猴免疫缺陷病毒抗体免疫梳检测方法的建立及应用  

Establishment and application of a new immune-comb assay for detection of serum antibody against simian immunodeficiency virus

作　　者：李丹丹 王绥家 陈平亚 张体银[2] 杨俊兴[3] 刘忠梅 高慎阳[5] LI Danda;WANG Suijia;CHEN Pingya;ZHANG Tiyin;YANG Junxing;LIU Zhongmei;GAO Shenyang(Technical Center of Hainan Entry-Exit Inspection and Quarantine Bureau,Haikou 570311,China;Technical Center of Fujian Entry-Exit Inspection and Quarantine Bureau,Fuzhou 350001;Technical Center of Animal and Plant Inspection and Quarantine,Shenzhen Entry-Exit Inspection and Quarantine Bureau,Shengzhen 518001;Technical Center of Heilongjiang Entry-Exit Inspection and Quarantine Bureau,Harbin 150001;Department of Animal Husbandry and Veterinary Medicine,Liaoning Medical University,Jinzhou 121001)

机构地区：[1]海南出入境检验检疫局检验检疫技术中心,海口570311 [2]福建出入境检验检疫局检验检疫技术中心,福州350001 [3]深圳出入境检验检疫局动植物检验检疫技术中心,广东深圳518001 [4]黑龙江出入境检验检疫局检验检疫技术中心,哈尔滨150001 [5]辽宁医学院畜牧兽医学院,辽宁锦州121001

出　　处：《中国实验动物学报》2018年第2期217-223,共7页Acta Laboratorium Animalis Scientia Sinica

基　　金：国家质检总局科技项目(No.2013IK031;No.2013IK051;No.2015IK089);福建省科技计划重点项目(No.2014N0001);海南省应用技术研究与开发专项项目(No.ZDXM20130025);广东检验检疫局科技计划项目(No.2011GDK44;No.2013GDK04)~~

摘　　要：目的以基因工程技术制备猴免疫缺陷病毒(SIV)的SIV30蛋白作为抗原诊断试剂,建立免疫梳方法(IC)用于特异性检测实验猴血清中抗SIV的抗体Ig G。方法应用原核表达载体p GEX-4T-1构建SIV SIV30基因的重组表达质粒,并在感受态细胞BL21中表达,将该重组蛋白纯化后作为诊断抗原,建立免疫梳标准化检测程序,并应用于临床检测。结果最佳抗原包被量为0.02 mg/m L;制备好的IC均能够特异性检测到相应的SIV阳性血清而不与其他病毒血清间发生交叉反应,表明该检测方法特异性强;敏感性分析结果表明,SIV30蛋白能够敏感地检测到1∶400倍稀释的SIV阳性血清;稳定性和重复性试验结果表明,同一样品重复检测3次,变异系数(CV)均小于10%;利用该检测方法在对10份可疑猴血清样品进行检测,IC与ELISA检测结果一致率为100.0%,Kappa=1.000。结论原核表达了SIV30蛋白,制备了IC,并应用于临床检测。该检测方法灵敏度高、特异性强、重复性好,具有良好的实用性。Objective SIV30 protein of simian immunodeficiency virus(SIV)was prepared by genetic engineering technique as an antigen diagnostic reagent,to establish an immune comb method for the specific detection of anti SIV IgG in monkey serum.Methods Recombinant expression plasmid of SIV SIV30 gene was constructed by prokaryotic expression vector pGEX-4T-1,and expressed in the competent BL21 cells.The recombinant protein was purified as a diagnostic antigen,and a standardized procedure for the detection of immune comb was established and applied for clinical detection.Results The optimum coating amount of antigen was 0.02 mg/mL.The prepared IC was able to specifically detect the positive serum of SIV.There was no cross reaction between the sera of other viruses.It showed a high specificity of the detection method.Sensitivity analysis showed that the SIV30 protein was able to detect 1∶400 times diluted SIV positive sera.The result of stability and repeatability test(the same sample was repeated 3 times)showed that the coefficient of variation(CV)was less than 10%.The serum samples of 10 suspicious monkeys were detected by this method,showing a consistent rate of comb method and ELISA test result of 100%,Kappa=1.000.Conclusions SIV30 protein is expressed in prokaryotic cells.The immune comb is prepared,and is successfullyl applied in clinical examination.It shows that the method has a high sensitivity,strong specificity,good reproducibility and practicability.

关 键 词：猴免疫缺陷病毒 SIV30蛋白 免疫梳 快速检测方法 抗体 

分 类 号：Q95-33[生物学—动物学]