基于p54的非洲猪瘟抗体胶体金检测方法建立

Development of P54 Based Colloidal Gold Detection Method for Detection of African Swine Fever Antibodies

为了建立一种能快速检测非洲猪瘟的免疫胶体金方法,本实验根据非洲猪瘟病毒(ASFV)的p54基因的成熟肽序列,设计了有终止子(p54)和无终止子(p54-his)的基因片段,并分别在pET30a载体中进行表达;将纯化后p54或p54-his免疫ASFV抗体阴性猪,制备相应多克隆抗体,4次免疫后用p54-his或p54检测多克隆抗体效价;根据胶体金免疫层析原理,用金颗粒标记纯化的p54抗原作为金标垫,SPA为检测线、p54多克隆抗体为质控线,优化条件,制作胶体金免疫层析试纸;最终用p54-his的多克隆抗体检验试纸条的检测可行性。结果显示,p54和p54-his均能在pET30a表达系统中可溶性表达,免疫猪后可产生特异性抗体,效价达10^(-6);以0.3 mg/mL p54抗原作金标垫、1 mg/mL SPA为捕捉抗原、0.5 mg/mL p54多克隆抗体为质控线制作的试纸条能有效检出p54-his抗体阳性血清。表明建立的胶体金试纸条能作为非洲猪瘟抗体的快速检测方法进一步推广应用。

In order to develop a colloidal gold method for rapid detection of African swine fever virus (ASFV),peptide fragments with terminator (p54) or without terminator (p54-his) were designed according to the published p54 gene sequence and expressed in pET30a vector in the prokaryotic expression.The purified p54 or p54-his was used to immunize pigs without ASFV antibodies.After4 times of immunization,p54-his or p54 was used to respectively detect the titers of polyclonal antibodies.According to the principle of colloidal gold immunochromatography,the purified p54 antigen labeled with gold particles was set as the gold standard pad and SPA as the detection line while anti-p54 polyclonal antibodies was set as the quality control line.The conditions were optimized to prepare colloidal gold strips.Finally,serum samples with anti-p54-his polyclonal antibodies were used to check the feasibility of the strips.The results showed that both p54 and p54-his were expressed in pET30a in soluble form and induced specific antibodies with titers of 10^(-6).The optimized reaction conditions included gold strips with 0.3 mg/mL p54 as gold standard pad,1 mg/mL SPA as capture antigen and0.5 mg/mL anti-p54 polyclonal antibody as quality control line.The results showed that the colloidal gold test strips could be applied as a rapid detection method for antibody detection of African swine fever.