西尼罗病毒多表位基因的融合表达及其免疫保护作用研究

目的研究西尼罗病毒（WNV）多表位基因的融合表达及其免疫保护作用。方法利用生物信息软件Biosun分析西尼罗全基因序列，确定表位基因片段并选择合适片段连接构成多表位基因。采用重叠PCR方法扩增该基因，构建多表位基因的原核重组表达载体pET-43a-M，进行融合表达和纯化。将表达蛋白加弗氏佐剂免疫小鼠并进行攻毒试验，观察其保护作用。结果分子克隆和构建了原核重组表达质粒pET-43a-M，表达和纯化了融合蛋白Nus-M，该蛋白免疫后的小鼠能够部分抵御西尼罗病毒的攻击。结论构建的西尼罗病毒多表位基因能够在原核细胞内表达，表达产物有较弱的免疫保护反应，为该病毒多表位抗原的串联及多表位疫苗研究提供了试验资料，但需要进一步改进。     

猪瘟病毒C株E~(rns)/E2嵌合基因表达蛋白单克隆抗体的制备及其生物学特性的鉴定

目的:制备抗猪瘟病毒(CSFV)C株 E~(rns)/E2嵌合基因表达蛋白的单克隆抗体(mAb),探讨~(rns)/E2蛋白的分子结构和生物学功能.方法:在大肠杆菌 Rosetta~(TM)2(DE_3)中表达了猪瘟病毒(CSFV)C株 E~(rns)、E2蛋白主要抗原域的嵌合基因CSFV E~(rns)/E2蛋白;以纯化复性后的CSFV E~(rns)/E2为免疫原,按常规方法免疫BALB/c小鼠,应用淋巴细胞杂交瘤技术制备抗CSFV E~(rns)/E2 mAb;利用间接ELISA 、Western blot、Dot blot方法鉴定mAb生物学特性.结果:获得了1株稳定分泌抗CSFV Erns/E2融合蛋白的单克隆杂交瘤细胞系C8株,该株mAb的细胞培养上清效价为 1:6 400,小鼠腹水效价 1:2×10~5.其免疫球蛋白亚类为IgG1.间接ELISA和Western blot结果显示该株mAb不与猪瘟C株细胞毒、pET-32a标签蛋白反应,仅与融合表达的 CSFVE~(rns)/E2蛋白存在特异性反应.Dot blot结果表明其识别的抗原表位为E2蛋白的 RSGLCPFDTSPV氨基酸序列.结论:获得了一株能特异识别E2蛋白抗原表位的 mAb,为进一步研究 CSFV 的分子结构及功能奠定了物质基础.     

猪瘟病毒E2糖蛋白抗原表位的预测、多肽合成及特性研究

目的：研究CSFV E2蛋白免疫原性肽的生物学特性。方法：应用计算机分析软件对猪瘟病毒＆糖蛋白的抗原表位进行了预测，结合＆基因的变异分析，人工合成了两条多肽(Pep1和Pep2)序列，与抗mE2蛋白的兔血清和抗mE2的8株单抗进行反应，并将两条多肽分别与载体蛋白(BSA)进行偶联和免疫家兔。结果：两条多肽均与兔的抗血清及单抗A11反应；多肽Pep2与单抗D5和D8反应。多肽Pep1与BSA载体蛋白偶联(Pep1-BSA)，免疫家兔后能够产生针对多肽Pep1的抗体。结论：两条多肽Bep1和Pep2中，只有多肽Pep1具有很好的反应原性和免疫原性。     

丙型肝炎病毒多表位抗原重组体免疫原性分析

目的：研究丙型肝炎病毒(HCV)多表位抗原重组体在大肠杆菌中的非融合表达及其免疫原性分析。方法：利用DNA重组技术将构建的HCV多表位抗原重组体克隆入原核表达载体pBV220并在BL-21中表达，非融合表达蛋白经SDS-PAGE检测，排阻层析纯化，利用ELISA和免疫印迹分析其抗原反应性。免疫昆明鼠和猕猴，并检测其抗体和特异性CTL(Cyto-toxic T lymphocyte)，在抗体转阴后再次用HCV病人血清攻击以检测其免疫应答能力。结果：HCV多表位重组体在pBV220／BL-21中表达量占菌体总蛋白量的15％。Western-blot和ELISA分析表明HCV多表位抗原能与HCV病人血清特异性结合，抗体水平和特异性CTL检测结果显示该抗原肽能诱导小鼠和猕猴产生较好的抗体水平和特异性CTL效应。用病人血清再次攻击后受试动物的抗体迅速阳转。结论：表达的多表位重组体具有特异的抗原反应性和免疫原性。     

抗猪囊尾蚴融合蛋白单表位抗体的抗原识别分析

抗猪囊尾蚴融合蛋白单表位抗体的抗原识别分析王俊霞１彭郁葱１孙树汉２本文采用抗原表位筛选法制备了抗猪囊尾蚴融合蛋白的单表位抗体，并用蛋白质转移技术分析了抗猪囊尾蚴融合蛋白单表位抗体特异性抗原，确定了具特异性的抗原。材料和方法一、猪囊尾蚴抗原取自然感染的...     

钩端螺旋体多抗原肽的构建及免疫性分析

目的 构建问号钩端螺旋体(简称钩体)主要外膜蛋白OmpL1、LipL21和LipL32优势抗原表位的串联基因及其表达系统,了解该重组蛋白的免疫活性.方法 采用噬菌体M13KE表面展示技术结合Western blot分析,鉴定了OmpLl、LipL21和LipL32的优势抗原表位,人工合成优势抗原表位串联基因并构建其原核表达系统.SDS-PAGE检测重组蛋白的表达情况;Western blot及ELISA鉴定重组蛋白的免疫活性.结果 该合成基因在原核表达系统中得到了有效表达,且表达产物主要以可溶性形式存在.Western blot和ELISA结果 显示该重组蛋白能与兔抗钩体全菌抗体及不同血清群的钩体病人血清中的抗体产生免疫反应.结论 本研究成功构建了钩体多表位串联基因及其表达系统,所表达目的 蛋白具有良好的免疫活性,且对不同血清群型抗体之间均有免疫原活性.     

HCV复合多表位抗原基因的克隆表达及其免疫学特性分析

目的构建丙型肝病炎病毒（HCV）截短C基因和多表位基因重组原核表达质粒,表达纯化融合蛋白,分析其免疫原性和抗原性。方法PCR方法扩增核心区羧基端部分缺失的基因片段Ct;合成HCVE2区模拟表位与NS3～NS57个表位基因Em;分别将Ct、Em克隆人原核表达质粒pQE30,筛选阳性重组质粒pQE30-CtEm,转化E．coli M15,IPTG诱导融合蛋白表达,薄层扫描分析表达蛋白;可溶性分析后用Ni^2＋-NTA凝胶亲和层析柱纯化、透析并浓缩融合蛋白;Westernblot分析纯化蛋白的特异性和抗原性;纯蛋白免疫小鼠后分析其免疫原性。结果成功构建了HCV复合多表位抗原基因的原核表达质粒pQE30-CtEm,目的基因可高效表达,表达产物主要以包涵体形式存在,Ni^2＋-NTA纯化可获得目的蛋白,纯化蛋白具有良好的抗原性和免疫原性。结论HCV复合多表位抗原基因融合蛋白可高效表达并得到纯化,该融合蛋白可作为HCV诊断抗原,也为丙型肝炎新型疫苗的研究提供了靶抗原。     

SARS冠状病毒膜蛋白膜内区的表达、纯化及抗原性初步研究

目的对SAILS冠状病毒膜蛋白膜内区基因片段进行克隆表达和表达产物的纯化复性,探讨该表达蛋白的抗原特性。方法克隆SAILS冠状病毒GD322株膜蛋白膜内区,在原核表达系统中表达His-融合蛋白,采用Western blot和酶联免疫吸附试验(ELISA)分析His-融合蛋白抗原特性。结果7份SARS临床诊断患者血清中5份血清能与His-融合蛋白反应,2份不与之反应。兔抗OCA3、229E血清及20份健康人血清皆不与His-融合蛋白反应。His-融合蛋白免疫动物可产生多克隆抗体。结论本研究获得的His-融合蛋白可与SARS临床诊断患者血清特异性结合,可免疫动物获得特异性多抗;但不与健康人血清及兔抗OCA3、229E血清发生特异性结合。     

汉滩病毒M片段噬菌体表位文库的构建及筛选

目的 通过构建汉滩病毒M片段的噬菌体展示文库，筛选汉滩病毒包膜蛋白的抗原表位。方法 DNaseⅠ随机消化的M片段与载体pComb3连接，转化宿主XL-1Blue，在辅助噬菌体VCSM13存在的条件下，获得M片段的随机噬菌体文库.利用纯化的恢复期病人血清对M片段噬菌体文库进行3轮淘洗，通过ELISA、序列分析对所获得的克隆进行鉴定。并对其中2个阳性克隆的目的片段进行了原核表达和免疫原性分析。结果 筛选获得的5个阳性噬菌体克隆均位于G1蛋白编码区，原核表达的2个阳性：充隆片段蛋白免疫兔，免疫兔血清能与病毒抗原发生特异反应。结论 本技术路线可用于汉滩病毒包膜蛋白抗原表位的研究，为汉滩病毒诊断试剂的研制、亚单位疫苗的设计提供了数据。     

SARS-CoV S蛋白表位的表达及鉴定

目的:制备SARS冠状病毒S蛋白串联表位重组蛋白,为SARS的防治提供新型抗原蛋白。方法:应用抗原表位分析软件分析S蛋白的表位。选取其中16个表位,设计并合成表位串联重组蛋白编码基因Z,构建其原核细胞表达重组体,在大肠杆菌BL21(DE3)表达该重组表位蛋白Z,应用Ni离子亲合层析法纯化重组蛋白Z做抗原,采用皮下注射法免疫新西兰白兔。斑点杂交法测定抗Z-血清中S蛋白的抗体,ELISA法检测Z蛋白的抗原性。结果:构建了S蛋白重组表位蛋白Z的原核表达体,在BL21菌中表达了Z蛋白,Z蛋白免疫新西兰白兔后获得了抗Z血清。斑点杂交分析显示,抗Z血清识别哺乳动物细胞中表达的S蛋白。ELISA检测结果显示,抗Z血清识别SARS冠状病毒抗原。结论:建立了制备SARS冠状病毒S蛋白重组表位蛋白的方法,为防治SARS提供了新型抗原蛋白Z。     

The protective immune response induced by B cell epitope of classical swine fever virus glycoprotein E2

Classical swine fever virus (CSFV) envelope glycoprotein E2 is a major protective immunogen responsible for eliciting neutralizing antibodies and conferring protective immunity against the virus. Based on the core sequence (TAVSPTTLR, 829-837 aa) of the B cell linear epitope of the CSFV E2 protein identified by Lin et al., two oligonucleotides MF and MR were synthesized and used to construct by PCR a gene cassette encoding a 15 amino acid polypeptide M (CTAVSPTTLRTEVVK), which spans 828-842 amino acids of E2. The gene cassette was fused in-frame to 3' terminal of glutathione S transferase gene (GST) of the prokaryotic expression vector pGEX-6p-1, resulting in the recombinant plasmid pGEX-M. After transformation into Escherichia coli BL21 a soluble fusion protein GST-M with expected size of 28 kDa was expressed after inducing with isopropyl-beta-d-thiogalactoside (IPTG). Enzyme-linked immunosorbent assay (ELISA) and Western blot analysis showed that the purified GST-M had good reactivity with swine anti-CSFV serum and rabbit anti-CSFV E2 serum. Further vaccination trials showed that the fusion protein GST-M could elicit effectively immune response protecting rabbits and pigs from virulent challenge. This study showed a possibility for developing epitope-based vaccines against CSFV.     

Effects of glycosylation on antigenicity and immunogenicity of classical swine fever virus envelope proteins

Classical swine fever virus (CSFV) harbors three envelope glycoproteins (E^rns, E1 and E2). Previous studies have demonstrated that removal of specific glycosylation sites within these proteins yielded attenuated and immunogenic CSFV mutants. Here we analyzed the effects of lack of glycosylation of baculovirus-expressed E^rns, E1, and E2 proteins on immunogenicity. Interestingly, E^rns, E1, and E2 proteins lacking proper post-translational modifications, most noticeable lack of glycosylation, failed to induce a detectable virus neutralizing antibody (NA) response and protection against CSFV. Similarly, no NA or protection was observed in pigs immunized with E1 glycoprotein. Analysis of E^rns and E2 proteins with single site glycosylation mutations revealed that detectable antibody responses, but not protection against lethal CSFV challenge is affected by removal of specific glycosylation sites. In addition, it was observed that single administration of purified E^rns glycoprotein induced an effective protection against CSFV infection.     

Baculovirus surface display of E(rns) envelope glycoprotein of classical swine fever virus

Classical swine fever virus (CSFV) causes significant losses in the pig industry in many countries. E(rns) is an envelope glycoprotein of CSFV which is known to induce virus-neutralizing antibodies and protective immunity in the natural host. In this study, one recombinant baculoviruses BacSC-E(rns) expressing histidine-tagged E(rns) with the transmembrane domain (TM) and cytoplasmic domain (CTD) derived from baculovirus envelope protein gp64 was constructed and its immunizing efficacy was evaluated in a mouse model. After infection, E(rns) was expressed and anchored on the plasma membrane of Sf-9 cells, as demonstrated by Western-blot and confocal microscopy. Immunogold electron microscopy demonstrated that the E(rns) glycoprotein was successfully displayed on the baculoviral envelope. Vaccine tests in animals showed that BacSC-E(rns) elicited significantly higher E(rns) antibody titers in the immunized mouse models than the control group. This demonstrates that the BacSC-E(rns) vaccine can be used potentially against CSFV infections. This is the first report demonstrating the potential of E(rns)-pseudotyped baculovirus as a CSFV vaccine.     

人tau蛋白外显子2和外显子3特异性抗体的制备

目的 制备人微管相关蛋白tau N端的外显子2和外显子3特异性多克隆抗体.方法 从人外周血DNA中获得tau蛋白外显子2及外显子3序列并连接至原核表达载体pGEX-2T,表达纯化重组融合蛋白GST-E2、GST-E3;以此融合蛋白免疫家兔及小鼠制备抗血清,通过ProteinG/A、偶联GST蛋白的溴化氰(CNBr)活化柱对抗血清进行亲和纯化;用Western Blot及ELISA方法确定所制备抗体的特异性和敏感性.结果 在大肠埃希菌中表达纯化出人tau外显子2和外显子3重组融合蛋白GST-E2和GST-E3,相对分子质量为29×103.免疫实验动物后获得抗血清,经系列纯化后Western Blot和ELISA检测显示,制备的抗人tau蛋白外显子2和外显子3抗体具有良好的特异性和免疫反应效价.结论 成功制备了4种人tau外显子2和外显子3特异性抗体,为进行tau蛋白在神经退行性疾病中的作用研究提供了基础.     

Comparison of the protective efficacy of recombinant adenoviruses against classical swine fever

Classical swine fever (CSF), which is caused by classical swine fever virus (CSFV), is a highly contagious and often fatal swine disease that is responsible for significant losses to the swine industry worldwide. Previously, we demonstrated that pigs immunized with a recombinant adenovirus (rAdV-E2) expressing the E2 glycoprotein of CSFV were protected against virulent CSFV; however, a few pigs showed a short-term fever and occasional pathological changes. To enhance the efficacy of the vaccine, we constructed two recombinant adenoviruses, namely, rAdV-E2UL49, which encodes the CSFV E2 gene fused with the UL49 gene from pseudorabies virus (PRV), and rAdV-optiE2, which expresses the codon-optimized CSFV E2 gene. With these viruses, we performed a comparative immunogenicity trial in rabbits and pigs and compared these recombinant adenovirus vaccines (rAdV-E2UL49 and rAdV-optiE2) with the one containing the wild-type E2 gene (rAdV-E2). In terms of antibody titers, IFN-γ production, lymphocyte proliferation, viral loads and clinical protection from the disease, rAdV-E2UL49 was more immunogenic and protective against C-strain CSFV in rabbits and Shimen strain CSFV in pigs than rAdV-optiE2 and rAdV-E2. Data from this study could assist in making decisions for further development of recombinant adenoviruses as vaccine candidates against CSF.     

Protection of pigs from lethal challenge by a DNA vaccine based on an alphavirus replicon expressing the E2 glycoprotein of classical swine fever virus

In a previous study, it has been shown that a Semliki Forest virus (SFV) replicon vectored DNA vaccine (pSFV1CS-E2) expressing the E2 glycoprotein of classical swine fever virus (CSFV) conferred full protection for pigs immunized three times with 600 microg of the vaccine. This study was designed to evaluate further the efficacy of the vaccine with lower dosage and fewer inoculations. Pigs were immunized twice with 100 microg of pSFV1CS-E2 (n=5) or control plasmid pSFV1CS (n=3), respectively, and challenged with virulent Shimen strain 6 weeks following the booster immunization. Pigs immunized with pSFV1CS-E2 developed high titers of specific neutralizing antibodies against CSFV after the booster, and the antibody titers increased rapidly upon challenge. The immunized animals showed no clinical symptoms except short-term fever and low-level viremia, whereas, the control pigs immunized with the control plasmid produced no detectable antibody prior to challenge, and showed obvious clinical signs following challenge, and two pigs died of illness. All control animals developed extended viremia as detected by nested RT-PCR and real-time RT-PCR. Severe pathologic lesions typical of CSFV infection were observed at necropsy. It is concluded that the alphavirus replicon-vectored DNA-based vaccine can be a potential marker vaccine against CSFV.     

Chimeric classical swine fever (CSF)-Japanese encephalitis (JE) viral replicon as a non-transmissible vaccine candidate against CSF and JE infections

A trans-complemented chimeric CSF-JE virus replicon was constructed using an infectious cDNA clone of the CSF virus (CSFV) Alfort/187 strain. The CSFV E2 gene was deleted, and a fragment containing the region encoding a truncated envelope protein (tE, amino acid 292-402, domain III) of JE virus (JEV) was inserted into the resultant plasmid, pA187delE2, to generate the recombinant cDNA clone pA187delE2/JEV-tE. Porcine kidney 15 (PK15) cells that constitutively express the CSFV E2p7 proteins were then transfected with in vitro-transcribed RNA from pA187delE2/JEV-tE. As a result, the chimeric CSF-JE virus replicon particle (VRP), rv187delE2/JEV-tE, was rescued. In a mouse model, immunization with the chimeric CSF-JE VRP induced strong production of JEV-specific antibody and conferred protection against a lethal JEV challenge. Pigs immunized with CSF-JE VRP displayed strong anti-CSFV and anti-JEV antibody responses and protection against CSFV and JEV challenge infections. Our evidence suggests that E2-complemented CSF-JE VRP not only has potential as a live-attenuated non-transmissible vaccine candidate against CSF and JE but also serves as a potential DIVA (Differentiating Infected from Vaccinated Animals) vaccine for CSF in pigs. Together, our data suggest that the non-transmissible chimeric VRP expressing foreign antigenic proteins may represent a promising strategy for bivalent DIVA vaccine design.     

Escherichia coli-derived envelope protein gD but not gC antigens of herpes simplex virus protect mice against a lethal challenge with HSV-1 and HSV-2

Immunization studies with HSV-1 and HSV-2 envelope proteins expressed in Escherichia coli were performed. After active immunization of mice with a gD-1 antigen (Leu₅₃-Ala₃₁₂) expressed as a fusion protein, the animals were protected from a lethal challenge with HSV-1 and HSV-2. In addition, antisera from rabbits immunized with the same gD-1 antigen also conferred passive immunity to mice against a challenge infection with either HSV-1 or HSV-2. In contrast to these successful gD-1 protection experiments, various gC-1 and gC-2 fusion proteins from E. coli failed to induce protective immunity. Moreover, the mice sera from immunized animals were not able to react with the authentic, glycosylated gC-1 and gC-2 envelope proteins, whereas sera raised against authentic gC-1 and gC-2 glycoproteins do recognize the gC fusion proteins from E. coli. These results indicate, that E. coli might represent an ideal system for expressing gD antigens as a possible component of a HSV vaccine, whereas gC antigen cannot be produced in an immunocompetent form in E. coli.     

Multiepitope vaccines intensively increased levels of antibodies recognizing three neutralizing epitopes on human immunodeficiency virus-1 envelope protein

A few neutralizing antibodies against human immunodeficiency virus-1 (HIV-1) envelope proteins have been shown to be highly effective at neutralizing different strains in vitro, and exist at very low levels in the sera of HIV-1-infected individuals. Based on our hypothesis that epitope vaccination may be a novel strategy for inducing high levels of antibodies against HIV-1, we prepared multiepitope vaccines using three neutralizing epitopes (GPGRAFY, ELDKWA and RILAVERYLKD) on HIV-1 envelope proteins. The PI [C-G-(ELDKWA-GPGRAFY)2-K] and PII (CG-GPGRAFY-G-ELDKWA-G-RILAVERYLKD) peptides were synthesized and conjugated to a carrier protein, bovine serum albumin (BSA). After vaccination, both the PI-BSA and PII-BSA multiepitope vaccines induced high levels of epitope-specific antibodies to the three neutralizing epitopes (antibody titre: 1 : 12,800-102,400). The recombinant glycoprotein 160 (rgp160) subunit vaccine induced strong antibody responses to rgp160, but only very weak epitope-specific antibody responses to the three epitopes. The epitope-specific antibodies were isolated from rabbit sera by single epitope-peptide-conjugated sepharose columns. A yield of 51 microg of epitope-specific antibodies/ml of serum (mean value) was obtained and identified to recognize these epitopes, while 0.35 microg of protein was isolated from 1 ml of pooled preserum by C-(ELDKWAG)4- or C-(RILAVERYLKD-G)2-K- and C-(GPGRAFY)4-sepharose columns. The levels of these epitope-specific antibodies induced in rabbits were much greater than 1 microg/ml, a level that is considered to confer long-term protection against some viruses. Moreover, these antibodies recognized the neutralizing epitopes on peptides and rgp41. Based on the fact that a very low level of ELDKWA epitope-specific antibodies exist in HIV-1-infected individuals, these results suggesting that synthetic epitope vaccines could induce high levels of multiepitope-specific neutralizing antibodies indicate a new strategy for developing an effective neutralizing antibody-based epitope/peptide vaccine against HIV-1.