骆驼斯氏副柔线虫病间接ELISA检测方法的建立
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摘要
为建立骆驼斯氏副柔线虫病间接ELISA(iELISA)诊断方法,本试验对骆驼斯氏副柔线虫半胱氨酸蛋白酶CPR基因进行重组表达,将获取的重组蛋白(rCPR)进行纯化和Western blotting检测,然后以纯化好的重组蛋白为抗原,通过棋盘滴定试验优化了抗原包被浓度、包被条件、抗体稀释度、酶标二抗稀释度、封闭液和封闭时间等,建立了骆驼斯氏副柔线虫病iELISA诊断方法,并对建立的iELISA检测方法进行了重复性、敏感性、特异性试验和临床检测。结果显示,抗原最佳包被浓度为8μg/孔,血清最佳稀释度为1∶50,酶标二抗最佳稀释度为1∶5 000,最佳包被条件为4℃包被过夜,最佳封闭条件为3%BSA封闭2 h。临界值为0.235,待检血清D_(450 nm)值>0.235则确定为阳性。重复性试验中变异系数均<10%,重复性较好;用该方法检测阳性血清的敏感性为96.3%;此方法仅与骆驼斯氏副柔线虫病阳性血清发生特异性反应,与感染了其他寄生虫的阳性血清无交叉反应,特异性为100%;对140份临床血清进行检测,阳性率为86.4%。综上可知,本试验成功建立了一种快速有效诊断骆驼斯氏副柔线虫病的iELISA方法。
In order to establish an indirect ELISA(iELISA) diagnostic method for detection of camel parabronemosis,in this experiment,the CPR gene of cysteine protease was recombinant expression,and the obtained recombinant protein(rCPR) was purified and detected by Western blotting.Using the purified recombinant protein as an antigen,the conditions of coating antigen,coating conditions,antibody dilution,dilution of enzyme-labeled secondary antibody,blocking solution and blocking time were optimized by checkerboard titration test,and the iELISA diagnostic method for detection of Parabronema skrjabini was established.Repeated,sensitive,specific and clinical tests were performed on the established iELISA assay.The results showed that the optimal coating concentration of the antigen was 8 μg/well,the optimal dilution of the serum and the enzyme-labeled secondary antibody were 1∶50 and 1∶5 000,respectively,and the optimal coating condition was 4 ℃ overnight.The optimal blocking condition was 3% BSA for 2 h.The cut-off value was 0.235,and the serum D_(450 nm) value>0.235 was determined to be positive.The coefficient of variation in the repeatability test was <10%,and the repeatability was good;The sensitivity of detecting positive serum by this method was 96.3%;This method only specifically reacted with the positive serum of Parabronema skrjabini,and did not cross-react with other parasite-positive sera,the specificity was 100%;140 clinical sera were tested and the positive rate was 86.4%.In summary,this experiment successfully established an iELISA method for rapid and effective diagnosis of camel parabronemosis.
In order to establish an indirect ELISA(iELISA) diagnostic method for detection of camel parabronemosis,in this experiment,the CPR gene of cysteine protease was recombinant expression,and the obtained recombinant protein(rCPR) was purified and detected by Western blotting.Using the purified recombinant protein as an antigen,the conditions of coating antigen,coating conditions,antibody dilution,dilution of enzyme-labeled secondary antibody,blocking solution and blocking time were optimized by checkerboard titration test,and the iELISA diagnostic method for detection of Parabronema skrjabini was established.Repeated,sensitive,specific and clinical tests were performed on the established iELISA assay.The results showed that the optimal coating concentration of the antigen was 8 μg/well,the optimal dilution of the serum and the enzyme-labeled secondary antibody were 1∶50 and 1∶5 000,respectively,and the optimal coating condition was 4 ℃ overnight.The optimal blocking condition was 3% BSA for 2 h.The cut-off value was 0.235,and the serum D_(450 nm) value>0.235 was determined to be positive.The coefficient of variation in the repeatability test was <10%,and the repeatability was good;The sensitivity of detecting positive serum by this method was 96.3%;This method only specifically reacted with the positive serum of Parabronema skrjabini,and did not cross-react with other parasite-positive sera,the specificity was 100%;140 clinical sera were tested and the positive rate was 86.4%.In summary,this experiment successfully established an iELISA method for rapid and effective diagnosis of camel parabronemosis.
引文
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[8] 赵治国,王海艳,郭铁筝,等.阿拉善双峰驼斯氏副柔线虫感染情况及病理组织学变化[J].动物医学进展,2012,33(11):56-58.ZHAO Z G,WANG H Y,GUO T Z,et al.Infection situation of Parabronema skrjabini and pathological lesions in Alashan bactrian camels[J].Progress in Veterinary Medicine,2012,33(11):56-58.(in Chinese)
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[12] KHALAFALLA R E,ELSEIFY M A,ELBAHY N M.Seasonal prevalence of gastrointestinal nematode parasites of sheep in Northern region of Nile Delta,Egypt[J].Parasitology Research,2011,108(2):337-340.
[13] MORGAN E,TORGERSON P,SHAIKENOV B,et al.Agricultural restructuring and gastrointestinal parasitism in domestic ruminants on the rangelands of Kazakhstan[J].Veterinary Parasitology,2006,139(1-3):180-191.
[14] 郑文青.骆驼斯氏副柔线虫病流行病学调查及生前PCR诊断方法建立[D].呼和浩特:内蒙古农业大学,2015.ZHENG W Q.Studies on epidemiological investigation and established the PCR antemortem diagnosis method of camel parabronemosis[D].Hohhot:Inner Mongolia Agricultural University,2015.(in Chinese)
[15] 赵学亮,王梦雅,孙柯,等.斯氏副柔线虫GP基因的克隆、抗原表位预测及生物信息学分析[J].中国畜牧兽医,2018,45(6):1645-1652.ZHAO X L,WANG M Y,SUN K,et al.Cloning,prediction of epitopes and bioinformatic analysis of GP gene in Parabronema skrjabini[J].China Animal Husbandry & Veterinary Medicine,2018,45(6):1645-1652.(in Chinese)
[16] 冯陈晨,王文龙,呼和巴特尔.骆驼斯氏副柔线虫转录组的高通量测序及分析[J].中国兽医学报,2017,37(4):671-675.FENG C C,WANG W L,HUHE-BATEER.Sequencing and analysis of the transcriptome of Parabronema skrjabini in camel[J].Chinese Journal of Veterinary Science,2017,37(4):671-675.(in Chinese)
[17] 王文龙,冯陈晨,王金玲,等.斯氏副柔线虫半胱氨酸蛋白酶Pj-CPR基因的克隆及原核表达[J].动物医学进展,2018,39(3):30-34.WANG W L,FENG C C,WANG J L,et al.Cloning and prokaryotic expression of Pj_CPR gene of Parabronema skrjabini[J].Progress in Veterinary Medicine,2018,39(3):30-34.(in Chinese)
[18] 柏丽,周俊文,李新海,等.中国6个双峰驼群体微卫星遗传多样性分析[J].中国畜牧兽医,2017,44(6):1734-1745.BAI L,ZHOU J W,LI X H,et al.Analysis on the genetic diversity of six bactrian camel populations in China using microsatellite marker[J].China Animal Husbandry & Veterinary Medicine,2017,44(6):1734-1745.(in Chinese)
[19] 浩斯娜,方舒,卓娜,等.骆驼尿辅助治疗疾病的研究进展[J].中国畜牧兽医,2018,45(11):3279-3285.HAO S N,FANG S,ZHUO N,et al.Research progress on camel urine adjuvant treatment of diseases[J].China Animal Husbandry & Veterinary Medicine,2018,45(11):3279-3285.(in Chinese)
[20] HEWITSON J P,GRAINGER J R,MAIZELS R M.Helminth immunoregulation:The role of parasite secreted proteins in modulating host immunity[J].Molecular & Biochemical Parasitology,2009,167(1):1-11.
[21] LOUISE VON G J,KURT B.Cysteine proteases as potential antigens in antiparasitic DNA vaccines[J].Vaccine,2011,29(34):5575-5583.
[22] DA SILVA M B,URREGO A J R,OVIEDO Y,et al.The somatic proteins of Toxocara canis larvae and excretory-secretory products revealed by proteomics[J].Veterinary Parasitology,2018,259:25-34.
[23] 冯陈晨.斯氏副柔线虫转录组学和蛋白质组学研究及免疫相关基因鉴定[D].呼和浩特:内蒙古农业大学,2017.FENG C C.Transcriptomic and proteomic analysis of Parabronema skrjabini and identification of immune-related genes[D].Hohhot:Inner Mongolia Agricultural University,2017.(in Chinese)
[24] 王钊,王好,钱爱东,等.新型免疫检测方法在五种寄生虫疾病诊断中的应用进展[J].中国兽药杂志,2015,49(7):63-69.WANG Z,WANG H,QIAN A D,et al.Application advances of new immune detection methods in diagnosis of five parasitic diseases[J].Chinese Journal of Veterinary Drug,2015,49(7):63-69.(in Chinese)
[25] SHRESTHA A,FREUDENSCHUSS B,SCHWARZ L,et al.Development and application of a recombinant protein-based indirect ELISA for the detection of serum antibodies against Cystoisospora suis in swine[J].Veterinary Parasitology,2018,258:57-63.
[2] 中华人民共和国国家统计局.2017中国统计年鉴[M].北京:中国统计出版社,2017.NATIONAL BUREAU OF STATISTICS OF THE PEOPLE’S REPUBLIC OF CHINA.China Statistical Yearbook of 2017[M].Beijing:China Statistics Press,2017.(in Chinese)
[3] 中国农业科学院兰州兽医研究所.人和动物寄生线虫图谱[M].北京:中国农业科学技术出版社,2003.LANZHOU VETERINARY RESEARCH INSTITUTE,CHINESE ACADEMY OF AGRICULTUL SCIENCES.Atlas of Parasitic Nematode of Human and Animal[M].Beijing:China Agricultural Science and Technology Press,2003.(in Chinese)
[4] 沈杰,黄兵.中国家畜家禽寄生虫名录[M].北京:中国农业科学技术出版社,2004.SHEN J,HUANG B.Chinese Livestock and Poultry Parasites List[M].Beijing:China Agricultural Science and Technology Press,2004.(in Chinese)
[5] 杨莲茹,杨晓野,刘珍莲,等.内蒙古地区骆驼斯氏副柔线虫(Parabronema skrjabini)病调查[J].内蒙古农业大学学报(自然科学版),2004,25(1):43-45.YANG L R,YANG X Y,LIU Z L,et al.Investigation of Parabronema skrjabini disease of camels in Inner Mongolia region[J].Journal of Inner Mongolia Agricultural University (Natural Science Edition),2004,25(1):43-45.(in Chinese)
[6] SEYED S H.Molecular characterization of the first internal transcribed spacer of rDNA of Parabronema skrjabini for the first time in sheep[J].Annals of Parasitology,2015,61(4):241-246.
[7] HASHEMINASAB S S,JALOUSIAN F,MESHGI B.Molecular and morphological characterization of Parabronema skrjabini of sheep and goats at three different geographical zones in Iran[J].Annals of Parasitology,2016,62(1):55-61.
[8] 赵治国,王海艳,郭铁筝,等.阿拉善双峰驼斯氏副柔线虫感染情况及病理组织学变化[J].动物医学进展,2012,33(11):56-58.ZHAO Z G,WANG H Y,GUO T Z,et al.Infection situation of Parabronema skrjabini and pathological lesions in Alashan bactrian camels[J].Progress in Veterinary Medicine,2012,33(11):56-58.(in Chinese)
[9] SHARKHUU T.Helminths of goats in Mongolia[J].Veterinary Parasitology,2001,101(2):161-169.
[10] ESLAMI A,NABAVI L.Species of gastrointestinal nematodes of sheep from Iran[J].Bulletin Dela Société de Pathologie Exotique,1976,69(1):92-95.
[11] MAGZOUB M,OMER O,HAROUN E,et al.Effect of season on gastrointestinal nematode infection in Saudi Arabian camels (Camelus dromedarius)[J].Journal of Camel Practice and Research,2000,7:107-108.
[12] KHALAFALLA R E,ELSEIFY M A,ELBAHY N M.Seasonal prevalence of gastrointestinal nematode parasites of sheep in Northern region of Nile Delta,Egypt[J].Parasitology Research,2011,108(2):337-340.
[13] MORGAN E,TORGERSON P,SHAIKENOV B,et al.Agricultural restructuring and gastrointestinal parasitism in domestic ruminants on the rangelands of Kazakhstan[J].Veterinary Parasitology,2006,139(1-3):180-191.
[14] 郑文青.骆驼斯氏副柔线虫病流行病学调查及生前PCR诊断方法建立[D].呼和浩特:内蒙古农业大学,2015.ZHENG W Q.Studies on epidemiological investigation and established the PCR antemortem diagnosis method of camel parabronemosis[D].Hohhot:Inner Mongolia Agricultural University,2015.(in Chinese)
[15] 赵学亮,王梦雅,孙柯,等.斯氏副柔线虫GP基因的克隆、抗原表位预测及生物信息学分析[J].中国畜牧兽医,2018,45(6):1645-1652.ZHAO X L,WANG M Y,SUN K,et al.Cloning,prediction of epitopes and bioinformatic analysis of GP gene in Parabronema skrjabini[J].China Animal Husbandry & Veterinary Medicine,2018,45(6):1645-1652.(in Chinese)
[16] 冯陈晨,王文龙,呼和巴特尔.骆驼斯氏副柔线虫转录组的高通量测序及分析[J].中国兽医学报,2017,37(4):671-675.FENG C C,WANG W L,HUHE-BATEER.Sequencing and analysis of the transcriptome of Parabronema skrjabini in camel[J].Chinese Journal of Veterinary Science,2017,37(4):671-675.(in Chinese)
[17] 王文龙,冯陈晨,王金玲,等.斯氏副柔线虫半胱氨酸蛋白酶Pj-CPR基因的克隆及原核表达[J].动物医学进展,2018,39(3):30-34.WANG W L,FENG C C,WANG J L,et al.Cloning and prokaryotic expression of Pj_CPR gene of Parabronema skrjabini[J].Progress in Veterinary Medicine,2018,39(3):30-34.(in Chinese)
[18] 柏丽,周俊文,李新海,等.中国6个双峰驼群体微卫星遗传多样性分析[J].中国畜牧兽医,2017,44(6):1734-1745.BAI L,ZHOU J W,LI X H,et al.Analysis on the genetic diversity of six bactrian camel populations in China using microsatellite marker[J].China Animal Husbandry & Veterinary Medicine,2017,44(6):1734-1745.(in Chinese)
[19] 浩斯娜,方舒,卓娜,等.骆驼尿辅助治疗疾病的研究进展[J].中国畜牧兽医,2018,45(11):3279-3285.HAO S N,FANG S,ZHUO N,et al.Research progress on camel urine adjuvant treatment of diseases[J].China Animal Husbandry & Veterinary Medicine,2018,45(11):3279-3285.(in Chinese)
[20] HEWITSON J P,GRAINGER J R,MAIZELS R M.Helminth immunoregulation:The role of parasite secreted proteins in modulating host immunity[J].Molecular & Biochemical Parasitology,2009,167(1):1-11.
[21] LOUISE VON G J,KURT B.Cysteine proteases as potential antigens in antiparasitic DNA vaccines[J].Vaccine,2011,29(34):5575-5583.
[22] DA SILVA M B,URREGO A J R,OVIEDO Y,et al.The somatic proteins of Toxocara canis larvae and excretory-secretory products revealed by proteomics[J].Veterinary Parasitology,2018,259:25-34.
[23] 冯陈晨.斯氏副柔线虫转录组学和蛋白质组学研究及免疫相关基因鉴定[D].呼和浩特:内蒙古农业大学,2017.FENG C C.Transcriptomic and proteomic analysis of Parabronema skrjabini and identification of immune-related genes[D].Hohhot:Inner Mongolia Agricultural University,2017.(in Chinese)
[24] 王钊,王好,钱爱东,等.新型免疫检测方法在五种寄生虫疾病诊断中的应用进展[J].中国兽药杂志,2015,49(7):63-69.WANG Z,WANG H,QIAN A D,et al.Application advances of new immune detection methods in diagnosis of five parasitic diseases[J].Chinese Journal of Veterinary Drug,2015,49(7):63-69.(in Chinese)
[25] SHRESTHA A,FREUDENSCHUSS B,SCHWARZ L,et al.Development and application of a recombinant protein-based indirect ELISA for the detection of serum antibodies against Cystoisospora suis in swine[J].Veterinary Parasitology,2018,258:57-63.
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