猴STLV1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 T-lymphotropic Virus 1

作　　者：李丹丹 王乃福 王绥家 刘忠梅 王昱 周晓黎 艾军 高慎阳 凌宗帅 李应国 LI Dan-dan;WANG Nai-fu;WANG Sui-jia;LIU Zhong-mei;WANG Yu;ZHOU Xiao-li;AI Jun;GAO Shen-yang;LING Zong-shuai;LI Ying-guo(Haikou Customs of People’s Republic of China,Haikou 570311,China;Tianjin Customs of People’s Republic of China,Tianjin 300457,China;Harbin Customs of People’s Republic of China,Harbin 150000,China;Chongqing Customs of People’s Republic of China,Chongqing 400020,China;Kunming Customs of People’s Republic of China,Kunming 650228,China;Jinzhou Medical University,Jinzhou 121001,China;Jinan Customs of People’s Republic of China,Jinan 250014,China)

机构地区：[1]中华人民共和国海口海关,海南海口570311 [2]中华人民共和国天津海关,天津和平300457 [3]中华人民共和国哈尔滨海关,黑龙江哈尔滨150000 [4]中华人民共和国重庆海关,重庆渝北400020 [5]中华人民共和国昆明海关,云南昆明650228 [6]锦州医科大学,辽宁锦州121001 [7]中华人民共和国济南海关,山东济南250014

出　　处：《中国兽医杂志》2020年第5期28-33,共6页Chinese Journal of Veterinary Medicine

基　　金：国家重点研发计划(2017YFF0210202);国家质检总局科技项目(2013IK031,2015IK089)。

摘　　要：以基因工程技术制备猴嗜T淋巴细胞病毒I型(STLV1)的STLV1-30蛋白作为诊断抗原,建立用于特异性检测实验猴血清中抗STLV1抗体的免疫梳方法。应用原核表达载体pGEX-4T-1构建STLV1 STLV1-30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。结果显示,最佳抗原包被量为0.02 mg/mL;制备好的免疫梳均能够特异性检测到相应的STLV1阳性血清而不与其他病毒血清间发生交叉反应;该方法能够敏感地检测到1∶400倍稀释的STLV1阳性血清;稳定性和重复性试验结果显示,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11份可疑猴血清样品进行检测,免疫梳方法与ELISA检测结果一致率为100.0%,Kappa=1.000。表明该检测方法具有灵敏度高、特异性强、重复性好等特性,可用于猴T淋巴细胞趋向性病毒I型抗体的检测。In this study,in order to develop a rapid assay for detecting the antibody of simian T-lymphotropic virus 1(STLV1),the immuno combarray(IC)was established using STLV1 recombinant STLV1-30 protein as antigen for specific detection of IgG antibodies of STLV1.The recombinant expression plasmid of STLV1 STIV-30 gene was constructed by using prokaryotic expression vector pGEX-4T-1 and expressed in the BL21 of receptive cells.The recombinant protein was purified as diagnostic antigen and standardized detection procedure of immune comb was established and applied to clinical detection.The results showed that the optimum coating amount of antigen was 0.02 mg/mL.The exstablished IC was able to specifically detect the positive serum of STLV1.The results of stability and repeatability tests showed that there was no cross reaction with the serum of other viruses,the method could sensitively detected 1∶400 time-diluted STLV1 positive serum.The experiment was repeated 3 times,and the coefficient of variation(CV)was less than 10%.For the detection of 11 suspected monkey serum samples using the immune detection method,the consistent rate with comb method and ELISA test was 100%,Kappa=1.000.It shows that the method has high sensitivity,strong specificity,good repeatability and good practicability.It can be used to detect typeⅠantibody of monkey T-lymphotropic virus.

关 键 词：猴嗜T淋巴细胞趋向性病毒I型 STLV1-30蛋白 免疫梳 快速检测方法 抗体 

分 类 号：S865.1[农业科学—野生动物驯养]