人乳头状瘤假病毒中和抗体滴度和ELISA法测定的小鼠血清抗体滴度的相关性研究

目的 探讨两种不同的报告基因纽扣珊瑚绿色荧光蛋白(Zoanthus sp.green fluorescent protein,ZsGreen)和分泌性碱性磷酸酶(secreted alkaline phosphatase,SEAP)测量的人乳头状瘤假病毒中和滴度之间的相关性,以及中和滴度和抗体滴度的相关性.方法 将密码子优化的人乳头状瘤病毒(HPV)衣壳蛋白L1、L2基因表达质粒和报告基因质粒共转染293FT细胞,48 h后收集细胞裂解上清,柱层析纯化假病毒,对假病毒滴度进行测定.采集免疫过候选HPV疫苗和Gardasil疫苗的小鼠血清,测量血清的中和滴度和抗体滴度.结果 经统计分析,这两种报告基因系统的假病毒检测的中和滴度结果高度相关(Spearman相关系数r=0.760),而且中和滴度和抗体滴度的相关度很高(Spearman相关系数r=0.577和0.741).结论 两种不同的报告基因ZsGreen和SEAP测量的HPV假病毒中和滴度之间,以及中和滴度和ELISA测定的抗体滴度之间高度相关,揭示了部分HPV疫苗预防病毒入侵的机制,为快速准确鉴定HPV-16和HPV-18候选疫苗的免疫保护效果奠定了基础.     

HPV16假病毒制作方法优化

目的为了优化HPV16假病毒制作方法。方法本实验用L1/L2结构蛋白质粒和p CMV-Gluc质粒,从融合试剂,结构质粒与报告质粒比例,细胞接种密度和收获时间等4个方面优化了HPV16假病毒的制作过程,ELISA验证不同抗体水平下假病毒检出效果。结果最优条件分别为E498b,结构质粒与报告质粒2:1,293FT细胞接种密度50%,收获时间48h或72h能够达到最佳包装效率。优化后的假病毒TCID50能够达到105.55,血清ELISA滴度分别为1:2000;1:8000;1:16000,中和实验ID50分别为90,270和810,具有很好的区分度。结论证明该优化方法现实可行,为HPV假病毒生产和中和抗体检测提供了一定的参考意义,为后续病毒试验提供了依据。     

人乳头瘤病毒(HPV)L2肽免疫小鼠诱导的抗体水平与保护作用的关系研究

目的 检测HPV-58 L2 11～200 AA肽在动物体内对HPV的保护效果,并分析疫苗诱导的抗体滴度或中和抗体滴度与疫苗保护作用之间的对应关系.方法 利用大肠杆菌表达HPV-58L2 11～200 AA肽,纯化目的 蛋白并与铝佐剂吸附后免疫小鼠,利用小鼠HPV-58假病毒感染模型检测不同剂量的免疫原对小鼠的保护作用.通过ELISA和假病毒中和抗体检测方法 检测免疫血清中的总抗体水平和中和抗体水平,分析具有保护作用的抗体或中和抗体滴度.结果 当蛋白免疫剂量为8μg时,能够完全保护小鼠不受HPV假病毒的感染.小鼠免疫血清中的中和抗体水平较低,利用已建立的假病毒中和试验方法 检测不到血清中的中和抗体.而利用ELISA检测血清中的总抗体水平结果 显示,免疫血清中的总抗体水平与疫苗的保护效果之间存在对应关系,当抗体滴度大于等于1:25 000时,小鼠体内检测不到荧光信号,能够保护机体不受假病毒的感染.结论 HPV-58 L2 11～200 AA肽能够保护小鼠不受假病毒的感染,L2 11～200 AA肽具有较好的疫苗发展前景,其引发的总抗体水平可以作为评价保护效果的间接指标.     

基于H5N1假病毒的体内中和试验模型的建立

目的建立H5N1假病毒的体内感染模型, 并对抗体FHA3的体内中和活性进行鉴定。方法依据A/Anhui/1/2005/H5N1毒株血凝素(hemagglutinin, HA)和神经氨酸酶(neuraminidase, NA)的序列信息, 构建重组表达质粒pcDNA3.1-HA5和pcDNA3.1-NA1, 并与质粒pNL4-3.Luc.R-E-共转染293T细胞制备H5N1假病毒上清, 电镜观察上清中假病毒颗粒形态。假病毒上清感染MDCK细胞后, 测定病毒滴度;经腹腔注射入BALB/c小鼠体内, 在感染后2、5、8、12 d进行生物发光成像, 检测假病毒体内感染情况。利用建立的小鼠感染模型, 评价抗体FHA3的体内功能活性。结果重组表达质粒pcDNA3.1-HA5和pcDNA3.1-NA1构建正确, 与pNL4-3.Luc.R-E-质粒共转染293T细胞可制备出高滴度的假病毒上清, 电镜下可见圆形的病毒颗粒。H5N1假病毒感染后, 小鼠体内发出较强的荧光, 而攻毒前给予抗体FHA3处理可减弱其荧光信号。结论成功构建出H5N1假病毒体内感染模型, 并证实抗体FHA3对假病毒的感染具有体内...     

α-珠蛋白基因的RNAi慢病毒载体的构建

目的构建α-珠蛋白基因RNAi重组慢病毒载体。方法合成靶序列Oligo DNA,退火形成双链DNA,克隆到慢病毒p LKO载体,重组载体进行酶切和测序鉴定。在脂质体的介导下将ps PAX2、pMD2.G及慢病毒载体三质粒共转染293T细胞,48h后收集细胞培养上清液。采用终点稀释法测定病毒滴度。结果酶切和测序证实重组载体构建成功。病毒的滴度为3.9×106TU/ml。结论成功构建α-珠蛋白基因RNAi慢病毒载体,为采用RNAi技术治疗β-地贫的研究创造了条件。     

胞嘧啶脱氨酶和血管内皮抑素基因慢病毒载体的构建与鉴定

目的 构建携带胞嘧啶脱氨酶(CD)和血管内皮抑素(ES)基因的慢病毒载体.方法 根据GenBank提供大肠杆菌源CD和ES基因序列设计、合成2个基因并构建与荧光蛋白融合的真核表达载体pEGFP-CD和pDS-RED-ES.酶切和测序鉴定后,将载体瞬时转染HEK293细胞,运用实时PCR检测CD和KS基因表达情况.把载体中基因片段EGFP-CD和DS-RED-ES通过BP/LR重组分别构建慢病毒载体,并利用293FT细胞进行慢病毒的包装.将病毒原液浓缩,并测定病毒滴度.结果 酶切、测序及PCR鉴定证实构建出慢病毒载体pLenti6.3-CD-EGFP和pLenti6.3- ES- Monomer - DsRed.感染293FT细胞后荧光显微镜下观察到GFP和DsRed的表达.浓缩慢病毒滴度分别为2.2× 108 TU/ml和6.5×107 TU/ml.结论 成功构建了CD和ES基因慢病毒载体.     

增殖抑制基因（HSG）RNAi 慢病毒载体的构建与鉴定

目的为研究HSG基因不同表达程度对肺癌细胞的影响,构建并鉴定HSG基因RNA干扰慢病毒表达载体,以建立HSG基因沉默的人肺癌细胞株。方法针对HSG mRNA设计了4条siRNA,并构建p GCSIL-GFP-HSG慢病毒质粒,通过测序鉴定。采用构建的p GCSIL-GFP-HSG,p Helper1.0和p Helper2.0共感染293T细胞,包装产生慢病毒,测定其滴度。将慢病毒转染人肺腺癌细胞株A549,通过real-time PCR分析HSG基因表达。结果测序结果显示,DNA序列与实验要求序列一致,提示插入人HSG基因RNAi序列正确。荧光显微镜观察转染慢病毒包装质粒后的细胞,见细胞生长良好,荧光强度强烈。测定病毒滴度为3×108TU/ml。real-time PCR分析提示RNA干扰病毒感染A549细胞株后,HSG基因表达显著下调。结论人HSG基因RNAi慢病毒载体构建成功,可用于肿瘤学的进一步应用。     

利用假病毒法检测重组人乳头瘤病毒双价(16/18型)疫苗的免疫原性

目的建立并验证重组人乳头瘤病毒双价(16/18型)疫苗中和滴度的检测方法 ,并且利用此假病毒法进行免疫原性研究。方法建立并优化以ZsGreen假病毒为基础的中和实验(ZsGreen法)测定血清中和滴度,比较荧光显微镜观察与微流式细胞分析法、流式细胞分析法的差异,并对专属性、精密度、耐用性、假病毒稳定性和不同细胞代次的影响进行了方法学验证。结果ZsGreen法的HPV16/18假病毒最佳接种量为1 600 TCID50,HPV16型与HPV18型无交叉,具有良好的专属性和精密度,采用不同细胞代次与不同批次假病毒进行检测,均能获得较好的重复性。结论 ZsGreen法假病毒中和滴度检测法准确可靠、重复性好,经方法学验证后此法适用于疫苗的免疫原性研究。     

人乳头瘤病毒L2 蛋白的病毒样颗粒疫苗研究

目的:构建乙肝病毒核心抗原(HBcAg)与人乳头瘤病毒(HPV)L2抗原的融合蛋白,在大肠杆菌中重组表达形成病毒样颗粒结构;通过小鼠模型检测HBc-L2融合蛋白的免疫原性,并研究免疫后获得的小鼠血清对HPV假病毒的中和效力。方法:通过DNA合成构建16型人乳头瘤病毒L2基因片段与HBcAg基因的融合基因,将其克隆至表达载体p ET9a并在大肠杆菌中进行HBc-L2融合蛋白表达;将经纯化、鉴定后的融合蛋白免疫BALB/c小鼠,用间接ELISA方法检测小鼠血清中针对L2抗原的抗体效价,并分别研究小鼠血清对16型和18型HPV假病毒的中和效力。结果:HBc-L2融合基因经大肠杆菌系统表达形成可溶性蛋白,经硫酸铵沉淀和CL-4B凝胶分离纯化后获得纯度80%的HBc-L2蛋白;分子筛高效液相色谱-多角度激光光散射(SEC-MALS)联用技术和透射电子显微镜的分析结果表明,HBc-L2融合蛋白在表达过程中自动组装形成稳定的病毒样颗粒;将纯化后的HBc-L2蛋白免疫BALB/c小鼠可获得针对L2抗原的高滴度抗体,且小鼠血清具有中和16和18型两种假病毒的中和抗体活性。结论:HBc-L2病毒样颗粒可以有效地增强L2抗原的免疫原性,并可刺激机体产生针对多型HPV的免疫保护力,是一个具有潜力的新型广谱HPV疫苗。     

小鼠IL-10重组慢病毒载体的构建和病毒包装

目的:构建小鼠IL-10重组慢病毒表达载体并进行包装,为进一步研究IL-10基因修饰树突细胞在哮喘免疫耐受中的作用提供实验基础。方法:小鼠IL-10基因片段经PCR扩增后,与酶切线性化的慢病毒载体进行定向连接,获得PGC-LV-IL-10重组慢病毒并转化细菌感受态细胞。克隆菌落行PCR鉴定,对阳性的重组质粒进行测序并转入293T细胞,荧光显微镜下观察GFP表达并行Western blot鉴定。将重组质粒与慢病毒包装系统一同转染293T细胞进行病毒包装,Real-time定量PCR法检测病毒滴度。结果:DNA测序结果及Western blot鉴定证实成功构建小鼠IL-10重组慢病毒载体,对其进行包装后测定慢病毒滴度为2×108TU/ml。结论:成功构建并包装小鼠IL-10重组慢病毒表达载体。     

Neutralization of HPV16, 18, 31, and 58 pseudovirions with antisera induced by immunizing rabbits with synthetic peptides representing segments of the HPV16 minor capsid protein L2 surface region

Neutralizing antibody against human papillomavirus (HPV) minor capsid protein L2 can cross-neutralize different HPV genotypes in vitro. To identify the segments containing the cross-neutralization epitopes of HPV16 L2, we characterized antisera obtained by immunizing two rabbits with each of the ten synthetic peptides of 14 to 20 amino acids (aa) long, which represents a part of the HPV16 L2 sequence from aa 14 to 144. The antisera against the peptides within the region from aa 18 to 144 efficiently bound to HPV16 L1/L2-capsids and neutralized HPV16 pseudovirions, indicating that the region is displayed on the surface of the capsids and contains several neutralization epitopes. Antiserum against the peptide from aa 18 to 38 (anti-P18/38) cross-neutralized HPV18. Anti-P56/75 cross-neutralized HPV18, 31, and 58. Anti-P61/75 and anti-P64/81 cross-neutralized HPV18 and 58. Anti-P96/115 and the antiserum induced by a mutant P96/115 (S and T at aa 101 and 112 were replaced with L and S, respectively) cross-neutralized HPV31 and 58. The mixture of equal volumes of three antisera, anti-P18/38, anti-P56/75, and anti-mutant P96/115, neutralized HPV16, 18, 31, and 58 more efficiently than anti-P56/75 alone, suggesting that there is a synergistic effect of antibodies on the cross-neutralization. The cross-neutralization appears to be correlated with conserved aa sequences among HPV types. The data in this study provide a basis for designing vaccine antigens effective against a broader spectrum of the high-risk HPVs.     

Induction of cross-neutralizing antibodies by sequential immunization with heterologous papillomavirus L1VLPs and its implications for HPV prophylactic vaccines

Sequential immunization with antigens from different strains of HIV-1, influenza viruses or dengue viruses induced cross-neutralizing antibodies and enhanced the antibody responses against previous antigens. The characteristics of neutralizing antibodies induced by sequential immunization with different types of human papillomavirus (HPV) L1 virus-like particles (L1VLPs) are unclear. In this study, mice were primed with one or two types (HPV-16 or HPV16/18) of L1VLPs, then boosted sequentially with HPV6/18/45/11/31/58 or HPV6/45/11/31/58 L1VLPs, and sera were analyzed with HPV pseudovirus-based neutralization assay. The results showed that neutralizing activities against earlier immunized vaccine types were enhanced gradually by subsequent immunizations, and low levels of neutralizing activities against nonvaccine types (HPV33/35/52/59/68) were also observed. After absorbing the immune sera with vaccine-type (HPV16/18/45) L1VLPs, neutralizing activities against tested priming and boosting types (HPV16/18/58) decreased significantly, and that against nonvaccine type (HPV-33) was also partially eliminated. Moreover, neutralizing activities against vaccine types (HPV16/58) were significantly reduced after absorbing with nonvaccine-type VLPs (HPV33/52). These data suggest that cross-neutralizing epitopes exist among different HPV L1VLPs. The cross-neutralizing activities against nonvaccine types and the enhanced neutralizing activities against earlier immunized vaccine types may result from sequential boosting with these cross-neutralizing epitopes. These observations support early vaccination with more types of L1VLPs derived from HPVs that cause a serious threat to the population.     

Neutralization of human papillomavirus (HPV) pseudovirions: a novel and efficient approach to detect and characterize HPV neutralizing antibodies

The development of vaccines against human papillomaviruses (HPVs) has long been hampered by the inability to grow HPVs in tissue culture and the lack of an efficient neutralization assay. To date, less than 10% of more than 100 different HPV types can be grown in athymic and "SCID" mouse xenograft systems or raft culture systems. Recently, the in vitro generation of HPV pseudovirions and their use in neutralization assays were demonstrated. The major shortcomings of the current approaches to HPV neutralization are the lack of HPV virions for most types for the xenograft methods and the time-consuming and inefficient generation of infective pseudovirions for the latter methods, which precludes their use in large-scale HPV clinical trials or epidemiological studies. We describe here a novel and efficient approach to generating pseudovirions in which HPV virus-like particles (VLPs) are coupled to the beta-lactamase gene as a reporter. We show that it is not necessary to encapsidate the reporter gene constructs into the pseudovirions. Using sera from human volunteers immunized with HPV-11 VLPs expressed in yeast, we demonstrate that our novel neutralization assay compares favorably with the athymic mouse neutralization assay. Furthermore, our assay was used to define neutralizing monoclonal antibodies to HPV-6, which were previously unknown.     

Characterization of common and rare human papillomaviruses in Portuguese women by the polymerase chain reaction,restriction fragment length polymorphism and sequencing

The present study aimed to provide additional information on the prevalence of mucosal human papillomavirus (HPV) types in Portuguese women by using polymerase chain reaction, restriction fragment length polymorphism and sequencing. HPV was detected in 15.5% (15/97) of the control samples, 23.5% (12/51) of atypical squamous cells of undetermined significance, 52.8% (28/53) of low‐grade lesions, 82.4% (28/34) of high‐grade lesions, and 100% (44/44) of carcinomas. Overall, 28 HPV types were detected: 11 high‐risk (HPV 16, 18, 31, 33, 35, 39, 45, 52, 56, 58, and 59), 3 probable high‐risk (HPV 53, 66, and 73), 6 low‐risk (HPV 6, 11, 44, 61, 70, and 81), and 8 unknown‐risk types (HPV 34, 62, 67, 71, 83, 84, 102, and 108). The most prevalent type was HPV 16, detected in 33.8% of women infected with HPV, followed by HPV 58 (9.2%), HPV 33 (7.0%), HPV 18 (6.3%), HPV 53 (5.6%), HPV 31 and 56 (4.9% each), HPV 6 (3.5%), and HPV 66 and 81 (2.8% each). Of 44 cervical carcinoma samples, 71% were associated with HPV 16 (60%) and HPV 18 (11.1%), followed by the high‐risk types 33 (11.1%), 35 (4.4%), 45 (4.4%), and 56 (2.2%), the probable high‐risk type 53 (4.4%) and the unknown‐risk type 67 (2.2%). This study provides information on the most common HPV types in Portuguese women and suggests that the current prophylactic HPV 16/18 vaccine may be useful for the prevention of cervical cancer in this population. J. Med. Virol. 82:1024–1032, 2010. © 2010 Wiley‐Liss, Inc.     

Papillomavirus pseudovirions packaged with the L2 gene induce cross-neutralizing antibodies

Background  

Current vaccines against HPVs are constituted of L1 protein self-assembled into virus-like particles (VLPs) and they have been shown to protect against natural HPV16 and HPV18 infections and associated lesions. In addition, limited cross-protection has been observed against closely related types. Immunization with L2 protein in animal models has been shown to provide cross-protection against distant papillomavirus types, suggesting that the L2 protein contains cross-neutralizing epitopes. However, vaccination with L2 protein or L2 peptides does not induce high titers of anti-L2 antibodies. In order to develop a vaccine with the potential to protect against other high-risk HPV types, we have produced HPV58 pseudovirions encoding the HPV31 L2 protein and compared their capacity to induce cross-neutralizing antibodies with that of HPV L1 and HPV L1/L2 VLPs.     

小鼠气性坏疽动物模型的建立

目的建立产气荚膜梭菌感染小鼠的后肢的气性坏疽动物模型,为早期诊断、了解气性坏疽的致病机理及治疗方法提供依据。方法小鼠随机分成4组,每组10只。三个实验组分别肌肉注射3.5×109、3.5×108和3.5×107cfu/ml浓度的产气荚膜梭菌（ATCC13124）菌液0.1ml,空白对照组肌肉注射生理盐水0.1ml,72h后观察小鼠感染情况,取伤口分泌物作革兰氏染色涂片,细菌血平板厌氧培养和荧光定量PCR方法定量检测。结果 3.5×109、3.5×108、3.5×107cfu/ml实验组和对照组在肌肉注射72h内的死亡率为90%、70%、10%和0%。各组平均存活时间依次为（20：43±11：12）h、（37：24±25：39）h、（68：36±10：45）h和（72：00±0：00）h,死亡小鼠出现了气性坏疽的症状,分泌物培养和镜检出产气荚膜梭菌;未死亡小鼠康复;空白对照组无任何症状。各组Ct值均值为21.21±2.69、28.45±2.74、32.49±2.87和0.00±0.00,组间P值均〈0.05。结论成功建立了不同浓度的产气荚膜梭菌在不同时间段内感染小鼠的后肢气性坏疽的动物模型,为了解气性坏疽的致病机理及治疗方法奠定基础。     

人16型乳头瘤病毒"假病毒"免疫保护作用的研究

目的评价构建的人16型乳头瘤病毒“假病毒”的免疫保护作用。方法利用杆状病毒表达系统在sf9昆虫细胞中表达组装了人16型乳头瘤病毒病毒样颗粒,将病毒样颗粒解聚后与真核表达质粒混合,重聚集成“假病毒”。用这种“假病毒”对小鼠进行免疫保护作用研究。结果小鼠经“假病毒”免疫后,可以在血清中检测到特异性的IgG,在阴道分泌物中检测到特异性的IgA,脾淋巴细胞可以检测特异性的CrrL活性。结论“假病毒”免疫能激活机体的免疫反应     

The 2010 global proficiency study of human papillomavirus genotyping in vaccinology

Accurate and internationally comparable human papillomavirus (HPV) DNA genotyping is essential both for evaluation of HPV vaccines and for effective monitoring and implementation of vaccination programs. The World Health Organization (WHO) HPV Laboratory Network (LabNet) regularly issues international proficiency studies. The 2010 HPV genotyping proficiency panel for HPV vaccinology contained 43 coded samples composed of purified plasmids of 16 HPV types (HPV types 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68a and 68b) and 3 coded extraction controls. Proficient typing was defined as detection in both single and multiple infections of 50 international units (IU) of HPV type 16 (HPV-16) and HPV-18 DNA and 500 genome equivalents (GE) for the other 14 HPV types. Ninety-eight laboratories worldwide submitted a total of 132 data sets. Twenty-four different HPV genotyping assay methods were used, with Linear Array being the most commonly used. Other major assays used were a line blot assay (Inno-LiPa), CLART, type-specific real-time PCR, PCR Luminex, and different microarray assays. Altogether, 72 data sets were proficient for detection of more than 1 type, and only 26 data sets proficiently detected all 16 HPV types. The major oncogenic HPV types, 16 and 18, were proficiently detected in 95.0% (114/120) and 87.0% (94/108) of data sets, respectively. Forty-six data sets reported multiple false-positive results and were considered nonproficient. A trend toward increased sensitivity of assays was seen for the 41 laboratories that participated in both 2008 and 2010. In conclusion, continued global proficiency studies will be required for establishing comparable and reliable HPV genotyping services for vaccinology worldwide.     

Visual detection of high-risk human papillomavirus genotypes 16, 18, 45, 52, and 58 by loop-mediated isothermal amplification with hydroxynaphthol blue dye

A simple, rapid, sensitive, qualitative, colorimetric loop-mediated isothermal amplification (LAMP) with hydroxynaphthol blue dye (HNB) was established to detect high-risk human papillomavirus (HPV) genotypes 16, 18, 45, 52, and 58. All initial validation studies with the control DNA proved to be type specific. The colorimetric type-specific LAMP assay could achieve a sensitivity of 10 to 100 copies at 63°C for 65 min, comparable to that of real-time PCR. In order to evaluate the reliability of HPV type-specific LAMP, the assay was further evaluated with HPV DNAs from a panel of 294 clinical specimens whose HPV status was previously determined with a novel one-step typing method with multiplex PCR. The tested panel comprised 108 HPV DNA-negative samples and 186 HPV-DNA-positive samples of 14 genotypes. The results showed that the sensitivity of HPV type-specific LAMP for HPV types 16, 18, 45, 52, and 58 was 100%, 100%, 100%, 100%, and 100%, respectively, and the specificity was 100%, 98.5%, 100%, 98.8%, and 99.2%, respectively, compared with a novel one-step typing method with multiplex PCR. No cross-reactivity with other HPV genotypes was observed. In conclusion, this qualitative and colorimetric LAMP assay has potential usefulness for the rapid screening of HPV genotype 16, 18, 45, 52, and 58 infections, especially in resource-limited hospitals or rural clinics of provincial and municipal regions in China.     

HIV合并HPV阳性标本中HPV的型别检测

目的探讨深圳地区人类免疫缺陷病毒（HIV）阳性人群中人乳头瘤病毒（HPV）感染分型情况,为HIV阳性人群中HPV感染的防治提供依据。方法运用荧光PCR方法和反向斑点杂交技术对HPV阳性患者进行HPV分型检测。结果在HIV感染者中。利用反向点杂交分型方法对40例HPV阳性标本进行HPV基因分型。其中单型感染有18例（45．0％）,混合感染有19例（47．5％）,共检出16种HPV型别,其中包括11种高危型（16、18、31、33、35、45、52、56、58、68、73型）和5种低危型（6、11、42、44、54型）。在16种型别中,感染率最高的为16型（25．0％）,其次为52型（17．5％）、58型（15．0％）。结论感染HIV人群中生殖器部位HPV的感染率较高,在HIV阳性人群中检出性病相关的高危HPV16／52／58亚型．对HIV阳性人群中HPV感染的防治有重要指导意义。