基于塔姆德病毒NP蛋白间接ELISA方法的建立  

Establishment of an indirect ELISA technique based on a nucleoprotein of the Tamdy virus

作　　者：古丽娜孜·沙都汉 美丽排提·玉素甫 阿布力米提·莫明 刘利平 孙素荣[1,2] 丁军涛 Gulinazi Shaduhan;Meilipaiti Yusufu;Abulimiti Moming;LIU Li-ping;SUN Su-rong;DING Jun-tao(College of Life Science and Technology Xinjiang University,Urumqi 830046,China;Xinjiang Key Laboratory of Biological Resources and Genetic Engineering)

机构地区：[1]新疆大学生命科学与技术学院,新疆乌鲁木齐830046 [2]新疆生物资源基因工程重点实验室

出　　处：《中国病原生物学杂志》2021年第9期991-996,共6页Journal of Pathogen Biology

基　　金：国家自然科学基金项目(No.81960369;81760365);新疆高校科研计划自然科学重点项目(No.XJEDU2019I002)。

摘　　要：目的原核表达和纯化Tamdy病毒(TAMV)核蛋白(NP),以此为抗原建立间接ELISA方法,以期用于TAMV感染血清流行病学调查。方法采用PCR法扩增NP基因,构建重组质粒(pET-32a-NP)并转化至E.coli BL21中诱导表达NP蛋白,15%SDS-PAGE鉴定和镍柱亲和层析纯化重组NP蛋白,以TAMV阳性羊血清为一抗,采用ELISA检测其抗原性。以纯化的pET-32a-rNP作为包被抗原,通过方阵滴定优化反应条件,建立检测TAMV血清抗体的间接ELSIA方法,评价其重复性、敏感性和特异性。检测采自新疆部分地区动物血清,采用建立的ELISA方法检测TAMV抗体。结果成功构建pET-32a-NP重组质粒和表达菌株pET-32a-rNP,重组蛋白大小约为72 ku并具有反应原性;ELISA优化试验确定的最佳包被浓度为4μg/ml、封闭液浓度为3%、血清稀释度为1∶100、血清孵育时间为100 min,酶标抗体稀释度为1∶3000,TMB显色时间为6 min;通过检测30份阴性样品确定临界值,当样品吸光充A450值≥0.268时判定为阳性;重复性试验结果表明,批内和批间变异系数(CV)<10%。敏感性试验表明,阳性血清稀释度达1∶600,具有良好的敏感性。特异性试验表明,该方法不与GTV、CCHFV和SFTSV发生交叉反应。取464份采自新疆不同地区的羊、狗和鼠血清标本进行ELISA检测,检出TAMV阳性血清68份,阳性率14.66%。结论建立的检测TAMV血清抗体的间接ELISA方法敏感、特异,且重复性良好,可用于TAMV的血清流行病学调查。Objective To express a nucleoprotein(NP) of the Tamdy virus(TAMV) in a prokaryotic expression system and to purify that NP in order to establish an indirect ELISA technique for seroepidemiological investigation of TAMV infections. Methods The NP gene was amplified using PCR, and the recombinant plasmid pET-32 a-NP was transformed into E. coli BL21 to induce the expression of the NP. The NP was identified using 15% SDS-PAGE and purified using Ni column affinity chromatography. The antigenicity of the NP was detected with ELISA using TAMV-positive sheep serum as the primary antibody. Using purified pET-32 a-rNP as the encapsulated antigen, an indirect ELISA technique for detection of serum antibodies against TAMV was established using square array titration to optimize the reaction conditions, and its repeatability, sensitivity, and specificity were evaluated. Animal serum was collected from several areas in Xinjiang, and TAMV antibodies were detected using the ELISA technique that was established. Results The recombinant plasmid pET-32 a-NP and the expression strain pET-32 a-rNP were successfully constructed. The size of the recombinant protein was about 72 ku and it was reactive. The optimal encapsulation concentration was 4 μg/mL, the concentration of blocking solution was 3%, serum dilution was 1:100, serum incubation time was 100 min, ELISA antibody dilution was 1:3 000, and the TMB color development time was 6 min. The critical value was determined by testing 30 negative samples, and the positive value was determined when the A450 value of the sample was greater than or equal to 0.268. A repeatability test indicated that the coefficient of variation(CV) between and within batches was 10%. A sensitivity test indicated that the dilution of positive serum reached 1:600, indicating good sensitivity. A specificity test indicated that the technique did not cross react with GTV, CCHFV, or SFTSV. A total of 464 serum samples were collected from sheep, dogs, and mice in different areas of Xinjiang, and 68 were found

关 键 词：Tamdy病毒 核蛋白 原核表达 间接ELISA 

分 类 号：R373.3[医药卫生—病原生物学]