非洲猪瘟CD2v截短蛋白的原核表达及间接ELISA方法的建立  

Prokaryotic expression of CD2v truncated protein of African swine fever virus and establishment of an indirect CD2v-ELISA

作　　者：胡巧云 黄小久 唐小明[1] 何世成[1] 林源[1] 彭志 石建 王卫国[1] 刘道新[1] 王昌建[1] HU Qiaoyun;HUANG Xiaojiu;TANG Xiaoming;HE Shicheng;LIN Yuan;PENG Zhi;SHI Jian;WANG Weiguo;LIU Daoxin;WANG Changjian(Hunan Center for Animal Diseases Control and Prevention,Changsha 410014,China;Hunan Sino-Clean Bio Ltd.,Changsha 410600,China)

机构地区：[1]湖南省动物疫病预防控制中心,湖南长沙410014 [2]湖南中净生物科技有限公司,湖南长沙410600

出　　处：《中国兽医学报》2022年第6期1103-1108,共6页Chinese Journal of Veterinary Science

基　　金：湖南省重点领域研发计划资助项目(2019NK2181);湖南省现代农业产业体系建设岗位专家基金资助项目(湘财预(2021)0002号)。

摘　　要：为建立非洲猪瘟(ASF)抗体检测方法,本研究根据ASFV HLJ18株基因组序列合成CD2v蛋白的胞外区段基因序列,连接至原核表达载体pET28a,构建表达质粒pET28a-CD2v。经测序鉴定正确后转化至大肠杆菌表达菌株BL21(DE3)pLysS,构建表达菌株p ET28a-CD2v/BL21(DE3)PLysS。经IPTG诱导和镍柱纯化后,获得纯化的CD2v蛋白并经SDS-PAGE和Westernblot鉴定。以纯化的CD2v蛋白作为包被抗原,通过优化反应条件建立检测CD2v抗体的间接ELISA方法,并评价该方法的特异性、敏感性和重复性及在临床血清中的应用。结果显示,经测序构建的表达质粒序列正确,CD2v蛋白表达经SDS-PAGE可见略小于25k Da的条带,Westernblot显示能与ASFV感染血清发生特异性反应。建立了ASFCD2v蛋白抗体检测方法CD2v-ELISA,抗原包被质量浓度为50μg/L,血清稀释倍数为1∶100,羊抗猪HRP-IgG稀释倍数为1∶100000,阴阳性临界值为0.26;与PRRSV、CSFV、PRV(g I)、PRV(gb)、FMDV血清不发生反应,具有良好的特异性;ASF阳性血清稀释至128000倍后仍检测为阳性,具有较高的敏感性;重复性结果显示,批间和批内变异系数均小于10%。使用CD2v-ELISA检测154份临床血清,阳性结果74份,阳性率为48.05%。与2个国产、2个进口商品化ASFV抗体试剂盒的检测结果比较,CD2v-ELISA方法跟其他方法的符合率较高,分别为95.45%,94.16%,88.31%,93.51%。因此,本研究成功建立了基于CD2v胞外区段抗原的ELISA方法,可用于ASF临床抗体的检测。In this study,the gene sequence of the extracellular region of CD2 v protein was synthesized according to sequence of ASFV HLJ18 strain.The gene sequence of the extracellular region of CD2 v protein was linked to the prokaryotic expression vector pET28 a to construct the expression plasmid pET28 a-CD2 v.After transformation,expression strain pET28 a-CD2 v/BL21(DE3) PLysS was constructed.The strain was induced by IPTG,then protein was purified by Ni-HTA affinity chromatography column.Coaling with purified CD2 v protein,an indirect ELISA method for detecting CD2 v antibody(CD2 v-ELISA) was established after optimizing the reaction conditions.The specificity,sensitivity and repeatability and its application in clinical serum were evaluated.The results showed that the CD2 v protein expressed in SDS-PAGE was a little less than 25 kDa.Western blot analysis showed that the CD2 v protein could react specifically with ASFV infected serum.A method for detecting CD2 v protein antibody was established.The optimum concentration of coating antigen was 50 μg/L and the dilution ratio of serum were 1:100 times and the dilution ratio of goat ant-swine HRP-IgG was 1:100 000.The critical value of negative and positive was 0.26.The CD2 v-ELISA did not react with PRRSV,CSFV,PRV(GI),PRV(GB) and FMDV serum,and had good specificity.It could detect ASFV positive serum after being diluted to 128 000,which showed high sensitivity.Repetitive results showed that the coefficients of variation in intro or inter batches were both less than 10%.CD2 v-ELISA was used to detect 154 clinical pig sera,and 74 of them were positive,with a positive rate of 48.05%.The coincidence rate of CD2 v-ELISA with other 4 detection kits were 95.45%,94.16%,88.31 % and 93.51 %,respectively.Therefore,an indirect ELISA method based on the extracellular region antigen of CD2 v was successfully established,which can be used for the detection of clinical antibodies against ASF.

关 键 词：非洲猪瘟 CD2v截短蛋白 间接CD2v-ELISA 

分 类 号：S855.3[农业科学—临床兽医学]