森林脑炎病毒prM-E蛋白在昆虫细胞中的表达及免疫活性测定

目的为了表达森林脑炎病毒prME蛋白,为森林脑炎快速诊断试剂的研制奠定基础。方法经过RTPCR扩增、重组转移载体构建、细菌内转座和昆虫细胞转染,以杆状病毒昆虫细胞表达系统成功地表达了森林脑炎病毒MDJ01株prME蛋白。结果从感染细胞上清中电镜观察到重组蛋白形成的球型颗粒,说明重组病毒感染细胞后产生病毒样表达颗粒(viruslikeparticlesVLPs),并且分泌至细胞外。免疫印迹试验和间接免疫荧光试验表明,表达的重组蛋白能够与抗森林脑炎病毒抗体特异结合,具有良好的抗原性。ELISA和间接免疫荧光染色证实,重组prME蛋白可以作为抗原用于检测患者血清特异性抗体。结论在昆虫细胞中表达的prME具有良好的抗原性,本研究为森林脑炎快速特异诊断试剂研制奠定了基础。     

人乳头瘤病毒16型晚期蛋白L1的表达及病毒样颗粒的装配

目的　在昆虫细胞中表达人乳头瘤病毒 16型 (HPV16 )哈尔滨地区分离株的晚期蛋白L1,并分离纯化由L1蛋白在细胞中自主组装成的病毒样颗粒 ,为HPV16预防性疫苗的研制奠定基础。方法　获得含有HPV16L1基因的重组杆状病毒并使目的蛋白L1在昆虫细胞中表达 ;对重组杆状病毒的扩增条件及L1蛋白的表达水平进行优化 ;电镜下观察昆虫细胞中晚期蛋白装配成病毒样颗粒的情况 ,经氯化铯密度梯度纯化病毒样颗粒 ,并经Westernblot进行验证。结果　获得了稳定表达L1蛋白的重组杆状病毒 ;以MOI值为 0 .2感染昆虫细胞可获得较高滴度的重组病毒 ,以MOI值为 10感染昆虫细胞可获得较高水平的L1蛋白表达 ;电镜下可观察到昆虫细胞核内直径为 5 5nm的病毒样颗粒 ;病毒样颗粒可通过氯化铯密度梯度离心获得。结论　获得人乳头瘤病毒哈尔滨地区分离株L1蛋白可在昆虫细胞中自主装配的病毒样颗粒并成功分离纯化。     

登革2型病毒ZS01/01株病毒样颗粒免疫原性研究

目的 研究登革2型病毒（Dengue virus type 2,DENV-2）病毒样颗粒（virus-Like particles,VLPs）的免疫原性.方法 利用已构建的DENV-2 ZS01/01株病毒样颗粒的表达质粒转染293T细胞,对分泌型VLPs进行大量培养并通过蔗糖密度梯度离心法对其进行纯化.纯化的VLPs经Western Blot及透射电镜观察等方法鉴定后免疫BALB/c小鼠.利用ELISA及中和试验等方法对体液免疫反应进行检测,ELISPOT法测定细胞免疫水平.结果 登革2型病毒样颗粒表达质粒转染哺乳动物细胞所得上清经蔗糖密度梯度离心后,电镜下可观察到类似于天然登革病毒的大小在45～55nm之间的病毒样颗粒.体液及细胞免疫检测结果显示登革2型VLPs可以刺激小鼠产生较高水平的登革E蛋白特异性抗体及一定水平的中和抗体,免疫小鼠脾淋巴细胞经体外刺激后IFN-γ水平显著升高.结论 登革2型病毒病毒样颗粒免疫BALB/c小鼠后可引起一定水平的细胞免疫及体液免疫反应,该研究结果为四价登革病毒样颗粒疫苗的研制奠定了基础.     

原核高效制备人乳头瘤病毒16型L1病毒样颗粒

目的通过原核表达系统高效制备人乳头瘤病毒(HPV)16晚期蛋白L1病毒样颗粒(VLPs)。方法构建HPV16L1基因序列优化前后的PET30aHPV16L1重组质粒,转化大肠杆菌Rosetta(DE3);用IPTG诱导目的基因表达,两步层析方法纯化HPV16L1蛋白;电镜下观察纯化产物形成VLPs的情况。结果成功构建大肠杆菌工程菌,高效可溶表达(目的蛋白约占总蛋白的38%)并纯化HPV16L1蛋白,纯度达95%以上,电镜下观察,发现纯化后的目的蛋白为直径50 nm左右,形态与天然病毒颗粒高度相似。结论在大肠杆菌原核系统中高效、简易地制备了HPV16L1VLPs,为诊断试剂和疫苗的研制奠定基础。     

登革Ⅰ型病毒E蛋白在293T细胞中的分泌表达

目的 分泌表达登革Ⅰ型病毒prM/E基因,为研究该蛋白的免疫学功能和特性奠定基础.方法 用RT-PCR法获得登革Ⅰ型病毒广州分离株全长prM/E基因,在prM基因前添加乙型脑炎病毒的信号肽或同时替换E基因羧基末端的20%为乙脑病毒E基因相应的部分,分别将其克隆入真核表达载体pcDNAS/FRT中,获得三种重组质粒DlprME-pc5,D1JsprME-pc5,D1JsprM80E20JE-pc5.用脂质体法分别将重组质粒DNA转入293T细胞,通过免疫荧光、Western印迹检测外源基因在真核细胞中的分泌表达.结果 用免疫荧光法检测到分别转染了三种重组质粒的293T细胞的胞质中均有登革Ⅰ型病毒蛋白的表达.Western印迹检测转染了D1prME-pc5重组质粒的293T细胞上清中没有特异蛋白条带,转染了经基因改造的重组质粒D1JsprME-pc5和D1JsprM80E20JE-pc5的细胞上清中均存在登革Ⅰ型病毒的特异蛋白条带.结论 转染了三种重组质粒的293T细胞均可表达登革Ⅰ型病毒prM/E蛋白,只有在prM基因前添加了信号肽的重组质粒转染后蛋白才获得分泌表达.     

人C-Met胞外区慢病毒穿梭载体构建及在293T细胞中的表达

本研究构建含人肝细胞生长因子受体(C-Met)胞外区基因的慢病毒表达载体,并将其转染293T细胞,真核表达并纯化获得人C-Met胞外段蛋白。采用RT-PCR扩增人C-Met胞外区(ED)基因,构建慢病毒表达载体p RRL-CMV-ED,磷酸钙法转染293T细胞,RT-PCR及Western blot法检测ED基因在293T细胞中mRNA的转录和蛋白表达。PCR扩增及测序结果表明成功构建了p RRL-CMV-ED慢病毒表达载体,PCR扩增的目的基因大小为2 700bp左右;转染p RRL-CMV-ED的293T细胞上清经镍柱亲和纯化,SDS-PAGE分析纯化产物在105kD处有明显蛋白条带;Western及ELISA结果显示,纯化蛋白为C-Met胞外区蛋白且能与肝细胞生长因子(HGF)特异性结合。本研究结果可能为制备C-Met单克隆抗体以及筛选阻断HGF/C-Met信号通路的中和抗体奠定基础。     

人细胞因子受体样因子3在真核及原核细胞中的表达

目的:观察CRLF3蛋白在293T细胞中的表达及亚细胞定位,并在大肠杆菌中表达和纯化了重组CRLF3蛋白.方法:将真核表达载体pCMV-myc-CRLF3瞬时转染293T细胞,转染24 h和48 h后免疫荧光染色,共聚焦显微镜观察蛋白表达及定位;构建原核表达质粒pGEX-4T-1-CRLF3,实现插入基因的融合表达,经GST亲合层析纯化蛋白.结果:CRLF3蛋白在293T细胞中高效表达,主要分布在细胞质和细胞膜;成功构建了表达CRLF3融合蛋白的原核表达载体,并在大肠杆菌内高效表达,纯化后得到Mr约为74 000的融合蛋白.结论:在真核细胞中过表达的CRLF3蛋白主要定位在细胞质和细胞膜;获得了重组GST-CRLF3融合蛋白,为进一步探讨CRLF3的功能奠定基础.     

大鼠微小RNA miR-16的克隆及其慢病毒表达载体的构建

目的 克隆微小RNA rno-miR-16,构建其慢病毒表达载体pLV-miR-16并包装成慢病毒颗粒,为进一步研究miR-16的功能奠定了实验基础.方法 从大鼠细胞基因组中用PCR 的方法扩增miR-16 的前体,构建了miR-16的重组表达载体pLV-miR-16,脂质体法将重组慢病毒载体和包装质粒混合物(pPACK-GAG、pPACK-REV和pVSV)共转染包装细胞293TN细胞,包装产生慢病毒,以293TN细胞绿色荧光蛋白(green fluorescent protein,GFP)的表达水平测定病毒滴度.结果 经PCR扩增检测阳性菌落和测序证实,成功构建携带大鼠miR-16基因重组慢病毒载体.倒置荧光显微镜下观察可见包装细胞293TN呈绿色荧光,并测得108＞ifu/ml.结论 成功构建大鼠慢病毒载体pLV-miR-16,为深入研究rno-miR-16的生物学功能奠定了基础.     

嵌合有HCV多中和抗原表位的乙肝S抗原病毒样颗粒的制备与鉴定

制备嵌合HCV多中和表位及HCV包膜蛋白E2的HBV S抗原病毒样颗粒(virus-like particle,VLP),并进行鉴定、纯化和浓缩。HEK293T细胞用DMEM培养至90%密度时,将构建的重组真核表达载体pCI-MEpS、pCI-E2S用脂质体法转染HEK293T细胞,48h后在培养上清中得到自我装配的嵌合HCV多中和表位及包膜蛋白E2的HBV病毒样颗粒(VLP-MEpS,VLP-M2S)。蔗糖密度梯度离心,透析浓缩后,电化学发光法进行HBsAg定量测定、电镜分析和Western blotting鉴定。制备出的嵌合病毒样颗粒VLP-MEpS和VLP-E2S经浓缩后其HBsAg定量最高达到3×10~4 ng/mL。实验表明我们成功制备出嵌合有HCV多中和抗原表位的HBV VLP,为进一步分析诱导的中和抗体研究奠定基础。     

猪囊尾蚴AgB基因的克隆表达及其免疫学特性分析

目的 克隆猪囊尾蚴 AgB 基因并在大肠杆菌中表达,为研制猪囊尾蚴病诊断试剂和基因工程疫苗奠定基础.方法 从猪囊尾蚴虫体中提取总 RNA,利用反转录聚合酶链式反应及重叠延伸 PCR 法扩增 AgB 完整序列,并克隆到原核表达载体 pGEX-4T-1,在大肠杆菌中诱导表达.通过 SDS-PAGE、Western-blotting检测AgB的表达和免疫反应活性.通过对包涵体的洗涤、纯化及变复性研究,纯化目的蛋白.采用ELISA法检测纯化蛋白检出猪囊尾蚴阳性血清率和免疫小鼠血清中抗猪囊尾蚴抗体的水平.结果 测序证实扩增出AgB基因序列并正确插入到原核表达载体.表达质粒在大肠杆菌BL21中高效表达.纯化的目的蛋白,具有良好的抗原性和免疫原性.结论 成功扩增了全长AgB基因并得到了高效表达,纯化了具有免疫性的重组蛋白,为进一步开展AgB的诊断试剂和重组疫苗研究奠定了基础.     

A recombinant particulate antigen of Japanese encephalitis virus produced in stably-transformed cells is an effective noninfectious antigen and subunit immunogen

A COS-1 cell line, stably transformed by a plasmid encoding the premembrane and envelope glycoproteins of Japanese encephalitis virus, produced a noninfectious recombinant antigen expressed as extracellular particles. Extracellular particles purified by equilibrium density centrifugation in sucrose gradients followed by electron microscopy were characterized as spherical particles with an average diameter of approximately 30 nm and a buoyant density of 1.15 g/cc. Purified extracellular particles were shown by western blot to contain premembrane, membrane and envelope proteins. The gradient-purified particles exhibited hemagglutination activity at the same pH optimum (6.6) as Japanese encephalitis virus. Recombinant antigen from cell culture fluid was concentrated by precipitation with polyethylene glycol and evaluated for immunogenicity in 8-10-week-old ICR mice. Groups of five mice received only one immunization of recombinant antigen with or without Freund's incomplete adjuvant. Mice immunized with recombinant antigen plus Freund's incomplete adjuvant elicited the highest anti-viral titers as determined by both enzyme-linked immunosorbent assay (ELISA) and plaque-reduction neutralization tests. The polyethylene glycol-concentrated recombinant antigen was also evaluated for use in IgM antibody-capture ELISA and indirect IgG ELISA. The IgM-capture ELISA results using recombinant antigen correlated well with the results of a similar test using Japanese encephalitis virus-infected mouse brain antigen for the analysis of serum samples from patients with symptoms of acute encephalitis. Similar IgG titers were observed in an indirect ELISA comparing recombinant antigen and purified Japanese encephalitis virus as plate-bound antigens. Based on these studies, this entirely safe, easily produced antigen that expresses authentic Japanese encephalitis virus envelope glycoprotein would provide an excellent alternative to standard viral antigens used in various ELISA formats.     

Enzyme-linked immunosorbent assay using recombinant antigens expressed in mammalian cells for serodiagnosis of tick-borne encephalitis

A recombinant plasmid that expresses the tick-borne encephalitis (TBE) virus premembrane (prM) and envelope (E) proteins in mammalian cells was constructed. Recombinant proteins retained antigenic and conformational structures similar to those of native virus proteins, and transfected cells released virus-like particles (VLPs), which were 1.13-1.14 g/ml in density and 20-30 nm in diameter, into the culture medium. Recombinant E proteins were used for the development of an enzyme-linked immunosorbent assay (ELISA) to detect TBE virus-specific IgM and IgG antibodies in serum. The results of this ELISA correlated well with the results of commercial ELISA, when tested with 95 serum samples from clinically TBE-suspected patients. In addition, ELISA using recombinant antigens showed no cross-reactivity against serum from Japanese encephalitis (JE) patients, despite the cross-reactivity shown by commercial ELISA systems. These observations indicated that this newly developed ELISA system could distinguish tick-borne encephalitis from Japanese encephalitis infection, and that it constitutes a useful and safe alternative to conventional ELISA systems.     

Noninfectious recombinant antigen for detection of St. Louis encephalitis virus-specific antibodies in serum by enzyme-linked immunosorbent assay

Proper surveillance of virus activity and a timely response to viral outbreaks depend upon the rapid diagnosis of viral infections. The immunoglobulin M (IgM) antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA) is a fast, sensitive test routinely used for the diagnosis of the medically important West Nile and St. Louis encephalitis flaviviruses. However, the suckling mouse brain-derived (SMB) antigen used in this assay is tedious to prepare and has a risk of exposing personnel to live virus and hazardous chemicals. We report the development of a St. Louis encephalitis virus (SLEV) noninfectious recombinant antigen that is a safe and easily produced alternative antigen for use in diagnostic assays. The expression plasmid pCB8SJ2, containing the premembrane and envelope structural protein-encoding regions of SLEV, was constructed to express secreted extracellular virus-like particles (VLPs) from CHO cells. Blind-coded human serum panels were assembled from patients having recent SLEV, West Nile virus (WNV), Powassan virus, or La Crosse encephalitis virus infections to assess the sensitivity and specificity of assays with SLEV VLP or SMB antigen. MAC-ELISAs with either antigen had comparable sensitivity for the detection of IgM antibodies against SLEV. Importantly, when these two antigens were tested against a human serum panel from patients having recent WNV or Powassan virus infections, the SLEV VLPs were less likely than SMB antigen to detect flavivirus cross-reactive IgM antibodies. An optimized IgG antibody capture ELISA (GAC-ELISA) with both WNV and SLEV VLPs was developed to circumvent the frequently observed higher background in the antigen-capture IgG-ELISA (ACG-ELISA). For the detection of IgG antibodies against WNV, the GAC-ELISA resulted in a statistically significant higher performance accuracy (P = 0.003) than the ACG-ELISA when the WNV VLP antigen was used in both assays. However, no statistical difference was observed in the assay performance of the GAC-ELISA with SLEV VLP or the ACG-ELISA with SLEV SMB antigen.     

Performance of immunoglobulin G (IgG) and IgM enzyme-linked immunosorbent assays using a West Nile virus recombinant antigen (preM/E) for detection of West Nile virus- and other flavivirus-specific antibodies

Focus Technologies developed an indirect immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) and a mu-capture IgM ELISA for the detection of West Nile virus (WNV)-specific antibodies based on a WNV preM/E protein recombinant antigen. Normal and disease state serum panels were used to assess the performance characteristics of the two WNV ELISA kits. Totals of 807 and 1,423 sera were used to assess the IgG ELISA and IgM ELISA kits, respectively. The Focus Technologies IgG ELISA had a sensitivity of 97.6% and a specificity of 92.1% (excluding non-WNV flavivirus sera). The comparative method for WNV IgG may lack sensitivity in detecting IgG in early WNV infection, so the specificity of the Focus IgG ELISA may be higher than 92.1%. When sera from patients either infected with or vaccinated against other flaviviruses were tested on the WNV IgG assay, 35% of the sera reacted as positive for WNV IgG. Yellow fever and Japanese encephalitis vaccinees were less reactive in the IgG ELISA than St. Louis and dengue fever patients. The Focus Technologies IgM ELISA had a sensitivity and a specificity of 99.3% (excluding the non-WNV flavivirus sera). The overall cross-reactivity for the IgM ELISA to flavivirus sera was 12%, with 31% of St. Louis encephalitis patients found to be WNV IgM positive and no yellow fever vaccinees found to be WNV IgM positive. In a selected population of 706 sera, 15 false-positive WNV IgM sera were identified. The use of a background subtraction method for the IgM ELISA eliminated all 15 false-positive results, giving a specificity of 100% for the Focus IgM ELISA.     

Amino-terminal amino acid sequences of structural proteins of three flaviviruses

N-terminal amino acid sequences of structural proteins of three flaviviruses, yellow fever, St. Louis encephalitis, and dengue-2 viruses, have been obtained. The glycoproteins of these three viruses are 52-60% conserved in the region sequenced, depending upon which pair of viruses are compared, and 40% of the amino acids are invariant in all three viruses. Thus, flaviviruses are closely related and have in all probability descended from a common ancestor. Furthermore, residues important in the secondary structure of proteins are conserved, suggesting that the overall conformation of the glycoproteins is the same in all three viruses while considerable variation in the primary sequence can be accommodated. The N-terminal regions of the nucleocapsid proteins of yellow fever and St. Louis encephalitis viruses show markedly less homology (25%) and this region is highly basic with one-quarter (yellow fever) or one-third (St. Louis encephalitis) of the residues being lysine or arginine. N-terminal sequences for the M protein of yellow fever and for NV2(GP19) of St. Louis encephalitis viruses are also reported.     

Development of an enzyme-linked immunosorbent assay for serological diagnosis of tick-borne encephalitis using subviral particles

The similarity of symptoms produced by tick-borne encephalitis (TBE) and Japanese encephalitis (JE) and the high degree of cross-reactivity between TBE and JE viruses by serological tests make the development of a differential diagnostic test a priority. In this study, recombinant prM/E proteins of TBE virus strain Oshima 5-10 expressed in mammalian cells resulted in the release of subviral particles (SPs) into the culture medium. Using the SPs as antigens, enzyme-linked immunosorbent assay (ELISA) systems were developed to detect TBE virus-specific IgM and IgG antibodies, designated SP-IgG and SP-IgM ELISAs, respectively. Of 83 serum samples from encephalitis patients in Khabarovsk, Russia, which were positive with the neutralization test (NT), 82 were positive by the SP-IgG ELISA, for a sensitivity of 98.8%, which was higher than that of a commercial ELISA kit. All 12 NT-negative samples were also negative by the SP-IgG ELISA (specificity, 100%). Of 17 patient samples that were NT-positive, 16 (94.1%) were positive by the SP-IgM ELISA. Of 15 paired serum samples that yielded equivocal results by NT, 11 had positive results with the SP-IgM ELISA, indicating a diagnosis of TBE infection. The SP-IgG and SP-IgM ELISAs showed no cross-reactivity with antibodies to the JE virus. The results indicate that these ELISAs will be useful for the detection of TBE-specific antibodies.     

Mutation analysis of the fusion domain region of St. Louis encephalitis virus envelope protein

The immune response to flavivirus infections produces both species-specific and flavivirus cross-reactive antibodies. The presence of cross-reactive antibodies complicates serodiagnosis of flavivirus infections, especially secondary infections caused by a heterologous virus. A successful public health response to the growing global threat posed by flaviviruses necessitates the development of virus-specific diagnostic antigens. The flavivirus envelope (E) glycoprotein is the principle antigen stimulating protective immunity during infection. Using recombinant St. Louis encephalitis virus-like particles (VLPs), we have identified amino acid residues involved in flavivirus cross-reactive epitope determinants. Most significant among the residues studied are three highly conserved amino acids in the fusion peptide: Gly104, Gly106, and Leu107. Substitutions of these residues dramatically influenced VLP secretion and cross-reactive monoclonal antibody reactivity. These results provide critical insight into the antigenic structure of the flaviviral E protein and toward development of species-specific diagnostic antigens that should improve both flavivirus diagnosis and estimates of disease burden.     

Visceral target organs in systemic St. Louis encephalitis virus infection of hamsters

Ultrastructural aspects of St. Louis encephalitis virus infection of the major extraneutral organs and tissues of suckling hamsters were examined. In the pancreas, both the exocrine and endocrine portions were equally affected by the virus. A feature apparently unique to flaviviruses was the accumulation of virus particles in all types of secretory granules in this organ. Virus particles were seen within myocardial fibers and within the smooth muscle cells and endothelial cells of small blood vessels of the heart. In the intestines, the lamina propria was the most severely infected, with virus particles accumulated in all cell types.     

Detection of human anti-flavivirus antibodies with a west nile virus recombinant antigen microsphere immunoassay

We report a new, suspended-microsphere diagnostic test to detect antibodies to West Nile (WN) virus in human serum and cerebrospinal fluid (CSF). The microsphere immunofluorescence assay can be performed in less than 3 h on specimens of 相似文献