非洲猪瘟病毒P30蛋白单克隆抗体制备、鉴定及阻断ELISA方法的建立

Preparation and Identification of Monoclonal Antibodies to P30 Protein and Establishment of Blocking ELISA to Detecting Antibodies Against African Swine Fever Virus

【背景】非洲猪瘟(ASF)于2018年8月在中国首次出现,对养猪业造成了巨大危害,损失惨重。目前尚无安全有效的疫苗用来预防ASF,于是建立快速特异的检测方法对于防控ASF提供了有效的手段。【目的】制备非洲猪瘟病毒(ASFV)特异性单克隆抗体,建立ASF快速特异性的检测方法。为ASF的检测和防控提供借鉴技术手段。【方法】构建表达载体pET-28a-P30,通过原核表达系统获得ASFV P30重组蛋白,以纯化的P30蛋白为抗原免疫BALB/c小鼠,经过细胞融合和细胞亚克隆制备出ASFV P30蛋白特异性杂交瘤细胞株;对P30蛋白进行截短表达,利用Western Blot和间接酶联免疫吸附试验(iELISA)鉴定单克隆抗体所对应的抗原表位;并利用制备的单克隆抗体建立非洲猪瘟阻断ELISA抗体检测方法。【结果】通过双酶切和PCR验证,结果显示构建出重组载体pET-28a-P30,经测序其序列未发生突变;IPTG诱导后,P30重组蛋白主要表达在包涵体中,分子量约为33 kD。纯化的P30蛋白与弗氏佐剂1:1混合免疫小鼠,3次免疫后,小鼠血清效价达到1:102400,说明表达的蛋白具有良好的免疫原性。经细胞融合和亚克隆,获得8株P30蛋白特异性杂交瘤细胞,Western Blot和间接免疫荧光试验(IFA)检测获得的8株单抗均具有良好的反应性。叠加试验显示8株单克隆抗体均针对相同的抗原位点;截短表达P30蛋白不同片段,选取制备的2-12B单克隆抗体与不同的截短P30蛋白反应,显示单克隆抗体的抗原表位区为187—194aa。利用2-12B单克隆抗体并经过条件优化,成功建立了ASF阻断ELISA抗体检测方法,检测了190份临床样品,并与商品化非洲猪瘟ELISA抗体检测试剂盒进行对比,两方法阳性符合率为90.91%,总符合率为96.32%。【结论】本研究成功获得ASFV P30蛋白,经过iELISA、Western Blot和IFA筛选出反应性良好的特异性单克隆抗体8株,抗原识别表位区为187—194aa。并利用制备的单克隆抗体建立了特异性高,敏感性好的ASFV阻断ELISA抗体检测方法,为ASF的检测及其防控提供了手段和支撑。

【Background】African swine fever(ASF)first appeared in China in August 2018,causing great harm to the pig industry and heavy losses.At present,there is no safe and effective vaccine to prevent ASF,so the establishment of rapid and specific detection method provides an effective means for the prevention and control of ASF.【Objective】To prepare specific monoclonal antibodies against African swine fever virus(ASFV)and establish a rapid and specific detection method for ASF.Provide technical means for the detection and prevention and control of ASF.【Method】The expression vector pET-28a-P30 was constructed and the rP30 protein was obtained by prokaryotic expression system.The purified r P30 protein was used as antigen to immunize BALB/c mice.The specific hybridoma cell line of ASFV P30 protein was prepared by cell fusion and cell subcloning.The truncated expression of P30 protein was performed,and Western Blot and indirect enzyme-linked immunosorbent assay(iELISA)were used to identify the antigen epitope corresponding to the monoclonal antibody.The prepared monoclonal antibody was used to establish the detection method of blocking ELISA antibody for ASF.【Result】The results of double digestion and PCR showed that the recombinant vector pET-28a-P30 was constructed,and the sequence was not mutated by sequencing.After IPTG induction,the recombinant P30 protein was mainly expressed in the inclusion body with a molecular weight of about 33 kD.The purified P30 protein was mixed with 1:1 Freund’s adjuvant to immunize mice.After three immunizations,the serum titer of mice reached 1:102400,indicating that the expressed protein had good immunogenicity.Eight P30 protein-specific hybridoma cells were obtained by cell fusion and subcloning.The eight monoclonal antibodies obtained by Western Blot and indirect immunofluorescence assay(IFA)showed good reactivity.The superposition test showed that all the 8 monoclonal antibodies had the same antigenic sites.Different fragments of P30 protein were truncated,and the prepared 2-12B monoclonal antibodies were selected to react with different truncated P30 proteins,showing that the antigenic epitope region of the monoclonal antibodies was 187-194aa.The ASF blocking ELISA antibody detection method was successfully established by using 2-12B monoclonal antibody and optimizing the conditions.190 clinical samples were detected and compared with commercial African swine fever ELISA antibody detection kit.The positive coincidence rate of the two methods was 90.91%,and the total coincidence rate was 96.32%.【Conclusion】In this study,ASFV P30 protein was successfully obtained.Eight specific monoclonal antibodies with good reactivity were screened by iELISA,Western Blot and IFA,and the antigen recognition epitopes were 187-194 aa.The monoclonal antibody was used to establish a high specificity and sensitivity ASFV blocking ELISA antibody detection method,which provided a means and support for the detection and prevention of ASF.