流行性乙型脑炎病毒

摘要： 目的表达流行性乙型脑炎病毒(EEBV)的囊膜蛋白(E蛋白),制备该蛋白特异性兔抗血清,为建立诊断EEBV感染的检测方法奠定基础。方法将pET32a-E质粒转化大肠杆菌BL21(DE3)细胞,以终浓度为0.5 mmol·L^(-1)的异丙基-β-D-硫代半乳糖苷于37°C下诱导蛋白表达8 h,在8 mol·L^(-1)尿素变性条件下,采用镍离子金属螯合亲和层析法对重组E蛋白进行纯化。透析复性后,以E蛋白为免疫原免疫新西兰兔制备兔抗血清,采用Western blot、酶联免疫吸附试验和免疫荧光分析法检测免疫血清中EEBV特异性抗体。结果成功表达了重组E蛋白,经纯化、复性后可从1 L培养物中获得23.8 mg具有生物活性的E蛋白,该蛋白能与抗EEBV单抗发生特异反应。免疫制备的兔抗血清能与E蛋白发生特异反应,抗体效价高达1×51 200;兔抗血清能与EEBV感染的细胞发生特异反应。结论成功表达了EEBV E蛋白并制备了特异性强、亲和力高的兔抗血清,为建立诊断EEBV感染的免疫学检测方法、有效防控流行性乙型脑炎奠定了基础。

摘要： 为了解流行性乙型脑炎病毒基因I型毒株SD12在感染C57BL/6小鼠体内的病毒分布,应用qRT-PCR方法检测该毒株在小鼠体内各组织的动态分布情况。结果显示腹腔感染组中2 dpi仅能在心脏和脾脏检测出病毒核酸,5~7 dpi能够在心脏、肝脏、脑部、盲肠和扁桃体检测到病毒核酸。血脑屏障受损的腹腔感染组中最早2 dpi在肠系膜淋巴结中检测到病毒核酸,3 dpi基本能在各组织中检测到病毒核酸,5~7 dpi在大脑中检测到大量病毒核酸。所有对照组没有检测到病毒阳性核酸。本研究表明基因I型流行性乙型脑炎病毒感染血脑屏障受损的C57BL/6小鼠后能迅速入侵各组织脏器,主要浸染肝脏、肺脏、肾脏、盲肠和大脑。拥有完整血脑屏障的小鼠对病毒具有一定抵抗能力,病毒在各组织中的含量相对比较低。

摘要： 为了研究不同激活状态的巨噬细胞对流行性乙型脑炎病毒(Japanese encephalitis virus,JEV)感染的影响,并明确NO的产生在此过程中的作用,利用细胞因子将小鼠巨噬细胞系RAW264.7诱导成不同激活状态的巨噬细细胞,通过TCID50、免疫荧光技术和实时荧光定量PCR方法比较JEV强毒株NJ2008在不同激活状态的巨噬细胞上的感染情况,并利用实时荧光定量PCR方法分析了调控NO产生的关键分子(iNOS和Agr1)的变化.结果显示,相对于PBS处理组,M1型巨噬细胞显著地抑制JEV的感染并伴随明显增加的iNOS/Arg1比值;而M2型巨噬细胞虽伴随有显著降低的iNOS/Arg1比值,但JEV在M2型巨噬细胞上的感染与PBS组差异无显著统计学意义.结果表明NO的上调表达在M1型巨噬细胞抑制JEV感染中起着主导作用.本研究为进一步揭示巨噬细胞在JEV感染中作用奠定基础.

摘要： 目的 调查云南省保山市蚊虫及其自然感染虫媒病毒状况,为蚊媒病毒病防控提供参考依据.方法2015年9月采用诱蚊灯法在保山市隆阳区农村人房和畜圈采集蚊虫标本,反转录聚合酶链反应(RT-PCR)用于检测蚊虫标本中黄病毒属、甲病毒属、布尼亚病毒属和流行性乙型脑炎病毒(乙脑病毒)等相关病毒核酸,阳性者进行C/prM基因序列测定、同源性和系统进化分析.结果 共捕获蚊虫3种8 202只,三带喙库蚊(Culex tritaeniorhynchus)、中华按蚊(Anopheles sinensis)和骚扰阿蚊(Armigeres subalbatus)构成比依次为88.50％(7 259只)、11.43％(937只)和0.07％(6只).蚊虫标本分30批进行RT-PCR检测,14批三带喙库蚊中检测到乙脑病毒核酸,并获得14株乙脑病毒C/prM基因核苷酸序列.同源性和进化分析表明,14株乙脑病毒与基因I型乙脑病毒同在一个进化分支,并与2007年保山市腾冲县和2010年德宏傣族景颇族自治州(芒市和瑞丽市)的基因I型乙脑病毒具有较近的进化关系.结论 三带喙库蚊为保山市的优势蚊种和乙脑病毒主要传播媒介,当地存在基因I型乙脑病毒流行,此为近10年保山市首次检测到基因I型乙脑病毒.%Objective To investigate the distribution of mosquitoes and their natural infection with mosquito-borne viruses in Baoshan of Yunnan province,and provide scientific evidence for the prevention and control of mosquito-borne diseases. Methods Mosquito samples were collected by using mosquito traps in the night from houses and livestock stables in villages in Baoshan in September 2015. The mosquito samples were used to detect the viral RNA with Reverse transcription-polymerase chain reaction (RT-PCR) using Flavivirus, Alphavirus, Bunyavirus and Japanese encephalitis virus (JEV) primers, and positive specimens were sequenced for C/prM gene for homology and phylogenetic analysis. Results A total of 8 202 mosquitoes in 3 species were collected in Longyang district of Baoshan. Culex tritaeriorhynchus, Anopheles sinensis and Armigeres subalbatus accounted for 88.50％ (7 259),11.43％ (937) and 0.07％ (6) of the total,respectively. Thirty mosquito batches were detected by using RT-PCR, and JEV RNA were detected from 14 batches of Cx. trtaeriorhynchus. The sequences of C/prM genes of the 14 JEV strains were obtained. Homology and phylogenetic analysis showed that the 14 JEV strains were in the same clade of genotype I (G-I ) strains of JEV from GenBank, indicating they belonged to G-I, and all the JEV strains detected were closely related to the JEV G-I strains from Tengchong county of Baoshan in 2007 and adjacent Mangshi and Ruili counties of Dehong in 2010. Conclusion Cx. tritaeriorhynchus was dominant mosquito species and the major transmitting vector of JEV in Baoshan. JEV G-I was confirmed to circulate in the area. It was first time to detect JEV G-I in Baoshan since 2007.

摘要： 目的 了解四川省的蚊、蠓以及相关虫媒病毒种类及分布.方法 在自然界使用灯诱法采集吸血昆虫标本,形态学分类后分装,液氮保存备用.蚊虫、蠓虫研磨上清液接种BHK-21细胞和C6/36细胞进行病毒分离和病毒基因检测.使用BioEdit 7.0.5.3、MEGA 6.0等软件对病毒序列进行分子生物学特征分析.结果 2016-2017年在四川省东南部3个县的7个自然村共采集到3属4种蚊虫17 019只,蠓虫12 700只.三带喙库蚊占蚊虫采集总数的79.4％(13 519/17 019),其次为骚扰阿蚊(11.1％,1 897/17 019),致倦库蚊占采集蚊虫标本总数的5.5％(930/17 019);中华按蚊最少(4.0％,673/17 019).蚊虫标本接种组织细胞后,在合江县采集的三带喙库蚊标本获得3株可以稳定传代的病毒分离株,经分子生物学实验鉴定为基因Ⅰ型流行性乙型脑炎(乙脑)病毒,在合江县采集的7批蚊虫中检测到乙脑病毒基因阳性.蠓虫标本接种细胞后未获得病毒分离物,在合江县采集的4批蠓虫标本经分子生物学检测,显示阿卡斑病毒基因阳性.结论 三带喙库蚊是四川省东南部3县的优势蚊种,四川省东南部存在经蚊虫传播的乙脑病毒及以蠓虫为媒介的阿卡斑病毒.%Objective To investigate the distribution patterns of mosquitoes,midges and related arboviruses in Sichuan province.Methods Blood-sucking insects were collected from houses and pens,using the ultraviolet lights.Mosquito samples were classified according to morphologic characteristics and then stored at liquid nitrogen.All samples were incubated with BHK-21 and C6/36 cells for virus isolation and then detected for their viral genes.Sequences of the virus were identified and analyzed by molecular biological software,such as BioEdit 7.0.5.3,MEGA 6.0.Results In total,17 019 mosquitoes from 3 genera and 4 species and 12 700 midges were collected from the southeast regions of Sichuan province in 2016 and 2017.Among them,79.4％ (13 519/17 019) belonged to Culex tritaeniorhynchus with 11.1％ (1 897/17 019) as Armigeres subalbatus,5.5％ (930/17 019) were Anopheles sinensis and 4.0％ (673/17 019) were Anopheles sinensis 3 virus strains that isolated from Culex tritaeniorhynchus were identified as type Ⅰ Japanese encephalitis virus.Seven pools of mosquitoes isolated from Hejiang county were identified Japanese encephalitis virus gene positive through PCR amplification.With 4 pool midges were detected positive for Akabane virus through PCR gene amplification while midges samples didn't have virus isolates.Conclusions Culex tritaeniorhynchus appeared the predominant species in the southeast regions of Sichuan.Japanese encephalitis virus transmitted by mosquitoes and Akabane virus by midges were prevalent in southeast Sichuan province.

摘要： Chinese scientists have isolated hundreds of Japanese encephalitis virus (JEV) strains from nature and studied their virulence in mice since 1949.They found that the JEV isolates in nature had apparently different virulence among them,especially the pheripheral virulence.This difference was caused by the effects of virus living circumstance such as the mosquito vectors,hosts and natural climate etc.Further studies demonstrated that the virulence of JEV could be reduced in laboratory by serial passages of the virus in mouse brain or different cell cultures especially in hamster kidney cells and the pheripheral virulence was reduced more rapidly.The technique of virus plaque purification was able to differentiate the higher and lower virulent viruses and can be used for selection of avirulent virus strain.By using different ways several attenuated virus strains have been selected and used as live vaccine for vaccination in humans,pigs and horses.Especially,they found that in vivo passage of the viruses in non-neural tissues of laboratory animals could further reduce residual virulence and promote their immunogenicity.By using this creative technology,a highly attenuated,immunogenic and genetically stable attenuated strain SA14-14-2 virus was selected.The attenuated live vaccine made by SA14-14-2 strain has been used worldwild since its licensure in 1989.And in 2013,the vaccine made by Chengdu Biological Institute obtained WHO prequalification.%1949年以来中国学者在自然界分离到数百株乙型脑炎(乙脑)病毒株,并对这些病毒株进行神经毒力的研究,发现毒株间存在明显的毒力差异,特别是神经外毒力,差异的原因与自然界环境如媒介、宿主和气候等有关.乙脑的显性和隐性感染则与病毒株的毒力强弱密切相关.对乙脑病毒的变异进行研究,发现病毒可以通过小鼠脑内或不同的细胞传代而毒力逐步减弱,特别是神经外毒力降低更快,并且可以通过空斑把强弱病毒分离从而挑选到低毒力的弱毒株.通过不同减毒试验,获得多株不同毒力的弱毒株用于人体、猪和马的免疫.特别是其中发现病毒通过实验动物体内的非神经组织传代,能够进一步降低病毒的残余毒力并提高其免疫性,应用该创新性技术成功筛选到1株高度减毒而稳定并保持高免疫性的SA14-14-2株,用该株病毒生产的活疫苗,自1989年取得生产许可证以来已在国内外广泛应用,证实其安全高效达到国际领先水平,并于2013年取得WHO的预认证.

摘要： 目的 探讨柴石退热颗粒对流行性乙型脑炎病毒CQ11-66的影响.方法 选取2015年5月～2016年12月在河南省中医院住院的流行性乙型脑炎患者150例,按随机数字表法将其分为两组,各75例.对照组行常规综合治疗,观察组在对照组基础上加用柴石退热颗粒.两组均治疗15d.治疗后观察两组临床疗效以及症状改善时间,比较两组患者住院时间以及治疗前后血清中神经元特异性烯醇化酶(NSE)、过氧化脂质(LPO)、白细胞介素-2(IL-2)含量.结果 观察组痊愈率明显高于对照组,差异有统计学意义(P＜0.05),观察组总有效率虽高于对照组,但差异无统计学意义(P＞0.05).观察组体温恢复正常时间、神智恢复正常时间及头痛、呕吐、抽搐缓解时间均明显短于对照组,差异均有统计学意义(P＜0.05).观察组平均住院时间明显短于对照组,差异有统计学意义(P＜0.05).治疗后,两组患者血清中NSE、LPO含量均明显低于治疗前,IL-2含量明显高于治疗前,差异均有统计学意义(P＜0.05).但两组治疗后血清中NSE、LPO、IL-2含量比较差异均无统计学意义(P＞0.05).结论 柴石退热颗粒对流行性乙型脑炎病毒CQ11-66有明显的治疗效果,能够明显缩短患者住院时间,加快症状改善,有效减轻患者痛苦,值得广泛推广.%Objective To investigate the effects of Chaishi Tuire Granules on epidemic encephalitis B virus CQ11-66.Methods One hundred and fifty patients with epidemic encephalitis B hospitalized in He'nan Traditional Chinese Medicine Hospital from May 2015 to December 2016 were selected and divided into two groups by random number table method,with 75 cases in each group.The control group was treated with routine comprehensive treatment,on basis of control group,the observation group was added with Chaishi Tuire Granules.Both groups were treated for 15 days.After treatment,the clinical efficacy and symptom improvement time of the two groups were observed,the hospital stay and the contents of serum neuron-specific enolase (NSE),lipid peroxidase (LPO),interleukin-2 (IL-2) before and after treatment between the two groups were compared.Results The cure rate in the observation group was significantly higher than that in the control group,the difference was statistically significant (P ＜ 0.05),the total effective rate in the observation group was higher than that in the control group,but the difference was not statistically significant (P ＞ 0.05).The time of body temperature returned to normal,time of intelligence returned to normal,and the relief time of headache,vomiting,convulsions in the observation group was all shorter than those of control group,the differences were all statistically significant (P ＜ 0.05).The average hospital stay of observation group was shorter than that of control group,the difference was statistically significant (P ＜ 0.05).After treatment,the contents of serum NSE,LPO in the two groups were all lower than those before treatment,the contents of IL-2 were higher than those before treatment,the differences were all statistically significant (P ＜ 0.05).But there were no statistically significant differences of the con tents of serum NSE,LPO,IL-2 after treatment between the two groups (P ＞ 0.05).Conclusion Chaishi Tuire Granules has obvious therapeutic effect for epidemic encephalitis B virus CQ11-66,which can obviously shorten hospital stay,speed up the improvement of symptoms,relieve the pain of patients effectively,it is worthy of extensive promotion.

摘要： 为构建流行性乙型脑炎病毒(JEV)E蛋白多表位疫苗,本研究将人工合成的优化后的E蛋白多表位肽(MP)序列克隆入酵母表达载体pPICZα-A,构建分泌型重组酵母表达质粒pPICZ-MP,经Sac Ⅰ酶切线性化后电转化导入毕赤酵母X-33,阳性重组子经甲醇诱导表达,亲和层析纯化重组蛋白(rMP).通过测定rMP免疫小鼠的抗体水平、抗体亚型、中和抗体和CTL,以评价该多表位疫苗在小鼠模型上的免疫原性.结果表明:成功构建了表达JEV E蛋白多表位抗原的重组酵母菌株X33 (pPICZ-MP),能分泌表达多表位肽rMP.纯化后的rMP免疫小鼠产生了较强的体液免疫和细胞免疫反应,其中和抗体水平、CTL活性均与活病毒疫苗组差异不显著(p＞0.05),且能保护小鼠免受致死量JEV的攻击.构建的JEV E蛋白多表位疫苗具有良好的免疫原性,能够刺激小鼠产生特异性的细胞免疫和体液免疫应答反应,为流行性乙型脑炎多表位疫苗研制提供新的思路.%This study aimed to construct a multi-epitope vaccine of Japanese encephalitis virus (JEV) envelope protein and evaluate its immunogenicity and protective efficacy in mice model.In this study,A codon optimized multiple-epitope peptide gene (MP) from E protein of JEV was cloned into the plasmid pPICZα-A to construct the recombinant expression vector pPICZ-MP.The resulting plasmid linearized with Sac Ⅰ was transformed into Pichiapastoris X-33 by electroporation.The positive clones were selected by Zeocin plates and confirmed by PCR.The supernatant from the shake flask culture was collected and purified with Ni-NTP column.The mice immunized with the recombinant multi-epitope peptide (rMP),and the level of antibody titers,antibody isotypes,neutralizing antibody and CTL were detection to evaluate the immunogenicity and protective efficacy in mice model.The results showed that rMP was highly immunogenic and was able to elicit high titer neutralizing antibodies and cell-mediated immune responses.Our findings indicated that the multi-epitope vaccine rMP might be an attractive candidate vaccine for the prevention of JEV infection.A multi-epitope vaccine of JEV was constructed successfully.Animal experiments showed that immunization with rMP was able to induced high humoral and cellular immune responses,and protect against lethal JEV challenge in mice.The neutralizing antibody level and CTL of mice immunized with the protein showed no significant difference with live JEV vaccine (p＞0.05).The results indicated that the rMP could induce strong immune responses,suggesting that it might be an attractive candidate vaccine for preventing JEV infection.

摘要： This study aimed to construct a multi-epitope vaccine of Japanese encephalitis virus (JEV) envelope protein and evaluate its immunogenicity and protective efficacy in mice model.In this study,A codon optimized multiple-epitope peptide gene (MP) from E protein of JEV was cloned into the plasmid pPICZα-A to construct the recombinant expression plasmid pPICZ-MP,which was linearized with Sac Ⅰ and transformed into Pichia pastoris X-33 by electroporation for expression.Then the recombinant multi-epitope peptide (rMP) were purified with Ni-NTP column from the recombinant P.pastoris X-33 culture medium.Mice were immunized with the rMP,and the immuno-responses were detected to evaluate the immunogenicity and protective efficacy of rMP in mice model.Animal experiments shown that the mice immunized with rMP had high humoral and cellular immune responses,and were protected against lethal JEV challenge.The neutralizing antibody level and CTL of mice immunized with the protein showed no significant difference with live JEV vaccine.The results indicated that the rMP was able to induce strong immune responses,suggesting that the rMP might be an promising subunite candidate vaccine for preventing JEV infection.%为构建流行性乙型脑炎病毒(JEV)E蛋白多表位疫苗,本研究将人工合成的优化后E蛋白多表位肽(MP)序列克隆于酵母表达载体pPICZα-A中,构建分泌型重组酵母表达质粒pPICZ-MP后电转化导于毕赤酵母X-33中,重组酵母菌组子经甲醇诱导表达重组蛋白(rMP).将其免疫小鼠后,通过测定rMP免疫小鼠的抗体水平、抗体亚型、中和抗体和CTL,以评价该多表位疫苗在小鼠模型的免疫原性.结果显示正确构建了表达JEV E蛋白多表位抗原的重组酵母菌株X33 (pPICZ-MP),并分泌表达多表位肽rMP;纯化后的rMP免疫小鼠产生了较强的体液免疫和细胞免疫反应,其中和抗体水平、CTL活性均与活病毒疫苗组无显著差异(p＞0.05),而且能保护小鼠免受致死量JEV的攻击.上述结果表明本研究以酵母菌表达的JEV E蛋白多表位疫苗具有良好的的免疫原性,能够刺激小鼠产生特异性的细胞免疫和体液免疫应答反应,为流行性乙型脑炎多表位疫苗研制提供了新的思路.

摘要： 通过分析GenBank数据库中25株不同流行性乙型脑炎病毒基因组序列，设计了3对特异性引物，组合成LAMP扩增引物组。对反应体系进行优化后建立了一步法反转录环介导等温扩增(RT-LAMP)检测方法。该检测方法检测时问短，从处理样品至结果判定可在2 h内完成。灵敏度比常规RT-PCR灵敏度高，可检测出103 PFU/0.2 ml乙型脑炎病毒。检测方法特异性强，对猪细小病毒、猪圆环病毒、伪狂犬病毒、猪瘟病毒等常见猪病病毒均无交叉反应。rn 结果判定不需要电泳检查，在扩增产物中加入SYBR Green I染料，可直接在可见光下观察或在紫外光下观察颜色变化。该方法简便快捷，省时省力，是一种适用于基层实验室快速准确诊断流行性乙型脑炎病毒的检测方法。

摘要： 目的：了解大连口岸蚊虫种类、生态习性及淡色库蚊、三带喙库蚊自然感染流行性乙型脑炎病毒情况。rn 方法：用帐诱法、灯诱法、采集幼虫等方法进行调查,并应用逆转录-聚合酶联反应技术(RT-PCR)进行乙脑病毒鉴定。rn 结果：大连口岸捕获蚊虫3属9种,其中按蚊属1种,伊蚊属4种,库蚊属4种.大连口岸蚊虫于5月出现,8月达到高峰,9月逐渐减少,10月消失。侵袭人类以淡色库蚊(占47.98%)、东乡伊蚊(占12.15%)和背点伊蚊(占10.41%)为多。不同场所蚊虫密度差异反映出蚊虫的不同嗜血习性,淡色库蚊、凶小库蚊和白纹伊蚊喜嗜人血;淡色库蚊同时喜嗜猪、牛血;三带喙库蚊偏嗜猪血;中华按蚊偏嗜牛血;背点伊蚊和刺扰伊蚊偏嗜猪、牛血。2007年8月中旬首先在三带喙库蚊中检出乙脑病毒,共检测70批次样本,阳性率为4.28%。rn 结论：淡色库蚊、东乡伊蚊、背点伊蚊是该地区危害人类的主要蚊种。大连口岸蚊虫自然感染乙脑病毒于8月中下旬开始,三带喙库蚊首先染毒.本调查为蚊虫防制提供了生态学依据,为预报流行性乙型脑炎的发生时间和流行强度提供了科学依据。

摘要： 为了解云南省流行性乙型脑炎(乙脑)病人感染乙脑病毒(JEV)基因型状况,在云南省采集脑炎病人急性期血清标本和脑脊液标本,用RT-PCR检测JEV核酸,阳性者进行JEV-PreM/C区基因序列测定,用核苷酸同源性和系统进化分析进行基因型鉴定.从11例脑炎病人脑脊液标本中检测到JEV核酸阳性3份(JB114、JB135和DLN24),而20例脑炎病人血清标本均为阴性.这3株病毒的PreM/C区核苷酸序列同源性分析显示,JB114、JB135与JEV基因Ⅰ型代表株同源性高达98.3％和99.2％,而与Ⅱ、Ⅲ、Ⅳ型仅为82.5％～84.6％;DLN24与基因Ⅲ型代表株同源性为94.6％,而与基因Ⅰ、Ⅱ、Ⅳ型分别为 85.0％、82.5％和80.4％.进化树分析显示,JB114和JB135位于基因Ⅰ型分支中,DLN24位于基因Ⅲ型分支上.结果表明,来自西双版纳的JB114和JB135病毒株属于基因Ⅰ型,来自大理的DLN24病毒株为基因Ⅲ型,说明云南省存在这2个基因型病毒的共同流行.

摘要： @@流行性乙型脑炎(Japanese encephalitis，JE)简称乙脑，是由乙型脑炎病毒引起的一种重要蚊媒性人兽共患传染病。JEV E蛋白是病毒粒子表面最重要的结构蛋白，其表面的抗原决定簇，具有血凝活性和中和活性。E蛋白与病毒的毒力、宿主范围、组织嗜性、膜融合、保护性免疫、血凝反应和血清特异性有关。单克隆抗体纯度高、专一性强、重复性好且能在动物体外或体内产生同质性抗体，以它作为诊断抗体能克服多克隆抗血清诊断时稳定性差及非特异性强等缺点，在生命科学领域中已经得到广泛的应用。

摘要： 本研究利用RT-PCR技术扩增了JEV E蛋白基因主要片段,将之克隆入大肠杆菌中进行高效表达,并利用间接ELISA对表达产物的抗原性进行检测.为进一步利用原核表达产物研制诊断试剂盒奠定基础.

摘要： 重组质粒pET-E转化宿主菌BL21,经1.0mmol/L IPTG诱导,外源基因以包含体的形式获得高效表达.通过Western blotting检测证明表达产物具有良好的抗原性.以纯化后表达产物作为诊断抗原包被酶标板建立了检测JEV抗体的间接ELISA方法.结果表明,抗原的最佳稀释度为1:2000,血清的最佳稀释度为1:200,阳性标准初步定为:OD/OD<,阴性血清2.

摘要： 重组质粒pET-E转化宿主菌BL21,经1.0mmol/L IPTG诱导,外源基因以包含体的形式获得高效表达.通过Western blotting检测证明表达产物具有良好的抗原性.以纯化后表达产物作为诊断抗原包被酶标板建立了检测JEV抗体的间接ELISA方法.结果表明,抗原的最佳稀释度为1:2000,血清的最佳稀释度为1:200,阳性标准初步定为:OD/OD<,阴性血清2.

摘要： 重组质粒pET-E转化宿主菌BL21,经1.0mmol/L IPTG诱导,外源基因以包含体的形式获得高效表达.通过Western blotting检测证明表达产物具有良好的抗原性.以纯化后表达产物作为诊断抗原包被酶标板建立了检测JEV抗体的间接ELISA方法.结果表明,抗原的最佳稀释度为1:2000,血清的最佳稀释度为1:200,阳性标准初步定为:OD/OD<,阴性血清2.

摘要： 重组质粒pET-E转化宿主菌BL21,经1.0mmol/L IPTG诱导,外源基因以包含体的形式获得高效表达.通过Western blotting检测证明表达产物具有良好的抗原性.以纯化后表达产物作为诊断抗原包被酶标板建立了检测JEV抗体的间接ELISA方法.结果表明,抗原的最佳稀释度为1:2000,血清的最佳稀释度为1:200,阳性标准初步定为:OD/OD<,阴性血清2.

摘要： 本发明公开了一种检测猪流行性乙型脑炎病毒和/或猪繁殖与呼吸综合征病毒的基因芯片和试剂盒。本发明基因芯片和检测试剂盒可以准确、有效的检测猪流行性乙型脑炎病毒和猪繁殖与呼吸综合征病毒，且特异性强、灵敏度高、耗时短，检测快速，应用前景良好。

摘要： 本发明公开了一种检测猪流行性乙型脑炎病毒、猪瘟病毒和猪繁殖与呼吸综合征病毒的试剂盒，它包括猪流行性乙型脑炎病毒、猪瘟病毒和猪繁殖与呼吸综合征病毒的基因扩增试剂以及检测用基因芯片。本发明检测试剂盒可以准确、有效的检测繁殖与呼吸综合征病毒、猪瘟病毒和猪流行性乙型脑炎病毒，且特异性强、灵敏度高、耗时短，检测快速，应用前景良好。

摘要： 本发明公开了一种检测猪流行性乙型脑炎病毒、猪瘟病毒和猪繁殖与呼吸综合征病毒的可视化基因芯片和试剂盒。本发明特定方法制备的可视化基因芯片，可以用肉眼直观地看到检测结果，不需要价格昂贵的激光共聚扫描仪，成本低廉，同时，灵敏度较高，与现有荧光标记方法的灵敏度相当，特异性强，耗时短，检测快速，临床应用前景良好。

摘要： 本发明公开了一种检测猪流行性乙型脑炎病毒、猪瘟病毒和猪繁殖与呼吸综合征病毒的基因芯片和试剂盒。本发明基因芯片和检测试剂盒可以准确、有效的检测猪流行性乙型脑炎病毒、猪瘟病毒和猪繁殖与呼吸综合征病毒，且特异性强、灵敏度高、耗时短，检测快速，应用前景良好。

摘要： 本发明涉及一种联合快速检测流行性乙型脑炎病毒、登革病毒和西尼罗病毒试剂盒，由主反应液和特异性引物组成。主反应液包括反应缓冲液、禽类成髓细胞瘤病毒反转录酶、核酸酶抑制剂、Bst DNA聚合酶、dNTP、硫酸镁、酣菜碱、焦碳酸二乙脂和处理水；特异性引物包括特异性扩增乙脑病毒引物、特异性扩增登革病毒引物和特异性扩增西尼罗病毒引物。本发明用环介导等温扩增技术，设计高度特异性引物，快速检测3种黄病毒，达到高效特异性扩增的目的。本发明提供了检测方法，利用紫外－可见光分光光度计检测反应体系的吸光度值，间接反应目的基因的DNA扩增情况。适于基层和现场快速检测，有较大的应用价值。

摘要： 本发明公开了一种检测猪流行性乙型脑炎病毒和/或猪繁殖与呼吸综合征病毒的基因芯片和试剂盒。本发明基因芯片和检测试剂盒可以准确、有效的检测猪流行性乙型脑炎病毒和猪繁殖与呼吸综合征病毒，且特异性强、灵敏度高、耗时短，检测快速，应用前景良好。

摘要： 本发明公开了一种检测猪流行性乙型脑炎病毒和/或猪瘟病毒的基因芯片和试剂盒。本发明基因芯片包括固相载体以及固定在固相载体上的探针；所述探针包括SEQ ID NO：1或2所示任意一个或者两个寡核苷酸片段，和/或SEQ ID NO：3或4所示任意一个或者两个寡核苷酸片段。本发明试剂盒包括前述基因芯片与扩增猪流行性乙型脑炎病毒和/或猪瘟病毒的基因的试剂。本发明基因芯片和检测试剂盒可以准确、有效的检测猪流行性乙型脑炎病毒和猪瘟病毒，且特异性强、灵敏度高、耗时短，检测快速，应用前景良好。

摘要： 本发明公开了一种检测猪流行性乙型脑炎病毒和/或猪繁殖与呼吸综合征病毒的可视化基因芯片和试剂盒。本发明特定方法制备的可视化基因芯片，可以用肉眼直观地看到检测结果，不需要价格昂贵的激光共聚扫描仪，成本低廉，同时，灵敏度较高，与现有荧光标记方法的灵敏度相当，特异性强，耗时短，检测快速，临床应用前景良好。

摘要： 本发明公开了一种检测猪流行性乙型脑炎病毒和/或猪瘟病毒的可视化基因芯片和试剂盒。本发明特定方法制备的可视化基因芯片，可以用肉眼直观地看到检测结果，不需要价格昂贵的激光共聚扫描仪，成本低廉，同时，灵敏度较高，与现有荧光标记方法的灵敏度相当，特异性强，耗时短，检测快速，临床应用前景良好。

摘要： 本发明公开一种高效检测猪流行性乙型脑炎病毒的引物及试剂盒，发明以改良后的LAMP技术为基因扩增反应原理，在反应体系里加入金纳米颗粒，吸附ssDNA和蛋白酶，抑制升温过程中的非特异性反应，达到热启动的目的，避免在升温过程中的非特异性反应；结合改良后的LAMP技术与微流控芯片技术，即可以快速给出准确的检测结果，又达到同一样品多个不同指标联检的目的，同时，反应试剂预埋于微流控芯片上，用户只需加入样品，操作简便。