牛结节性皮肤病病毒抗体竞争ELISA检测方法的建立

Establishment of a competitive ELISA method for detecting antibodies against lumpy skin disease virus

为建立高效检测牛结节性皮肤病病毒(lumpy skin disease virus, LSDV)的血清学方法,本研究设计优化LSDV RNA聚合酶30亚基(RPO30)蛋白的密码子,构建其原核表达质粒pET-25b-RPO30,并进行诱导表达和阴离子纯化。纯化后的RPO30蛋白可以被LSDV的阳性血清识别,具有良好的反应原性。用纯化的RPO30蛋白免疫BALB/c小鼠,并将免疫小鼠的脾细胞和骨髓瘤细胞SP2/0进行细胞融合,筛选到能够稳定分泌抗体且具有竞争效果的杂交瘤细胞株1H1。以重组RPO30蛋白为包被抗原,经反应条件的优化,建立了基于RPO30蛋白的竞争ELISA(cELISA)抗体检测方法。本研究建立的cELISA检测方法的最适抗原包被质量浓度为1 mg/L,用含5%牛血清白蛋白(BSA)的PBST作为封闭液37℃孵育2 h,待测血清的最适稀释度为1∶2,1H1抗体稀释度为1∶2 000,兔抗鼠HRP-IgG稀释度为1∶4 000,最佳显色条件是37℃反应15 min。当检测样品的1-S/P≥0.55,判定结果为阳性;当检测样品的1-S/P<0.55,判定结果为阴性。利用该方法检测了LSDV、牛传染性鼻支气管炎病毒(IBRV)、牛支原体(M.bovis)、牛病毒性腹泻病毒(BVDV)、牛布鲁杆菌(B.abortus)、口蹄疫病毒(FMDV)阳性血清,结果显示除了LSDV检测结果阳性外,其他病原的检测结果都是阴性,说明本方法特异性强。利用本试验建立的cELISA检测有免疫背景的临床血清200份,结果显示,其可用于临床疫苗免疫效果的评估。本研究基于重组RPO30蛋白建立的cELISA方法操作简单、特异性强、敏感性高、重复性好,为LSDV的检测和预防提供了可靠的技术支持。

In order to establish an efficient serological detection method for bovine lumpy skin disease virus(LSDV),the prokaryotic expression plasmid pET-25b-RPO30 was constructed after the optimization of the protein codon of RNA polymerase 30 subunit(RPO30)of LSDV.The recombinant RPO30 was expressed and purified using anion purification method,which was recognized by the positive serum of bovine LSDV in a Western blot assay,and has good reactivity.The purified RPO30 protein was used to immunize the BALB/c mice.The splenocytes from the immunized mouse were fused with myeloma cells SP2/0.After screening and subcloning,a hybridoma cell line 1H1 with stable antibody secretion was obtained.A competitive ELISA(cELISA)antibody detection method was established by optimizing reaction conditions using recombinant RPO30 protein as the coating antigen.The optimal conditions for the cELISA method were established as follows:using 1 mg/L rRPO30 as the coating antigen in a 100μL volume overnight at 4℃;blocking with 1%BSA at 37℃for 2 h;the optimal dilution of serum to be tested was 1∶2;the optimal dilution of the 1H1antibody was 1∶2000;and that of rabbit anti-mouse HRP-IgG was 1∶4000;the optimal color development condition was to react at 37℃for 15min.In addition,when the cutoff value was set to 0.55,the consistency between the values of sensitivity and specificity and the identification of serum samples were the largest.Therefore,the cut-off value of PI was set to 0.55.Therefore,samples with 1-S/P<0.55were considered negative,and samples with 1-S/P≥0.55were considered positive.The cross-reactivity with other serum samples including bovine infectious rhinobronchitis virus(IBRV),Mycoplasma bovis,bovine viral diarrhea virus(BVDV),bovine brucella,and foot-and-mouth disease virus(FMDV)were negative by cELISA.A total of 200clinical serum samples with immune background was confirmed using cELISA,the results showed that it can be used to evaluate clinical vaccine immunity.It is the first to establish cELISA method based on recombinant RPO30protein,which is a simple,specific,sensitive,and reproducible serological diagnostic method,providing reliable technical support for the detection and prevention of LSDV.