摘要
通过对布鲁菌优势抗原eryA基因进行原核表达,在获得目的蛋白的基础上建立以该蛋白为包被抗原的间接ELISA方法。构建原核表达载体pET-30a-eryA,并将其转入大肠杆菌BL21(DE3),IPTG诱导表达,镍柱亲和纯化eryA重组蛋白,SDS-PAGE鉴定纯化蛋白,Western-Blotting检测反应原性后建立间接ELISA方法。反应条件优化后,抗原包被浓度为0.3125μg/mL,37℃包被2 h;封闭液为5%猪源明胶,37℃作用2 h;血清稀释度为1∶100,37℃作用1 h;酶标抗体稀释度为1∶8000,37℃作用45 min。血清稀释度为1∶1600时,仍然检测为阳性,证明建立的间接ELISA方法灵敏性良好;其他菌种及病毒经过特异性检测,均为阴性,证明建立的间接ELISA方法特异性良好;通过重复性检测,批间及批内变异系数均小于10%,证明建立的间接ELISA方法重复性良好。检测临床血清样品,建立的方法与试管凝集试验总符合率为95.7%,证明该方法可初步应用于临床诊断。
In this study,the prokaryotic expression of Brucella dominant antigen eryA gene was used to obtain the target protein.On this basis,an indirect ELISA method using this protein as the coating antigen was established.The prokaryotic expression vector pET-30a-eryA was constructed and transformed into E.coli BL21(DE3)to induce expression with IPTG.The indirect ELISA method was established after the eryA recombinant protein was purified by Ni column affinity,identified and purified by SDS-PAGE,and detected by Western-Blotting on reactogenicity.After optimizing the reaction conditions,the antigen coating concentration was 0.3125μg/mL,which was coated at 37℃for 2 h.The blocking solution was 5%gelatin from porcine skin,which was reacted at 37℃for 2 h.The dilution of serum was 1∶100,which was reacted at 37℃for 1 h.The antibody dilution was 1∶8000,and the reaction was performed at 37℃for 45 min.When the serum dilution was 1∶1600,and the test was still positive,which proved that the method was sensitive.The specificity of other strains and viruses was negative,which proved that the specificity of indirect ELISA was good.After repetitive test,the coefficients of variation between and within batches were both less than 10%,which proved that the repeatability was good.The total coincidence rate between the established method and the tube agglutination test in detection of clinical serum samples was 95.7%,which proved that the method could be preliminarily applied to clinical diagnosis.
作者
程家海
宋前进
郭昊
侯丽娜
杨超
王凤雪
关平原
温永俊
CHENG Jia-hai;SONG Qian-jin;GUO Hao;HOU Li-na;YANG Chao;WANG Feng-xue;GUAN Ping-yuan;WEN Yong-jun(College of Veterinary Medicine,Inner Mongolia Agricultural University,Key Laboratory of Clinical Diagnosis and Treatment of Animal Diseases,Ministry of Agriculture and Rural Affairs,Hohhot010018,China)
出处
《畜牧与饲料科学》
2020年第6期7-12,25,共7页
Animal Husbandry and Feed Science
基金
内蒙古自治区科技计划项目“布鲁菌病免疫方法及新型可鉴别疫苗的开发”(201702165)。
作者简介
程家海(1997-),男,硕士研究生,主要研究方向为动物传染病学;通信作者:温永俊(1979-),男,教授,博士,博士生导师,主要研究方向为动物疫苗与分子免疫学;通信作者:关平原(1961-),男,教授,博士,博士生导师,主要研究方向为动物传染病的诊断与防控。
