绿色荧光蛋白标记重组人偏肺病毒的制备

目的 人偏肺病毒(human metapneumovirus,hMPV)可致人上、下呼吸道感染.研究利用反向遗传学技术,以带绿色荧光蛋白(GFP)标记的hMPV NL/1/00全长cDNA质粒及4种辅助质粒pCITE-N、pCITE-P、pCITE-M2.1和pClTE-L为基础,在体外制备GFP标记的重组hMPV病毒(命名为rhMPV NL/1/00 GFP).方法 采用LipofectAMINE 2000将带GFP标记的hMPV NL/1/00全长cDNA质粒以及蛋白表达质粒pcITE-N、pCITE-P、pCITE-M2.1和pcITE-L共转染BSR-T7细胞,3 d后取BSR-T7细胞上清液感染Vero-E6细胞,1-4 d后观察Veto-E6细胞出现明显细胞病变(CPE)和绿色荧光,持续观察至第10天.收集病毒上清用于病毒滴度检测.提取培养上清的病毒RNA并通过RT-PCR方法扩增病毒N基因、F基因和G基因验证所获重组病毒.结果 Vero-E6细胞接种1～4 d后观察到明显CPE和绿色荧光,随后CPE加重,荧光信号加强,持续至感染后10 d;第1、5、10、15和20代病毒的滴度波动于105.0～106.5TCID50/ml;RT-PCR检测出符合预期大小的910 bp(N)、450 bp(F)和980 bp(G)条带,经卜述片段cDNA序列测定和比对表明获得的重组病毒与hMPV NL/1/00原型病毒序列一致.rhMPV NL/1/00 GFP重组病毒在体外传代20代后,遗传信号和荧光信号均稳定.结论 采用反向遗传学技术成功拯救了具有感染性的重组hMPV病毒,为hMPV感染发病机制及疫苗研究奠定了基础.     

BALB/c和SCID小鼠人偏肺病毒感染特点的比较研究

目的 比较研究人偏肺病毒(hMPV)在BALB/c小鼠和SCID小鼠肺内复制动力学和肺病理特点.方法 GFP-rhMPV滴鼻感染BALB/c小鼠和SCID小鼠,于感染后3、5、7、9、14d处死小鼠并无菌获取心、肝、脾、肺、肾和脑用于病毒分离和病理检查,空斑形成法检测病毒滴度,RT-PCR和real-time PCR法检测hMPV mRNA表达.结果 GFP-rhMPV滴鼻感染BALB/c小鼠和SCID小鼠后第5天肺组织均分离到病毒.感染GFP-rhMPV的BALB/c和SCID小鼠在感染后第5天肺组织病毒滴度均达峰值,分别为(5.25±1.69)×104 PFU/g和(5.83±1.21)×105 PFU/g.SCID小鼠在感染后第14天肺组织内病毒滴度仍可达(4.25±1.04) ×101 PFU/g,此时BALB/c鼠肺内已不能分离到病毒,但可检测到GFP-rhMPV F蛋白mRNA表达.感染后第5天所有小鼠的心、肝、脾、肾和脑组织内均不能分离到病毒,也无法检测到hMPV F蛋白mRNA表达.肺组织病理改变在感染后第5天最明显,为典型的间质性肺炎改变,BALB/c小鼠组肺组织病理评分略低于SCID小鼠组,差异无统计学意义.结论 GFP-rhMPV滴鼻感染后只能在小鼠肺内复制.同BAL B/c小鼠相比,SCID小鼠感染GFP-rhMPV后肺内病毒滴度高,复制时间久,但病理损伤无明显差异.     

应用细菌同源重组法快速构建和制备重组腺病毒

目的：利用大肠杆菌细菌同源重组构建重组腺病毒载体并在293细胞制备高滴度重组病毒。方法：自细胞周期相关激酶真核表达载体pCR31-CCRK中酶切出CCRK基因，亚克隆至带有增强型绿色荧光蛋白（EGFP）表达盒的腺病毒穿梭质粒pAdTrack-CMV中，形成转移质粒pAdTrack-CMV-CCRK，采用电穿孔或化学转化法在大肠杆菌BJ5183内与腺病毒骨架质粒pAdEasy-1同源重组，得到重组腺病毒载体pAd-CCRK。以pAd-CCRK为模板，经DNA测序正确后，用Pac Ⅰ酶切线性化pAd-CCRK，转染293细胞，包装成重组病毒颗粒，荧光显微镜观察转染细胞EGFP的表达，采用PCR的方法对重组腺病毒进行鉴定。将重组病毒上清感染RAW细胞，荧光显微镜下观察感染细胞重组病毒的表达。结果：成功地构建了携带CCRK基因的重组腺病毒载体并制备出高滴度重组病毒，重组病毒能在体外高效表达。结论：应用细菌内同源重组能够快速构建腺病毒载体，可高效制备均一的高滴度重组病毒，为CCRK基因功能的研究奠定了基础.     

Effect of recombinant adeno-associated virus transforming growth factor-beta 1 on the infection activity of bone marrow mesenchymal stem cells

背景：目前常用基因载体具有一定缺陷性,无法直接在体内应用。应用腺相关病毒作为转化生长因子β1载体促进软骨修复的研究报道较少。 目的：构建重组转化生长因子β1腺相关病毒,测定病毒滴度,并测定重组病毒对骨髓基质干细胞的感染活性。 方法：PCR方法扩增转化生长因子β1基因,构建重组骨架质粒pAAV-转化生长因子β1-绿色荧光蛋白,与包装质粒pAAV-RC、辅助质粒pAAV-Helper共转染AAV-293细胞包装重组腺相关病毒rAAV-转化生长因子β1-绿色荧光蛋白。应用该重组病毒感染AAV-HT1080细胞测定病毒滴度并鉴定。并感染体外培养的兔骨髓基质干细胞,检测重组病毒感染骨髓基质干细胞的效率及活性。 结果与结论：转化生长因子β1扩增产物测序结果正确,重组骨架质粒pAAV-转化生长因子β1-绿色荧光蛋白双酶切后可见位于1.3 Kb附近转化生长因子β1条带。收获病毒滴度5.2×1011 v.g/mL。鉴定重组病毒rAAV-转化生长因子β1-绿色荧光蛋白包装成功。重组病毒感染骨髓基质干细胞后可于荧光显微镜下观测到绿色荧光蛋白的表达,感染效率达42%。结果证实,成功构建重组腺相关病毒rAAV-转化生长因子β1-绿色荧光蛋白,能够高效感染兔骨髓基质干细胞。     

小鼠人偏肺病毒感染模型的建立

目的 建立人偏肺病毒(hMPV)感染小鼠模型,了解病毒肺内复制规律及所致病理改变,为hMPV感染免疫病理机制研究及新型防治手段开发奠定基础.方法 BALB/c小鼠经滴鼻感染荧光标记的重组hMPV,于感染后1、3、5、7、9、16 d处死小鼠并无菌获取肺组织用于病毒分离和病理检查,改良噬斑形成法检测hMPV滴度,RT-PCR法检测hMPV mRNA表达.结果 小鼠滴鼻感染hMPV后肺组织分离到病毒;肺组织病毒滴度在感染后5 d达到高峰(5.16±1.09)×105PFU/g,感染后第9大仍能检测到病毒(2.79±1.22)×102PFU/g;感染后16 d肺组织仍可检测到hMPV mRNA;病理改变在感染后3～7 d最明显,为典型的间质性肺炎改变.结论 hMPV感染BALB/c小鼠模型建立成功,可用于hMPV感染的免疫病理机制研究.     

运用流式细胞术快速检测汉滩病毒滴度

目的:建立用流式细胞仪快速测定汉滩病毒滴度的方法.方法:汉滩病毒76-118株感染Vero-E6细胞,以汉滩病毒核蛋白单克隆抗体(mAb) 3G1为一抗,FIX标记的羊抗小鼠抗体为二抗,用流式细胞术(FCM)检测阳性细胞率,评价感染后不同时间点,以及不同病毒接种量的阳性细胞率,并与间接免疫荧光法对比.结果:感染后36 h阳性细胞百分率为(10.06±0.42)%,最低检测的病毒滴度为100 TCID50/ML.结论:相对于传统的空斑实验及间接免疫荧光滴定法,FCM是一种简单、快速、有效检测汉滩病毒滴度的方法.     

荧光定量PCR用于重组杆状病毒鉴定及病毒滴度检测的研究

目的：建立一种高效、简便的荧光实时定量PCR方法,用于重组杆状病毒鉴定及病毒滴度的检测.方法：利用Bac-to-Bac载体系统在E.coli菌株DH10 Bac中构建重组杆状病毒穿梭质粒（Bacmid）和在昆虫细胞中构建含人IL-18基因的重组杆状病毒,纯化的重组Bacmid作为PCR检测的标准模板,由昆虫细胞中收获的病毒母液用于空斑测定和病毒DNA提取.以10倍梯度稀释的重组Bacmid作为标准模板,进行荧光定量PCR扩增IL18基因片段并绘制标准曲线,然后以提取的重组杆状病毒DNA作为模板,采用同样体系进行实时PCR反应检测.同时,以琼脂糖空斑法测定病毒母液的滴度.结果：成功构建了重组杆状病毒并建立了病毒滴度的实时荧光PCR检测方法.运用标准模板进行的PCR反应显示该方法的线形范围为101～108拷贝,病毒母液的DNA拷贝浓度（vg/ml）值约为空斑检测的滴度pfu/ml值的10倍.结论：荧光定量PCR方法可灵敏快速地鉴定重组杆状病毒,并在较大的线性范围内检测重组杆状病毒滴度,较之空斑法更准确地反映了重组杆状病毒的实际数量.     

表达miR-10α的重组腺病毒的构建与鉴定

目的构建表达miR．10a的重组腺病毒（adenovirus,Ad）载体。方法用红色荧光蛋白mCherry基因替换穿梭质粒pDC316-EGFP—U6的增强型绿色荧光蛋白（enhanced green fluorescent protein,EGFP）报告基因。利用表达siRNA的策略,合成编码miR-10a的长链DNA序列,双链退火后克隆至pDC316,mCherry—U6获得pDC316-mCherry—U6-miR-10α。pDC316-mCherry-U6-miR-10α与骨架质粒pBHGlox△E1,3Cre共转染HEK293细胞,包装成复制缺陷型重组腺病毒Ad-miR-10α空斑形成实验纯化、扩增、滴定重组腺病毒。Ad-miR-10α感染HeLa细胞后在荧光显微镜下观察mCherry表达,用RT—qPCR检测miR-10α表达。结果构建的pDC316-mCherry—U6-miR-10α序列正确,荧光显微镜下可见mCherry的表达,重组腺病毒Ad—miR-10α滴度为1．8×10^7pfu／mL,感染HeLa细胞后miR-10α表达较mock高40倍。结论成功构建了表达miR-10α的首组腺病毒．siRNA表达策略可用于miRNA的人工表达。     

携eGFP及表达人gp100-TCR基因的慢病毒表达载体的构建与鉴定

目的构建携eGFP的人gp100-TCR基因慢病毒表达载体。方法应用基因重组手段,从pGCsamgp100APB质粒上扩增出人gp100-TCR片段,将其插入到pLentiLox3.7(pLL3.7)慢病毒表达载体中,同时将其U6启动子替换为EF-1a启动子,形成慢病毒表达载体pLL3.7-gp100TCR-pEf-1a。将其与包装质粒混合,采用磷酸钙转染法转染293FT细胞,包装产生慢病毒,荧光显微镜观察GFP蛋白的表达水平。检测病毒滴度。结果 PCR、酶切及测序表明慢病毒表达载体pLL3.7-gp100TCR-pEf-1a构建成功。转染后的293FT细胞在荧光显微镜观察可见强绿色荧光,形成的慢病毒可感染293FT细胞,包装的慢病毒浓缩后滴度为4.5×106 TU/ml。结论成功构建eGFP和gp100-TCR共同表达的慢病毒表达载体,为gp100-TCR在肿瘤治疗中的功能研究提供了有效平台。     

重组慢病毒表达载体介导HIV-1 Vpr蛋白调控KSHV潜伏感染的初探

目的 利用慢病毒载体系统包装含人类免疫缺陷病毒1型(HIV-1)病毒蛋白R(Vpr)的重组慢病毒,使之感染原发性渗出性淋巴瘤(PEL)细胞BCBL-1,并检测Vpr蛋白对细胞中卡波济肉瘤相关疱疹病毒(KSHV)潜伏感染与裂解性复制的影响.方法 从实验室先前构建的重组真核表达质粒pCI-neo-Vpr中扩增出Vpr基因,插入到pHAGE-CMV-MCS-IzsGreen载体中构建成重组慢病毒质粒pHAGE-Vpr,利用脂质体将其与包装质粒psPAX2及包膜质粒pMD2.G共转染293T细胞,荧光显微镜观察293T细胞中绿色荧光蛋白(GFP)的表达,293T细胞培养上清经0.45 μm滤器过滤后即获得重组病毒悬液.慢病毒系列稀释后感染293T细胞,荧光计数法测定病毒滴度,逆转录PCR(RT-PCR)检测Vpr基因在293T细胞中的转录.以感染复数(MOI)为1的病毒量感染靶细胞BCBL-1,荧光显微镜观察靶细胞中GFP的表达,并利用RT-PCR和和免疫印迹(Western blot)技术分别检测Vpr基因在靶细胞中的转录和表达情况,同时检测KSHV裂解期基因复制与转录激活子(Rta)mRNA转录及蛋白表达.结果 经酶切鉴定、核酸序列测定和293T细胞中GFP表达证实成功包装了携带HIV-1Vpr基因的重组慢病毒,滴度为4×107TU/ml.重组慢病毒感染BCBL-1细胞后,细胞中有GFP表达,RT-PCR和Western blot均能够在相应位置检测到目的 基因Vpr的条带,并且Vpr降低了KSHV RtamRNA转录及蛋白表达水平.结论 重组慢病毒介导的HIV-1 Vpr蛋白过表达能够抑制KSHV裂解性复制、增强病毒的潜伏感染.     

Development and optimization of a direct plaque assay for human and avian metapneumoviruses

The genus Metapneumovirus within the subfamily Pneumovirinae and family Paramyxoviridae includes only two viruses, human metapneumovirus (hMPV) and avian metapneumovirus (aMPV), which cause respiratory disease in humans and birds, respectively. These two viruses grow poorly in cell culture and other quantitation methods, such as indirect immuno-staining and immuno-fluorescent assays, are expensive, time consuming, and do not allow for plaque purification of the virus. In order to enhance research efforts for studying these two viruses, a direct plaque assay for both hMPV and aMPV has been developed. By optimizing the chemical components of the agarose overlay, it was found that both hMPV with a trypsin-independent F cleavage site and aMPV formed clear and countable plaques in a number of mammalian cell lines (such as Vero-E6 and LLC-MK2 cells) after 5 days of incubation. The plaque forming assay has similar sensitivity and reliability as the currently used immunological methods for viral quantitation. The plaque assay is also a more simple, rapid, and economical method compared to immunological assays, and in addition allows for plaque purification of the viruses. The direct plaque assay will be a valuable method for the quantitation and evaluation of the biological properties of some metapneumoviruses.     

Infectivity assays for the Autographa californica nuclear polyhedrosis virus using Spodoptera littoralis cells

Tissue culture ID50 and plaque assays for the Autographa californica nuclear polyhedrosis virus, using the Spodoptera littoralis cell line HPB-SL, were developed. Direct comparison using these assay methods showed that these cells were as susceptible to infection as the more commonly used Spodoptera frugiperda cell line IPLB-SF-21. Both infectious tissue culture supernatants or virus isolated directly from polyhedra could be titrated. It was important to use low cell seeding densities in the assays so that clear centres of infection formed. Dose-response curves indicated that one infectious particle was capable of initiating an infection. Virus could be cloned using either method even though, for the plaque assay, plates had been stained. The tissue culture ID50 assay was performed using 96-well plates and required an incubation period of about 10 days. The plaque assay used a simple nutrient agarose overlay and an incubation period of 5-6 days. Easily countable plaques of 0.3-1.2 mm diameter were detected after staining with iodonitrote-trazolium chloride. The plaques comprised areas of inhibited cell division and round or dead cells. Most plaques contained only some cells with polyhedra and yields averaged about 1/cell. Occasionally plaques or infected wells were found in which no polyhedra could be seen. These infectivity assays are therefore not dependent on polyhedra formation.     

Plaque assay for avian metapneumovirus using a Japanese quail fibrosarcoma cell line (QT-35)

A plaque assay for detecting, isolating and titrating avian pneumoviruses using a Japanese quail fibrosarcoma cell line (QT-35) is described. Plaques are produced after application of either an agarose or carboxymethyl-cellulose (CMC) overlay onto cell monolayers infected with representative avian metapneumoviruses which belong to subgroup A, B or C. Virus plaques can be easily visualized by light microscopy or after staining. The parameters affecting plaque appearance include: cell seeding concentration, virus strain, overlay composition and incubation time following infection. Optimal conditions for plaquing involve seeding QT-35 cells at 40,000 cells per cm(2) when using a 1.5% CMC overlay or 100,000 cells per cm(2) when using a 1.0 or 0.8% agarose overlay. In both cases, cell monolayers are infected with virus 24 h post-seeding and clearly visible plaques develop in 6 days. Due to the robust nature of these cells, the incubation time can be extended to a maximum of 13 days after infection in order to produce larger plaques.     

A simplified plaque assay for respiratory syncytial virus--direct visualization of plaques without immunostaining

Due to their generally small size, detection of respiratory syncytial virus (RSV) plaques commonly relies on immunostaining using either polyclonal antisera or a pool of monoclonal antibodies against the surface fusion glycoprotein. Disadvantages include the costs of the antibodies and substrate, and microscopic examination is often needed for counting of plaques. By optimizing many parameters, greatly improved plaque assays for RSV A2, Long and RSV B 18537 strains using Vero or HEp-2 cells have been developed. Although many groups use Vero cells for plaquing, after optimization plaques were much larger in HEp-2 cells. Both cell types needed to be confluent at infection and agarose was more suitable than carboxymethyl cellulose for the overlay. Overlay medium for HEp-2 was DMEM/F12, 0.3% agarose, and for Veros it was Liebovitz L15, 5% fetal calf serum, 0.3-0.5% agarose. After 5-7 days, monolayers were fixed with 1% formal saline overnight, agarose was removed and monolayers were stained with 0.05% neutral red in water. Plaques were readily visible by eye and plates could be dried and stored permanently. Parainfluenza virus type 3 also formed large plaques in HEp-2 cells under the conditions optimal for RSV.     

人偏肺病毒感染人肺上皮细胞后Toll样受体的表达及其功能

目的 观察人偏肺病毒(human metapneumoviras)感染人肺上皮细胞后Toll样受体(TLR)表达变化及其信号通路的功能,探讨hMPV诱导气道炎症的部分机制.方法 hMPV感染体外培养的人肺上皮细胞株A549,检测病毒在A549细胞中的生长曲线,并通过RT-PCR,real-timeRT-PCR方法检测细胞TLR mRNA的表达,ELISA检测细胞培养上清IFN-α及TNF-α的表达.结果 (1)hMPV可在A549细胞中复制,感染后3 d达高峰10~(5.2)TCID_(50)/ml.(2)RT-PCR结果提示:hMPV感染A549细胞6 h后大部分TLR的表达均上调.(3)定量PCR结果提示:hMPV感染A549细胞后TLR3-4、TLR7-9的表达增高,且有时间依赖性,而紫外灭活的hMPV刺激细胞后TLR表达无明显变化.(4)ELISA结果提示hMPV感染后24 h,IFN-α及TNF-α的表达均明显升高.结论 人偏肺病毒感染A549细胞后可上调TLR表达,其诱导的炎性反应与部分TLR介导的信号转导途径有关.     

Optimal conditions for titration of SV40 by the plaque assay method

The parameters of the Simian Virus 40 (SV40) plaque assay on African green monkey kidney cells were optimized for reproducibility and maximum plaquing efficiency. Plaques were visible as early as 8 days postinfection; maximum titers were obtained with a 10- to 11-day incubation period. Titers read 12-16 days postinfection were not significantly higher than those observed after 10-11 days. Adsorption volumes greater than 0.1 ml/60 mm Petri dish decreased plaque forming units (PFUs) detected. Times greater than 60 min for adsorption of virus to the cell monolayer did not significantly increase the titer; adsorption times less than 60 min resulted in decreased titers. Under standard conditions, 3 ml of overlay medium containing 0.8% agar was applied following virus adsorption and again on days 5 and 10. Concentrations of fetal calf serum (FCS) in the overlay medium of 2.5 to 7.5% gave equal plaque formation. FCS concentrations of 1 and 10% resulted in slightly decreased and increased plaquing efficiencies respectively. Of the reagents tested, agar or agarose containing overlay media produced plaques of maximum number and size. An overlay of methyl cellulose resulted in the same number of plaques, but their size was reduced by approximately 70% relative to those observed in agar; thus longer incubation times were required. Gum tragacanth overlay medium was actually inhibitory to plaque development. DEAE-dextran, dextran sulfate, or DMSO added to agar overlay medium did not enhance plaque number or size, nor did they shorten the incubation period required for their detection.     

Prevalence of human metapneumovirus in hospitalized children with respiratory tract infections in Tianjin,China

Human metapneumovirus (hMPV) has recently been recognized as an important respiratory pathogen, especially in children. At present, our understanding of the characteristics of hMPV from China is very limited. Nasopharyngeal aspirates were taken from 310 hospitalized pediatric patients. Twenty (6.5%) of them were infected with hMPV, and they all developed pneumonia. Sixty five percent (13/20) of the cases were under 12 months. Phylogenetic analysis of F gene fragments indicated that three sub-genotypes of hMPV(A2a/A2b, B1,B2) circulated in Tianjin and A2b was the predominant subtype. The Vero-E6 cell line was better than LLC-MK2 for hMPV isolation. Three hMPV strains were successfully isolated using the Vero-E6 cell line.     

Seroprevalence of human metapneumovirus (hMPV) in the Canadian province of Saskatchewan analyzed by a recombinant nucleocapsid protein-based enzyme-linked immunosorbent assay

Human metapneumovirus (hMPV) is a newly identified respiratory virus associated with respiratory tract infection in both adults and children. Previous reports showed that infection of hMPV appeared to be ubiquitous. To determine the seroprevalence of hMPV, a total of 576 human sera from patients in Saskatchewan, Canada, were screened by enzyme-linked immunosorbent assay (ELISA) based on expression of the nucleocapsid (N) protein of hMPV in recombinant baculovirus. The recombinant N protein with a molecular mass of 43.5 kDa was abundantly produced in insect cells. Moreover, the recombinant N proteins of the prototype viruses for the two major groups of hMPV have cross-antigenicity. The seropositive rate for each age group was 13.5% (13/96) (0-5 years), 26.1% (25/96) (6-10 years), 32.3% (31/96) (11-15 years), 99.0% (95/96) (16-30 years), 91.7% (88/96) (31-60 years), and 93.8% (90/96) (61+ years), respectively. The data indicated that exposure to hMPV is a common phenomenon. The ELISA based on recombinant baculovirus produced N protein of hMPV provides a useful tool for seroepidemiological study of this virus.