猪传染性胃肠炎病毒N蛋白原核表达和间接ELISA检测方法的建立

试验构建了pET28a-TGEV-N重组表达质粒,完成TGEV N蛋白的表达和纯化,通过SDS-PAGE和Western blotting分析,表明表达产物N蛋白可被猪TGEV阳性血清识别。用TGEV N蛋白作为诊断抗原,建立了检测TGEV血清抗体的间接ELISA检测方法,该方法批内、批间重复试验变异系数均小于5%;检测血清的特异性为100%,假阳性率为0,假阴性率为5%,与7种常见猪病血清无交叉反应。针对临床血清,用该方法检测结果与中和试验结果相比,符合率为100%。该方法能够快速、有效地检出TGEV抗体,重复性和特异性好,为标准化诊断试剂盒的开发奠定了基础。     

Establishment of Double PCR Detection Method of Transmissible Gastroenteritis Virus (TGEV) and Porcine Epidemic Diarrhea Virus (PEDV)

Two pairs of primers used to respectively amplify transmissible gastroenteritis virus (TGEV) S gene and porcine epidemic diarrhea virus (PEDV) M gene were designed to develop a method of differential diagnosis of TGEV and PEDV.The established double PCR could detect S gene of TGEV with the length of 299 bp and M gene of PEDV with the length of 437 bp.Negative results using CSFV,PCV2,PRRSV and PRV as control were obtained.The detection limit of this method was 10⁴ copies/μL.68 clinical samples collected from swine farm were submitted to detect TGEV and PEDV,and the result showed that the established double PCR method with the characteristics of high sensitivity and high specificity could be widely used in clinical diagnosis and epidemiological investigation.     

猪传染性胃肠炎病毒和猪流行性腹泻病毒二重PCR检测方法的建立

通过对猪传染性胃肠炎病毒(TGEV)S基因和猪流行性腹泻病毒(PEDV)M基因进行序列分析,本试验利用DNAStar软件分别设计2对特异性引物,扩增片段长度分别为299和437 bp,建立一种针对TGEV和PEDV感染的二重PCR鉴别诊断方法.该方法能同时检测到TGEV和PEDV,而对猪瘟病毒(CSFV)、猪圆环病毒2型(PCV2)、猪繁殖与呼吸综合征病毒(PRRSV)、猪伪狂犬病病毒(PRV)等均无扩增,其检测TGEV、PEDV的极限为10⁴ 拷贝/μL;用该方法对临床收集的68份疑似病毒性腹泻仔猪粪便和肠道组织样本进行检测,结果表明本试验建立的二重PCR方法具有特异性强、灵敏度高等特点,能用于临床诊断及流行病学调查.     

小反刍兽疫病毒N蛋白原核表达与间接ELISA检测方法的建立

小反刍兽疫病毒(PPRV)N蛋白是病毒粒子的主要结构蛋白,也是诊断小反刍兽疫的主要靶蛋白。为获得高效表达的可溶性N蛋白,并建立基于N蛋白的小反刍兽疫的间接ELISA检测方法,通过优化密码子、优化表达条件等条件探索,在大肠埃希菌表达系统中获得了高效表达的可溶性N蛋白,进一步基于N蛋白建立了间接ELISA检测方法,该方法对其他相关的羊类病原无交叉反应,其组内与组间变异系数均低于9%,具有良好的重复性。对480份临床血清样品进行检测,同时用法国IDVET竞争ELISA试剂盒进行比较,符合率达到98.33%。本研究为进一步开发成熟的小反刍兽疫抗体检测试剂盒奠定了基础。     

Prokaryotic Expression of Thymidine Kinase Gene of Duck Plague Virus and Establishment of an Indirect ELISA Based on the Protein

In the study,an indirect ELISA was developed using the purified thymidine kinase (TK) protein as antigens to detect the antibody of duck plague virus (DPV).The TK gene of DPV was amplified by PCR using specific primers designed according to the sequence of TK gene in GenBank.Using gene recombination technology,the TK gene was cloned and inserted into prokaryotic expression vector pET-32a and the recombinant plasmid was identified by PCR,double enzyme digestion and sequence analysis.The positive recombinant plasmid was then transformed into E.coli BL21 (DE3) and induced by IPTG.The expressed protein was purified by gel extraction and analyzed by Western blotting.Then an indirect ELISA was established to detect DPV antibody by using the purified recombinant TK as the coating antigen.The other assay conditions were also optimized.The recombinant plasmid was constructed successfully and Western blotting detected the target protein,TK recombinant protein could be recognized by positive serum,and the result indicated that the recombinant protein had good antigenicity.The optimum reaction conditions were as follow:The optimal dilution of enzyme labelled antibody was 1:200,the optimal dilutions of antigen and antibody were 1:400 and 1:200,reaction time was 60 min,most prefer blocking solution was 5% BSA,closed for 60 min,chromogenic time was 10 min.The method had good stability and high sensitivity,and it could provide a reliable method for the clinical detection,immunization surveillance,and prevention and cure of DP.     

鸭瘟病毒胸苷激酶基因的原核表达及其蛋白间接ELISA检测方法的建立

本研究旨在以表达纯化的鸭瘟病毒胸苷激酶(thymidine kinase,TK)重组蛋白作为包被抗原,建立检测鸭瘟病毒抗体的间接ELISA方法。根据GenBank上收录的鸭瘟病毒TK基因序列设计出1对引物,采用PCR扩增出鸭瘟病毒TK基因。将TK基因与原核表达载体pET-32a连接,通过PCR、双酶切和测序鉴定,将阳性重组质粒转化至大肠杆菌BL21(DE3)工程菌,IPTG诱导表达。采用切胶纯化的方法纯化蛋白,Western blotting分析鉴定表达产物。用纯化的TK重组蛋白作为包被抗原,并优化其他条件,最终建立检测鸭瘟病毒抗体的间接ELISA方法。结果显示,重组质粒构建成功,经Western blotting检测得到目的蛋白,TK重组蛋白能被阳性血清识别,说明该重组蛋白具有较好的抗原性。确定了最佳反应条件:酶标二抗的最佳稀释度为1:200,最佳抗原、抗体的稀释度分别为1:400和1:200,抗原抗体作用时间为60 min,最佳封闭液为5% BSA,最佳封闭时间为60 min,最佳显色时间为10 min。结果表明,该方法具有良好的稳定性及较高的敏感性,为鸭瘟的检疫、监测和防制工作提供了一种有效的手段。     

猪瘟病毒石门株NS3蛋白的原核表达及其抗体检测间接ELISA的建立

为配合猪瘟新型疫苗的研发,建立了猪瘟病毒NS3蛋白抗体检测间接ELISA,以期达到有效区分新型疫苗免疫猪与自然感染猪(包括常规疫苗接种猪)的目的。以接种猪瘟病毒(CSFV)石门株的PK-15细胞为模板提取总RNA,经特异性PCR扩增获得长度为2 049bp的CSFV NS3基因,将其克隆至插入了具有自聚集自切割功能短肽的原核表达载体pET-32a(+),在大肠埃希菌Rosetta(DE3)中优化表达CSFV石门株NS3基因。Western blot分析表明重组蛋白NS3具有反应原性。将纯化的重组蛋白NS3作为包被抗原建立检测CSFV NS3抗体的间接ELISA,以美国爱德士(Idexx)猪瘟病毒抗体检测试剂盒抗体检测结果为标准,对502份血清样品进行检测。结果表明,所建立方法的特异性为96.9%,敏感性为89.7%,总符合率为95.8%,为猪瘟新型疫苗的推广应用提供了血清学检测方法。     

非洲猪瘟病毒p30基因的原核表达及间接ELISA抗体检测方法的建立

为建立检测血清中非洲猪瘟病毒（African swine fever virus,ASFV）抗体的间接ELISA方法,本试验将ASFV p30基因进行原核表达,采用SDS-PAGE和Western blotting方法对重组蛋白进行表达鉴定和免疫原性分析,随后以纯化的重组蛋白为包被抗原,经条件优化、特异性试验、敏感性试验和重复性试验,建立一种血清中ASFV抗体的检测方法。结果显示,ASFV p30基因成功克隆到原核表达载体pET-32a （+）中,获得pET-32a-p30重组质粒;转化大肠杆菌BL21（DE3）感受态细胞进行诱导表达,得到P30重组蛋白,重组蛋白大小约为42 ku,主要以包涵体形式存在;Western blotting结果显示,纯化后的蛋白具有良好的免疫原性;以纯化的P30重组蛋白为包被抗原,建立了检测ASFV抗体的间接ELISA方法,通过方阵试验对间接ELISA方法进行优化,最终确定了抗原最佳包被浓度为1.2 μg/mL,待检血清最佳稀释倍数为1：100,最佳封闭液为1% BSA,酶标抗体最佳稀释度为1：4 000,以此建立的ASFV间接ELISA方法临界值为0.322。本方法仅与ASFV阳性血清发生特异性反应,与猪瘟病毒、猪繁殖与呼吸综合征病毒、口蹄疫病毒、伪狂犬病病毒、猪圆环病毒2型及猪流行性腹泻病毒阳性血清均无交叉反应,具有较强的特异性。该方法检测ASFV阳性血清灵敏度可达到1：1 600;批内重复性和批间重复性变异系数均<10%。本试验建立的间接ELISA方法具有良好的特异性、灵敏度和重复性,可初步应用于ASFV抗体的检测。     

丝状支原体山羊亚种LPPA蛋白原核表达及间接ELISA方法的建立

【目的】建立基于丝状支原体山羊亚种(Mmc)膜脂蛋白LPPA的间接ELISA方法。【方法】扩增Mmc LPPA基因并将其克隆至pCold-Ⅰ载体,构建重组质粒pCold-Ⅰ-LPPA,测序鉴定正确后转化大肠杆菌DH5α感受态细胞,经IPTG诱导表达后纯化得到Mmc LPPA重组蛋白,采用SDS-PAGE验证该重组蛋白是否表达,并采用Western blotting和传统经典的琼脂扩散血清学试验分析其与Mmc阳性血清的反应原性。以该重组蛋白为包被抗原,建立Mmc重组LPPA蛋白的间接ELISA抗体检测方法,对该方法进行反应条件优化后开展特异性、重复性试验,并将其初步应用于184份山羊血清样本。【结果】通过PCR扩增得到Mmc LPPA基因,成功构建了重组质粒pCold-Ⅰ-LPPA,经诱导表达后得到Mmc LPPA重组蛋白,SDS-PAGE结果显示,获得了大小约23 ku的LPPA重组融合蛋白;琼脂扩散及Western blotting试验证明该蛋白反应原性良好。ELISA方法反应条件优化结果显示,LPPA抗原蛋白的包被浓度为2.0μg/100μL,血清稀释度为1∶300,3%BSA封闭...     

Establishment of Indirect ELISA Method for Detecting Recombinant Truncated N Protein of Porcine Epidemic Diarrhea Virus

In order to establish an indirect ELISA to detect antibody of porcine epidemic diarrhea virus (PEDV).The experiment using the recombinant and purified truncated N protein as antigen expressed in E.coli BL21(DE3),the indirect ELISA was named rnPED-ELISA.The recombinant truncated N protein antigen showed no cross-reaction with the positive sera of other 7 kinds of swine diseases,CV%of intro-batch duplicativity test and inter-batch duplicativity test were less 13%;Sensitivity and specificity of rnPED-ELISA relative to SN were 93.33% and 90.00%,respectively;rnPED-ELISA compared with TSZ PEDV antibody diagnosis Kit,91.67% concordance was obtained.200 serum samples were detected by this method,the total masculine ratio was 69.5%.Therefore,this rnPED-ELISA based on recombinant truncated N protein antigen had good sensitivity and specificity,could afforded a simple and rapidmeans for assessment of vaccination in the field and investigation of PED epidemiology.     

PEDV流行株重组截短N蛋白间接ELISA检测方法的建立

为了建立猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)抗体检测的间接ELISA方法,本研究以纯化的原核表达的PEDV截短N蛋白作为包被抗原,建立了PEDV抗体检测的间接ELISA方法,将该方法命名为rnPED-ELISA。该抗原不与其他常见的7种猪病的阳性血清发生交叉反应,批内和批间重复性试验的变异系数均小于13%;rnPED-ELISA相对于血清中和试验(SN)试验的敏感性为93.33%,特异性为90.00%;rnPED-ELISA与TSZ全病毒抗体检测试剂盒的符合率达91.67%。采用rnPED-ELISA方法检测200份临床样品,PEDV抗体阳性检出率为69.5%。本试验建立的rnPED-ELISA方法具有良好的敏感性和特异性,可为免疫猪群抗体监测和猪流行性腹泻流行病学调查提供一种快速、简便的血清学诊断方法。     

Prokaryotic Expression and Immunogenicity Analysis of N Protein from Vesicular Stomatitis Virus

This study was aimed to clone and express the specific antigen nucleoprotein (N) of vesicular stomatitis virus (VSV), and then purify and analyze its immunogenicity. Based on published VSV genome N gene sequence in GenBank, two kinds of N genes of VSV with different serotypes were synthesized, respectively. After sequence analysis, one pair of specific primers was designed and synthesized, N gene fragment with about 1 300 bp length was amplified by PCR, and subcloned into pCold Ⅰ expression vector. The recombinant N protein was induced with IPTG and purified by Ni-NTA. The results of SDS-PAGE showed that the N gene was successfully expressed in E. coli and the molecular weight of protein was 50 ku; The results of Western blotting showed that this recombined protein specifically reacted with polyclonal antibody serum of VSV. The recombinant vector with VSV-IND and VSV-NJ were successfully constructed, and the N protein was solubly expressed in E. coli, and the purified protein demonstrated promising immunogenicity.     

水泡性口炎病毒N蛋白原核表达及免疫原性分析

本研究旨在克隆和表达水泡性口炎病毒（vesicular stomatitis virus,VSV）特异性抗原N蛋白,进而纯化并分析其免疫原性。根据GenBank中已发表的VSV基因组N基因序列,分别合成VSV两种不同血清型的N基因,经序列对比分析后,设计合成1对特异性引物,PCR扩增获得约1 300 bp的N基因片段,将目的片段亚克隆至pCold Ⅰ原核表达载体中,经IPTG诱导表达后,采用Ni-NTA树脂亲和层析法纯化重组N蛋白。SDS-PAGE分析表明,N基因在大肠杆菌中得到表达,蛋白大小约为50 ku;Western blotting检测结果表明,该重组蛋白与VSV多克隆抗体发生特异性反应。本试验成功构建了VSV-IND和VSV-NJ的原核表达载体,实现了N蛋白在大肠杆菌中的可溶性表达,纯化后的重组蛋白具有良好的免疫原性。     

基于非洲猪瘟病毒截短p72蛋白的间接ELISA抗体检测方法的建立

【目的】建立非洲猪瘟病毒（African swine fever virus,ASFV）ELISA抗体检测方法。【方法】以重组截短p72（p72s）蛋白作为检测抗原,通过方阵滴定法,确立ELISA的抗原、待检血清和酶标抗体的最佳工作浓度,优化抗原包被、酶标板封闭、血清/酶标抗体反应及底物显色等工作条件;应用受试者工作特征（receiver operating characteristic,ROC）曲线阈值评价标准,确定方法的阴阳性临界值;检测方法的特异性、敏感性、批内与批间重复性;最后分别用建立的ELISA方法和商品试剂盒检测124份血清,比较两者的阳性检出率,并通过Western blotting验证ELISA的检测结果。【结果】ELISA方法的抗原最佳包被浓度为0.5 μg/mL,待检血清与酶标抗体的最适稀释度分别为1∶100和1∶5 000;反应条件为：抗原于37 ℃孵育1 h后经4 ℃包被酶标板过夜、于37 ℃封闭1 h或室温封闭2 h、待检血清和酶标抗体分别于37 ℃反应60和45 min及加入底物后于室温避光显色20 min;阴阳性血清的判定标准为：当待检血清的D_{450 nm}值≥0.365时,判定为阳性;当D_{450 nm}值＜0.365时,判定为阴性。该方法只与ASFV阳性血清发生特异性结合,与猪瘟病毒、伪狂犬病病毒、猪繁殖与呼吸综合征病毒等病毒抗血清均无交叉反应;能检测到ASFV抗血清中的总蛋白量为0.091~0.153 mg/mL,低于商品试剂盒的最低检测量（0.110~0.554 mg/mL）;批内和批间重复性试验的平均变异系数分别为4.70%与5.125%。对临床血清样本检测时,建立方法和商品试剂盒的阳性检出率分别为75.81%（94/124）和32.26%（40/124）。进一步验证表明,Western blotting与ELISA的检测结果完全一致。【结论】建立了基于ASFV-p72s蛋白的ELISA抗体检测方法,该方法特异、敏感和重复性稳定,能够用于ASF临床诊断与流行病学调查,具有极大的开发应用潜力。     

牛传染性鼻气管炎病毒截短gB基因原核表达与间接ELISA诊断方法建立

用DNAstar-Protean分析牛传染性鼻气管炎病毒gB蛋白的亲水性、抗原性和表面展示概率,据IBRVgB全基因序列设计引物,PCR扩增编码385-550位氨基酸的基因序列。将目的片段克隆,酶切鉴定并测序鉴定后,定向连接到pET-28b载体上,转化表达菌DE3并诱导表达。经SDS-PAGE分析,获得大小约为21.4 ku的目的蛋白,与预测值相符。纯化后的蛋白浓度约为2.0 mg/mL,纯度为95.27%。间接ELISA及Western blotting证明,表达的目的蛋白具有抗原性。以该蛋白作为诊断抗原建立的间接ELISA诊断方法,确定的抗原包被量为50μg/mL,血清的最佳稀释倍数为50。与进口IBRV全毒ELISA抗体诊断试剂盒比较,特异性为92.3%、敏感性为93.8%、符合率为93.3%;试验结果表明该诊断方法具有良好的特异性和敏感性。     

猪脑心肌炎病毒抗体间接ELISA检测方法的建立及应用

以猪脑心肌炎病毒(Encephalomyocarditis virus,EMCV)VP1重组蛋白为包被抗原,建立了检测EMCV血清抗体的间接酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA).经优化,获得间接ELISA的最佳反应条件为抗原包被浓度0.625 μg/mL,待...     

非洲猪瘟病毒p22蛋白表达、多克隆抗体制备及间接ELISA方法的建立

【目的】试验旨在表达与纯化非洲猪瘟病毒(African swine fever virus, ASFV)的结构蛋白p22,将其作为包被抗原建立ASFV抗体的间接ELISA检测方法,用于诊断非洲猪瘟。【方法】将ASFV p22编码基因KP177R的截短体(24―145位氨基酸)克隆至原核表达载体pET-32a(+)中,将重组质粒pET-32a-p22转化大肠杆菌BL21(DE3)感受态细胞,经0.1 mmol/L IPTG诱导表达5 h,利用镍柱亲和纯化p22蛋白,并进行Western blotting鉴定。利用p22蛋白免疫BALB/c小鼠制备抗血清,并以含p22全长基因的真核表达质粒pCAGGS-EGFP-fp22转染HEK293T细胞为抗原基质,利用间接免疫荧光(IFA)鉴定抗血清的反应性。以重组p22蛋白为包被抗原,优化最佳抗原包被浓度、待检血清稀释度、封闭条件、抗原抗体反应时间、酶标二抗工作浓度等参数,建立ASFV抗体间接ELISA检测方法,并对临床猪血清样品进行检测。【结果】ASFV p22截短蛋白在大肠杆菌中高水平表达,蛋白产量为0.85 mg/100 g菌体;p22蛋白具...     

猪传染性胃肠炎病毒S基因在昆虫细胞中的表达

为获得具有天然活性的猪传染性胃肠炎病毒(TGEV)重组S蛋白,制备针对猪传染性胃肠炎S蛋白A、D抗原位点的单克隆抗体及建立快速抗体检测方法,本研究将TGEV S基因A、D抗原位点经PCR扩增并克隆入p Fast Bac HBM-TOPO载体,经转座、转染后获得重组杆状病毒,并对重组杆状病毒进行间接免疫荧光(IFA)及Western blot分析,结果显示,TGEV S基因A、D抗原位点在杆状病毒中成功表达,其表达产物为TGE诊断试剂的制备、基因工程疫苗的研制奠定了基础。     

Prokaryotic Expression of PEB1A Protein of Campylobacter jejuni and Establishment of an Indirect ELISA

In order to develop an indirect ELISA method to detect Campylobacter jejuni antibody, PEB1A gene of Campylobacter jejuni was cloned and amplified by PCR, the prokaryotic expression vector pET32a-PEBIA was constructed, and then transferred into the expression strain E.coli BL21 (DE3), and obtained about 47 ku of soluble protein. Western blotting result showed that the expressed recombinant protein had good biological activity, an indirect ELISA method for detecting antibody against Campylobacter jejuni was developed using expressed PEB1A protein as coating antigen,and detected its specificity, sensitivity, repeatability, respectively. The results showed that the established method for detection of Campylobacter jejuni antibody critical value was 0.3424. This method only specifically reacts with Campylobacter jejuni positive sera, and had no cross-reactivity with other antiserum and strong specificity. In addition, the coefficients of variations in both inter-and intra-assay were less than 5% indicating that it had good repeatability and stability. The establishment of this method could be applied to the rapid detection of Campylobacter jejuni in serum, and provided basis for further prevention and control of Campylobacter jejuni diarrhea.     

空肠弯曲菌PEB1A蛋白的原核表达及间接ELISA方法的建立

为了建立检测血清中空肠弯曲菌（Campylobacter jejuni）抗体的间接ELISA方法,试验通过PCR扩增并克隆空肠弯曲菌PEB1A基因,构建原核表达载体pET32a-PEB1A,将该表达载体转化大肠杆菌BL21（DE3）感受态细胞,诱导表达获得约47 ku的可溶性目的蛋白,通过Western blotting证明所表达的重组蛋白具有良好的生物学活性。以纯化后的重组蛋白作为包被抗原,建立空肠弯曲菌抗体间接ELISA方法,对其特异性、敏感性、重复性进行检测。结果表明,本试验建立的空肠弯曲菌抗体间接ELISA检测方法临界值为0.3424,本方法仅与空肠弯曲菌阳性血清发生特异性反应,与其他抗血清无交叉反应,具有较强的特异性,批内、批间的重复性试验变异系数均<5%,具有较好的重复性和稳定性。该方法的建立可应用于血清中空肠弯曲菌抗体的快速检测,为进一步防制空肠弯曲菌腹泻提供依据。