be made up to a year in advance of the peak of influenza season because of production constraints. ? Effective collaboration and coordination between human and animal influenza networks is increasingly recognized as an essential requirement for the improved integration of data on animal and human viruses, the identification of unusual influenza A viruses infecting human, the evaluation of pandemic risk and the selection of candidate viruses for pandemic vaccines. ? Training workshops, assessments and donations have led to significant increases in trained laboratory personnel and equipment with resulting expansion in both geographical surveillance coverage and in the capacities of NICs and other laboratories. This has resulted in a significant increase in the volume of information reported to WHO on the spread, intensity and impact of influenza. In addition, initiatives such as the WHO Shipment Fund Project have facilitated the timely sharing of clinical specimens and virus isolates and contributed to a more comprehensive understanding of the global distribution and temporal circulation of different viruses. It will be important to sustain and build upon the gains made in these and other areas. ? Although the haemagglutination inhibition (HAI) assay is likely to remain the assay of choice for the antigenic characterization of viruses in the foreseeable future, alternative assays - for example based upon advanced recombinant DNA and protein technologies - may be more adaptable to automation. Other technologies such as microtitre neuraminidase inhibition assays may also have significant implications for both vaccine virus selection and vaccine development. ? Microneutralization assays provide an important adjunct to the HAI assay in virus antigenic characterization. Improvements in the use and potential automation of such assays should facilitate large-scale serological studies, while other advanced techniques such as epitope mapping should allow for a more accurate assessment of the quality of a protective immune response and aid the development of additional criteria for measuring immunity. ? Standardized seroepidemiological surveys to assess the impact of influenza in a population could help to establish well-characterized banks of age-stratified representative sera as a national, regional and global resource, while providing direct evidence of the specific benefits of vaccination. ? Advances in high-throughput genetic sequencing coupled with advanced bioinformatics tools, together with more X-ray crystallographic data, should accelerate understanding of the genetic and phenotypic changes that underlie virus evolution and more specifically help to predict the influence of amino acid changes on virus antigenicity. ? Complex mathematical modelling techniques are increasingly being used to gain insights into the evolution and epidemiology of influenza viruses. However, their value in predicting the timing and nature of future antigenic and genetic changes is likely to be limited at present. The application of simpler non-mechanistic statistical algorithms, such as those already used as the basis of antigenic cartography, and phylogenetic modelling are more likely to be useful in facilitating vaccine virus selection and in aiding assessment of the pandemic potential of avian and other animal influenza viruses. ? The adoption of alternative vaccine technologies - such as live-attenuated, quadrivalent or non-HA-based vaccines - has significant implications for vaccine virus selection, as well as for vaccine regulatory and manufacturing processes. Recent collaboration between the GISRS and vaccine manufacturers has resulted in the increased availability of egg isolates and high-growth reassortants for vaccine production, the development of qualified cell cultures and the investigation of alternative methods of vaccine potency testing. WHO will continue to support these and other efforts to increase the reliability and timeliness of the global influenza vaccine supply. ? The WHO GISRS and its partners are continually working to identify improvements, harness new technologies and strengthen and sustain collaboration. WHO will continue in its central role of coordinating worldwide expertise to meet the increasing public health need for influenza vaccines and will support efforts to improve the vaccine virus selection process, including through the convening of periodic international consultations.     

猪流感H1N1亚型NP基因杆状病毒表达载体构建及其在昆虫细胞中的表达

目的获得研发A型猪流感病毒ELISA检测试剂盒和制备NP蛋白单克隆抗体的抗原物质。方法根据已报道的猪流感病毒H1N1亚型NP基因序列(GenBank登录号:GQ422386)人工合成NP基因,通过XbaⅠ和EcoRⅠ特异性酶切,将NP基因克隆于昆虫杆状病毒表达载体pFastBacHTB,经PCR、酶切、测序鉴定后,获得携带NP基因的重组质粒pFast-BacHTB-NP。该重组质粒转化含有杆状病毒穿梭载体的DH10Bac感受态细胞,经抗生素、PCR筛选,获得转座的杆粒Bac-mid-NP。在脂质体介导下转染sf9昆虫细胞,获得重组杆状病毒,再感染细胞,收获目的蛋白。结果通过SDS-PAGE和Westernblotting分析表明该蛋白得到表达,且具有良好的生物活性,大小约为57KD。结论实验结果表明已成功构建了携带NP基因的重组质粒pFastBacHTB-NP和转座的杆粒Bacmid-NP,转染后在sf9昆虫细胞上成功的表达。