Seroprevalence of Bovine Parainfluenza Virus Type 3 (bPI-3V) in Ruminants from Grenada

Respiratory viral infections are known for serious economic losses in ruminants. Bovine parain-fluenza 3 virus (bPI-3V) a member of Respirovirus genus in association with other respiratory viruses causes respiratory disease complex in ruminants. The aim of this study was to estimate the seroprevalence of bPI-3V in non vaccinated cattle, sheep and goats from Grenada. Sera were collected randomly from 60 sheep, 60 goats and 60 cattle from all six parishes of Grenada. Sera were tested for antibodies to bPI-3V using an indirect Enzyme Linked Immunosorbant Assay (ELISA) kit. Antibodies to bPI-3V were detected in 13.4% (Confidence Level (CL): 95%;Confidence Interval (CI): 4.76% to 22.02%) in cattle;16.7% (CL: 95%;CI: 7.26% to 26.14%) in sheep and 11.7% (CL: 95%;CI: 3.57% to 19.83%) in goats. There was statistically no significant difference in prevalence (p > 0.05) of antibodies to bPI-3V in cattle, sheep and goats in Grenada. This is the first report on seroprevalence of bPI-3V in ruminants in Grenada, West Indies.

Evaluation of an attenuated vaccine candidate based on the genotype C of bovine parainfluenza virus type 3 in albino guinea pigs

Bovine parainfluenza virus type 3（BPIV3） is considered as one of the most important respiratory tract pathogens of both young and adult cattle, and widespread among cattle in the world. BPIV3 was first reported in China in 2008 and four strains of BPIV3 were isolated from Shandong Province, known as genotype C（BPIV3c）. Pathogen investigations had shown that BPIV3 c infection was very common among cattle in China. To date, BPIV3 can be classified into genotypes A, B and C based on genetic and phylogenetic analysis. Serological survey also demonstrates that BPIV3 infection is widespread in China, however, there is still no available vaccine for BPIV3 prevention in China nowadays. In the present study, the BPIV3 c strain SD0835 was continuously passaged on Madin-Darby bovine kidney（MDBK） cells for hundreds of times, and the pathogenicity of passage 209 was reduced in guinea pigs. The passage 209 of BPIV3 c strain SD0835 was used as a live vaccine candidate to immunize the guinea pigs. The vaccination results revealed that two vaccinations could induce excellent serum neutralizing antibody responses as well as proliferation of T lymphocytes. The vaccinated guinea pigs were well protected against challenge with a low passage of BPIV3 c strain SD0835. Additionally, the percentages of CD4~＋ and CD8~＋ T cell subsets of animals in vaccinated group increased after immunization; T cell subsets on day 2 after challenge in both groups decreased, and the decline of CD4~＋ and CD8~＋ T cell subsets levels of four guinea pigs in vaccinated group was relatively moderate, comparing with that of the control group. These data support further testing of the attenuated virus as an effective candidate vaccine.

Domain Ⅲ of Dengue Virus Serotype 2 Envelope: Expression at High Levels in Escherichia coli and Competitive Inhibition of Virus Entry

Obejective The domainⅢof dengue virus type 2 envelope was cloned and expressed in Escherichia coli and the inhibited effects of recombinant protein on virus was detected. Methods In this study, the domainⅢ(DⅢ) protein of the dengue virus type-2 (DENV-2) envelope (E) antigen was expressed in Escherichia coli by fusion with a carrier protein. The protein was puriifed using enzymatic cleavage and afifnity puriifcation. Rabbit immunization and antibody detection was carried out. Inhibition of DENV-2 infection was observed by DENV-2 EDⅢprotein and its immunity rabbits serum. Results The recombinant expression DENV-2 EDⅢ protein plasmid was constructed successfully. After isopropyl thiogalactoside induction, a speciifc soluble 29 kD protein was obtained, and the expression product accounted for 68.87%of the total protein of the cell lysate. Western blot demonstrated the reactivity of the recombinant protein with his-tag and DENV (Ⅰ-Ⅳ) monoclonal antibodies. The protein was puriifed using enzymatic cleavage and affinity purification. The purified recombinant EDⅢ protein inhibited the entry of DENV-2 into BHK-21 cells. DENV-2 plaque neutralization assays were carried out using serially diluted antibodies against EDⅢprotein. At a 1︰16 dilution, the antibodies produced at least 90%neutralization of the DENV-2 virus. Furthermore, the antibodies continued to exhibit high neutralization effects (approximately 80%) until the anti-EDⅢantibody titer reached 1︰1 024. Conclusions DENV-2 EDⅢwas cloned and expressed successfully. DENV-2 EDⅢprotein could be useful in the development of inexpensive dengue vaccine. The data also suggested that DENV-2 employed an attachment molecule or receptor for its entry into C6/36 mosquito cells.

Mechanism of Japanese encephalitis virus genotypes replacement based on human,porcine and mosquito-originated cell lines model

Objective:To examine the multiplication efficiency Japanese encephalitis virus(JEV)genotype Ⅰ(G Ⅰ) and genotype Ⅲ(GⅢ) of different cell lines which originated from human,porcine,mosquitoes in order to prove mechanism of JEV G Ⅰ replacement JEV GⅢ since it emerging in nature recent decades.Methods:The mixture of Gi and GⅢ JEV isolates was inoculated on human rhabdomyosarcoma(RD).pig kidney epithelial(PS) and Aedes albopictus C6/36 clone(C6/36) which originated from human,porcine and mosquitoes,respectively.Plaque assays were performed to calculate virus titer and real-time RT-PCR with GⅠand GⅢspecific primer sets to quantify the number of GⅠ and GUI RNA copies.Results:The highest virus titer reached at the 3rd day of post infection when G Ⅰand GⅢ mixture was inoculated on RD and PS and that of C636 was at the 4^(th)day.JEVs were amplified and maintained by C6/36 cells after 10 passages whereas that by RD and PS only limited within 8 and 6 passages,respectively.GⅠ strain amplified and maintained more efficiently on C6/36 and PS but not RD.whereas GⅢ strain amplified and maintained more efficiently on RD.Conclusions:There is a correlation between the multiplication efficiency of GⅠ and GⅢ JEV strains when these two genotype strains co-infected on different cell lines with the predominance of GⅠstrains in C6/36 and PS and the limited detection of G 1 strains in RD cells proving a possible mechanism of shift JEV genotypes in nature recent decades since GⅠ emerging.

Positional effect of mutations in 5'UTR of hepatitis C virus 4a on patients' response to therapy

AIM： To investigate the effects of mutations in domain Ⅲ of the hepatitis C virus （HCV） internal ribosome entry sequences （IRES） on the response of chronic HCV genotype 4a patients to interferon therapy.METHODS： HCV RNA was extracted from 19 chronic HCV 4a patients receiving interferon/ribavirin therapy who showed dramatic differences in their response to combination therapy after initial viral clearance. IRES domain Ⅲ was cloned and 15 clones for each patient were sequenced. The obtained sequences were aligned with genotype 4a prototype using the ClustaIW program and mutations scored. Prediction of stem-loop secondary structure and thermodynamic stability of the major quasispecies in each patient was performed using the MFOLD 3.2 program with Turner energies and selected constraints on base pairing.RESULTS： Analysis of RNA secondary structure revealed that insertions in domain Ⅲ altered WatsonCrick base pairing of stems and reduced molecular stability of RNA, which may ultimately reduce binding affinity to ribosomal proteins. Insertion mutations in domain - were statistically more prevalent in sustained viral response patients （SVR, n = 14） as compared to breakthrough （BT, n = 5） patients.CONCLUSION： The influence of mutations within domain Ⅲ on the response of HCV patients to combination therapy depends primarily on the position, but not the frequency, of these mutations within IRES domain Ⅲ.

YCB-Ⅲ对烤烟农艺性状和病毒病防治的影响

采用土壤改良剂(YCB-Ⅲ)与农药配施,在减施农药总用量的同时不降低(或提升)对烤烟病毒病的防治效果,且改善烤烟的农艺性状,提高烤烟的产量与品质。从不同处理对烤烟旺长期农艺性状的影响看,T4和T5对烤烟的株高、茎围、有效叶片数、鲜叶重和开片度均达到显著水平。相对于T1,T2、T3、T4和T5的YCB-Ⅲ施用量分别增加了25%、50%、75%和100%,农药施用量分别减少了0.00%、25%、50%和50%,但是防治效果均大于74%,处于同一水平。研究表明,YCB-Ⅲ的施用量525 kg/hm~2、4%嘧肽霉素水剂(KV_1)有效成分施用量120 g/hm~2、0.5%香菇多糖水剂(KV_2)有效成分施用量12.5 g/hm~2为最佳推荐量。

D-D、AT-Ⅲ与乙型肝炎患者严重程度的相关性分析

目的:探究血浆D-二聚体(D-dimer,D-D)、抗凝血酶Ⅲ(Antithrombin III,AT-Ⅲ)与乙型肝炎患者严重程度的相关性。方法:选取2020年12月~2023年12月期间我院收治的60例乙型肝炎患者的临床及随访资料进行研究。根据病情严重程度将患者分为轻度组(n=22)、中度组(n=26)、重度组(n=12)。比较三组患者D-D、AT-Ⅲ水平的差异。采用Spearman相关系数分析D-D、AT-Ⅲ与乙型肝炎患者严重程度的相关性。结果:重度组患者的D-D水平明显高于轻度组、中度组;重度组患者的AT-Ⅲ水平低于轻度组、中度组;中度组患者的D-D水平高于轻度组,AT-Ⅲ水平低于轻度组(P<0.05)。Spearman相关分析结果显示,患者血浆D-D与乙型肝炎患者病情的严重程度呈正相关(r=0.721,P<0.05);AT-Ⅲ水平与乙型肝炎患者病情严重程度呈负相关(r=-0.685,P<0.05)。结论:D-D和AT-Ⅲ水平与乙型肝炎患者病情的严重程度存在明显的相关性。D-D水平升高和AT-Ⅲ水平降低可能是乙型肝炎患者病情加重的重要标志。

新生儿和小婴儿Ⅲ型副流感病毒肺炎55例

目的探讨新生儿和小婴儿Ⅲ型副流感病毒（PIV3）肺炎的临床特点，提高对该病的诊治水平。方法2005年8月-2007年7月依据肺炎临床诊断标准和实验室间接免疫荧光法诊断并住院治疗的新生儿和小婴儿PIV3肺炎55例，对该组患儿的临床特点进行回顾性分析，包括患儿的性别、年龄、症状、体征、肺部X线特点、实验室检查结果、治疗情况及预后等。结果肺炎患儿55例中小婴儿占80％（44／55例），新生儿占20％（11／55例）。季节分布以春夏最多。城郊病例占74．5％（41／55例），城区病例占25，5％（14／55例）。均有咳嗽，刺激性咳嗽或痉挛性咳嗽34例（61．8％），咳嗽同时呛奶39例（70．9％）；发热26例（47．3％），多为短期轻度发热；喘息14例（25．5％）。肺部细湿哕音20例，其中伴喘鸣8例，有喘鸣音无湿啰音6例，肺部呼吸音粗伴痰鸣音21例。肺外表现主要有轻度吐奶、腹泻；胸片示右肺受累18例，左肺2例，双肺35例。病程1～2周16例，〉2～3周28例，〉3周11例，病程最长者32d。55例患儿中42例（76．4％）未使用抗生素，患儿均未接受抗病毒治疗。55例均临床痊愈出院，其中35例出院2周内随访，未见到肺部严重受损。结论春夏是新生儿和小婴儿PIV3肺炎发病高峰，病程长，临床表现为刺激性咳嗽，右肺易受累，并发症少是其主要特征，临床特点和实验室检查相结合有利于准确诊断和治疗。

甘草抗病毒有效部位体外抗副流感病毒(Ⅲ型)作用的研究

目的:研究甘草抗病毒有效部位(GC3-1-4)体外抑制副流感病毒(Ⅲ型)的作用。方法:采用煎煮法和柱层析法提取分离得到甘草抗病毒有效部位(CC3-1-4)。以病毒唑为阳性对照药,采用细胞病变抑制实验观察CC3-1-4在Hela细胞中对副流感病毒(Ⅲ型)的抑制作用。结果GC3-1-4半数申毒浓度(TQ50))为144．17μg／ml;抑制副流感病毒(Ⅲ型)的半数有效浓度(EC50)为12.82μg/ml,治疗指数(TI)为11．25;GC3-1-4对副流感病毒(Ⅲ型)的抑制作用存在量效反应关系;在感染后6h以內给药,GC3-1-4对副流感病毒(Ⅲ型)均有抑制作用。结论:GC3-1-4在Hela细胞中对副流感病毒(Ⅲ型)有抑制作用。

猪乙型脑炎病毒E-Ⅲ抗原域蛋白间接ELISA的建立及应用

该研究利用纯化后的流行性乙型脑炎病毒(Japanese encephalitis virus,JEV)E-Ⅲ抗原域蛋白,将其作为包被抗原,建立了间接ELISA检测方法。试验的最佳反应条件为:最佳抗原包被浓度为17.5μg/mL,血清抗体稀释度为1∶4,酶标二抗稀释度为1∶5 000,封闭液和稀释液用含0.4%BSA的PBST。采用该方法对127份临床样品进行检测,结果显示,与商品试剂盒检测结果比较符合率达到90.00%。因此,该研究建立的流行性乙型脑炎间接ELISA检测方法具有良好的稳定性和可重复性,并具有较高的特异性和敏感性。

病毒唑体外抗副流感病毒(Ⅲ型)作用的实验研究

目的:观察病毒唑体外对副流感病毒( 型)的抑制作用。方法:采用细胞病变抑制实验,观察病毒唑在Hela细胞中对副流感病毒( 型)的抑制作用。结果:病毒唑半数中毒浓度(TC50 )为1783.86μg/ m l;抗副流感病毒( 型)的半数有效浓度(EC50 )为11.5 3μg/ ml,治疗指数(TI)为15 4 .71;病毒唑对副流感病毒( 型)的抑制作用存在明显的量效反应关系(P<0 .0 1) ;在感染病毒后0～2 4 h,每隔2小时加一次药,病毒唑的5 0、2 5、12 .5、6 .2 5 μg/ ml组对副流感病毒( 型)均有抑制作用(P<0 .0 1) ;病毒唑对副流感病毒( 型)有预防和中和作用(P <0 .0 1)。结论:病毒唑在Hela细胞中对副流感病毒( 型)有明显的抑制作用,其作用机制是多途径的。

葡萄卷叶病毒Ⅲ RT-PCR检测技术研究

利用ＲＴ－ＰＣＲ技术,对葡萄卷叶病毒株系Ⅲ进行了检测。采用的技术流程为:通过提取葡萄叶片或韧皮部总ＲＮＡ,获得病毒的ＲＮＡ,反转录合成ｃＤＮＡ,经ＰＣＲ扩增,获得病毒ｃＤＮＡ的特征片段,并进行了序列分析。对提取的总ＲＮＡ进行了完整性和纯度检测,确定了良好的ＲＮＡ获得方法。结果表明,通过ＲＴ－ＰＣＲ扩增的片段序列与病毒源序列的同源性高达９９．３%,说明采用本研究确定的方法检测葡萄卷叶病毒株系Ⅲ结果可靠。

一株野鸭源基因Ⅲ型新城疫病毒主要生物学特性及基因组序列测定分析

为研究分离自黑龙江三江自然保护区的野生绿头鸭粪拭子中的一株新城疫病毒(命名为Mallard/CH/HLJ383/06)主要生物学特性及基因组序列,本实验对病毒F基因序列进行测定,结果表明该病毒分离株属于NDV基因Ⅲ型,病毒的1日龄雏鸡脑内致病指数(ICPI)为1.81,高于NDV中等毒力病毒株ICPI值,预示该病毒分离株毒力有增强趋势。15日龄SPF鸡致病性试验表明,该病毒分离株可导致雏鸡发病但不死亡,致病性仍低于与其ICPI值相近的基因Ⅶ型毒株。对该病毒分离株进行全基因组序列分析表明:该病毒分离株与Ⅰ系疫苗病毒Mukteswar、江苏2株基因Ⅲ型分离株(JS/7/05/Ch,JS/9/05/Go)结构蛋白氨基酸同源性高达99.0%～99.7%;与疫苗株Mukteswar相比,3株基因Ⅲ型病毒分离株在F蛋白中只有1个共同的氨基酸位点变异(A203T);HN蛋白中存在15个变异氨基酸,但位置各不相同;L蛋白中变异位点最多,为28个,其中有4个变异位点为3个分离株所共有。以上结果初步表明,Mallard/CH/HLJ383/06株可能是由疫苗株Mukteswar在野禽、家禽生态系统中传播进化而来,并由于宿主或免疫压力而发生毒力变异;分离株L蛋白氨基酸位点的变异,可能是导致病毒株毒力返强主要因素之一。

抗凝血酶Ⅲ活性检测在HBV相关慢加急性肝衰竭中的意义

目的分析HBV相关慢加急肝衰竭患者(HBVACLF)抗凝血酶原Ⅲ(AT-Ⅲ)活性变化,研究其与患者肝功能及凝血功能指标潜在相关性。方法选取住院患者中诊断明确的HBV-ACLF患者80例和作为对照的慢性乙型肝炎(CHB)患者48例,发色底物法分析患者AT-Ⅲ活性水平,采用Spearman秩相关分析其与血清总胆红素水平(TBIL)、白蛋白(ALB)、丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)、凝血酶原活动度(PTA),PT国际标准化比值(PTINR)及终末期肝病模型(MELD)评分的相关性,运用受试者工作特征(ROC)曲线评价AT-Ⅲ能否作为影响HBV-ACLF临床预后的潜在实验室检测指标。结果与CHB患者相比,HBV-ACLF患者AT-Ⅲ活性水平降低,其它肝功能和凝血功能检测指标除ALB和PTA减少外均增加,差异有统计学意义(P<0.05)。HBV-ACLF患者AT-Ⅲ活性水平与TBIL、PT-INR、PTA均具有相关性,HBV-ACLF患者预后分组中AT-Ⅲ活性水平与MELD评分呈负相关,ROC曲线结果显示,采用AT-Ⅲ预测HBV-ACLF患者临床预后的曲线下面积为0.686,敏感性为75.0%,特异性为52.9%,界值为16.4%;采用MELD评分预测HBV-ACLF患者临床预后的曲线下面积为0.698,敏感性76.5%,特异性55.9%,界值为28.5;AT-Ⅲ联合MELD评分预测HBV-ACLF患者临床预后的曲线下面积为0.756,敏感性为62.5%,特异性为88.2%,界值为AT-Ⅲ:20.1%,MELD评分:25。结论AT-Ⅲ与HBV-ACLF患者肝功能损伤严重程度、病情进展相关,临床联合MELD评分在预测HBV-ACLF患者临床预后方面具有一定的参考价值。

抗凝血酶Ⅲ活性与慢性乙型肝炎病毒感染者肝纤维化程度的相关性

目的分析抗凝血酶Ⅲ(antithrombinⅢ,AT-Ⅲ)活性与慢性乙型肝炎病毒(hepatitis B virus,HBV)感染者肝纤维化程度的相关性。方法选取2017年6月至2019年11月在安徽医科大学第二附属医院行肝组织活检的102例慢性HBV感染者为研究对象,检测患者丙氨酸氨基转移酶(alanine aminotransferase,ALT)、AT-Ⅲ活性及D-二聚体水平,根据AT-Ⅲ活性将患者分为减低组(<75%,27例)和正常组(75%~125%,75例),比较两组间ALT和D-二聚体水平的差异。根据肝组织病理进行肝脏炎症活动度分级(G)和纤维化分期(S),比较不同肝脏炎症活动度分级及纤维化分期患者AT-Ⅲ活性的差异。AT-Ⅲ活性与肝脏炎症活动度和肝纤维化分期的相关性采用Spearman秩相关检验。结果 AT-Ⅲ活性减低组和正常组患者ALT水平(中位数:60 U/L vs 39 U/L)差异有统计学意义(z=-2.56,P=0.01);D-二聚体水平(中位数:0.21 mg/L vs 0.25 mg/L)差异无统计学意义(z=-1.42,P=0.16)。肝脏炎症活动度G1、G2、G3分别有62例、34例、6例,AT-Ⅲ活性分别为83.00%(78.00%,90.05%)、(79.19±17.88)%、(77.02±18.16)%,差异无统计学意义(H=1.338,P=0.512)。肝纤维化程度S1期、S2期、S3期、S4期分别有52例、23例、16例、11例;AT-Ⅲ活性分别为(84.45±9.87)%、85.40%(74.00%,96.00%)、(77.56±14.03)%、(66.61±25.84)%,差异有统计学意义(H=13.886,P=0.003),其中S1期和S2期患者AT-Ⅲ活性均显著高于S4期患者(t=2.255,P=0.046;z=-2.670,P=0.008)。Spearman相关性分析表明,AT-Ⅲ活性与肝脏炎症活动度无显著相关性(r=-0.44,P=0.253),而与纤维化程度呈负相关(r=-0.296,P=0.002)。结论 AT-Ⅲ活性与慢性HBV感染者肝纤维化程度相关,可能成为一种新的肝脏病理的无创诊断指标。

重组黄热病毒E蛋白结构域Ⅲ作为亚单位疫苗的抗原性和免疫原性

目的:表达纯化黄热病毒(YFV)囊膜蛋白(E蛋白)结构域Ⅲ,研究其作为亚单位疫苗预防YFV、日本脑炎病毒(JEV)感染的可能。方法:扩增YFVE蛋白结构域Ⅲ(YFDⅢ)的cDNA片段333bp,将其连接到原核表达载体pET-32a(+)中,构建原核表达载体pET-YFDⅢ,转化感受态大肠杆菌Rosetta(DE3),IPTG诱导表达重组YFDⅢ;用纯化的YFDⅢ免疫新西兰兔和BALB/c鼠,检测相关抗体滴度。结果:在大肠杆菌中可溶性表达了YFDⅢ融合蛋白,表达量约占菌体蛋白的50%;Western印迹及ELISA分析表明,纯化的YFDⅢ具有良好的抗原性和免疫原性;利用纯化的YFDⅢ免疫新西兰兔,获得了高达1∶4×105滴度的抗YFV抗体和1∶2×104滴度的抗JEV抗体;利用纯化的YFDⅢ免疫BALB/c鼠,获得了1∶7×104滴度的抗YFV抗体和1∶2×103滴度的抗JEV抗体。结论:重组YFDⅢ有较好的免疫原性,具有开发成亚单位疫苗的潜能。

葡萄卷叶伴随病毒Ⅲ宁夏分离物外壳蛋白基因的克隆及序列分析

用酶联免疫吸附法对宁夏贺兰山东麓地区不同葡萄品种进行卷叶伴随病毒Ⅲ(GLRaVⅢ)的测定,检测率为37.1%,与田间自然发病率调查结果基本一致.设计GLRaVⅢ外壳蛋白(CP)基因序列引物对,进行RT-PCR扩增,获得1.1 kb的特异片段.其中,CP基因长942 bp,推导的编码蛋白含有313个氨基酸.通过对GLRaVⅢCP基因同源性比较,结果表明,GLRaVⅢ宁夏分离物的CP基因与四川分离物SL-10 CP基因的亲缘关系最近,核苷酸和所编码的氨基酸序列同源性分别为98.8%和98.1%;与巴西分离物PET-4和智利分离物C1-817 CP基因亲缘关系较远,核苷酸序列同源性低于95.0%,所编码的氨基酸序列同源性低于97.0%,认为GLRaVⅢ株系间的CP基因存在差异.

FcγRⅢ在乙型肝炎病毒胎盘感染中作用的研究

目的研究FCγRⅢ在乙型肝炎病毒胎盘感染中的作用。方法收集早孕、中孕,足月胎盘各15例,进行免疫荧光检测FcγRⅢ的表达情况;并随机选取经免疫组化PV-9000法检测为HBsAg(+)的足月胎盘15例,进行免疫荧光双标检测HBsAg/FcγRⅢ的分布。结果早孕绒毛组织未见FcγRⅢ表达,中孕胎盘有11例表达,其中最早的为孕17w,足月胎盘均有明显表达;HBsAg/FcγRⅢ在胎盘屏障各层细胞上可分布于同一部位。结论孕17w后胎盘组织开始表达FCγRⅢ,使胎盘具有转运IgG的功能:HBV可能是以免疫复合物的形式经由滋养细胞表面FCγRⅢ的介导而感染胎盘的。

新城疫病毒基因Ⅲ型强毒JS/7/05/Ch的拯救

新城疫病毒Ⅰ系苗Mukteswar株为中等毒力,野外分离株JS/7/05/Ch的基因组核苷酸与其具有高于99%的相似性,但其毒力却明显强于前者。为了给基因Ⅲ型NDV的遗传进化机制研究提供基础,本研究成功构建了JS/7/05/Ch株的反向遗传平台。首先根据GenBank上公布的基因Ⅲ型新城疫病毒JS/7/05/Ch株基因组全序列设计并合成8对引物,RT-PCR扩增目的片段后,分别依次亚克隆至pCR2.1载体中构建含有JS/7/05/Ch全长基因组cDNA的克隆质粒pJS/7/05/Ch,然后再通过特异性酶切位点将JS/7/05株全长基因组cDNA转移到TVT7R(0.0)转录载体中,成功构建出含JS/7/05/Ch株基因组全长cDNA的转录质粒pTVT/JS705。然后将该质粒与3个辅助质粒pCI-NP、pCI-P和pCI-L共转染BSR-T7/5细胞,转染60h后将该细胞及其上清接种鸡胚;血凝(HA)试验和RT-PCR结果表明JS/7/05/Ch株新城疫病毒被成功拯救。

猪乙型脑炎病毒EDⅢ蛋白的原核表达、纯化及活性检测

在大肠杆菌BL21(DE3)中表达乙脑病毒(JEV)EDⅢ蛋白、镍柱纯化并检测表达蛋白。采用RT-PCR方法扩增EDⅢ基因片段,构建重组表达载体p ET-28a-EDⅢ并转化到BL21(DE3)菌,经IPTG诱导蛋白表达,表达可溶性产物经NiNTA亲和层析纯化,最后通过SDS-PAGE和Western-Blot检测表达蛋白。结果成功构建原核表达载体p ET-28a-EDⅢ;EDⅢ蛋白在BL21(DE3)菌已表达,经Ni-NTA获得纯化蛋白,并经Western-Blot检测具有反应活性。