非洲猪瘟病毒无标签p30-ELISA抗体检测方法的建立及应用

Establishment and Application of an Indirect ELISA Antibody Detection Method Based on African Swine Fever Virus Tag-free p30 Protein

非洲猪瘟(African swine fever,ASF)是由非洲猪瘟病毒(African swine fever virus,ASFV)引起猪的一种急性、热性、出血性、高度接触性传染病,临床症状以败血症、皮炎和关节炎为特征,高发病率和高死亡率。为建立临床检测ASFV抗体的间接ELISA检测方法,本研究扩增了ASFV-CP204L基因,通过pET-30a原核表达系统表达p30蛋白,使用Ni-NTA纯化表达产物,通过肠激酶切除外源性蛋白,得到无His-组氨酸标签的p30蛋白,以此为诊断抗原,建立间接ELISA方法。结果显示:表达的无标签p30重组蛋白大小约为30 ku,与ASF阳性猪血清具有较好的反应原性;确定ELISA抗原包被浓度为1μg·mL^(-1),根据ROC曲线下面积确定S/P值>0.398判定为阳性,批内、批间变异系数均<10%;与PCV2、CSFV、PRV-gE、PRRSV阳性血清无交叉反应与INGENASA商品化试剂盒总符合率为97.78%。用该方法分别检测标准阳性血清、动物感染试验血清和收集的区域性临床血清644份,该方法最低可检测到1∶512倍稀释的标准阳性血清样品;检测感染动物血清,其中80%(4/5)的试验动物在第10天抗体为阳性。644份临床猪血清样品中抗体阳性率为7.61%,其中,母猪、后备母猪、仔猪、保育猪和育肥猪抗体阳性率分别为3.03%、0%、4.94%、7.55%和28.7%。本试验建立的ASFV-p30间接ELISA方法具有良好的特异性、灵敏度和重复性,可应用于ASFV的抗体检测,为ASF的诊断和流行病学调查提供了技术手段。

African Swine fever(ASF),caused by infection with the ASF virus(ASFV),is one of the most serious viral diseases affecting pigs.It is featured by acute,febrile,hemorrhagic,high morbidity and mortality,etc.The aim of this paper is to develop an efficient and clinically convenient indirect ELISA antibody detection method for ASFV.Here,the ASFV-CP204L gene was amplified from positive samples,and the p30 protein was expressed through the pET-30a prokaryotic expression system.The expressed product was further purified with Ni-NTA,and the His protein was removed by enterokinase.Thus,an indirect ELISA method was established based on the purified recombinant tag-free p30 protein.An approximate 30 ku fusion p30 tag-free protein was observed.The results showed that the protein expressed in this study has good reactogenicity with ASF-positive pig serum.It was finally determined that the ELISA antigen coating concentration was 1μg·mL^(-1).The positive value(S/P)was more than 0.398 by the area under the ROC curve.The coefficients of inter-and intra-batches were less than 10%,indicating the good repeatability of the method.This method can detect at least 512-fold diluted standard positive serum samples.In the detection of experimentally infected sera,80%of the samples were seroconversion on the 10th day.In this study,a total of 644 pig serum samples were collected.The results showed that the positive rates were 7.61%.Among them,the positive rates of antibodies in sows,gilts,piglets,nursery pigs and fattening pigs were 3.03%,0%,4.94%,7.55%and 28.7%,respectively.The indirect ELISA method based on the tag-free ASFV-p30 protein has good specificity,sensitivity and reproducibility,and can be initially applied to the detection of antibodies of ASFV.It provides a technical tool for epidemiologically serological investigation of ASF.