副溶血弧菌耐热直接溶血素DAS-ELISA检测方法的建立

Establishment of a DAS-ELISA detection method for thermostable direct hemolysin of Vibrio parahaemolyticus

目的建立副溶血弧菌耐热直接溶血素的双抗体夹心ELISA(double antibody sandwich ELISA,DAS-ELISA)检测方法。方法本研究通过PCR技术扩增副溶血弧菌耐热直接溶血素基因序列,并将其克隆至pET-28a载体后进行原核表达,对表达蛋白进行纯化和鉴定,随后用高纯度的重组蛋白免疫新西兰白兔制备多克隆抗体,得到抗体后利用分子互作测定抗体的亲和力,并利用该抗体建立检测副溶血弧菌耐热直接溶血素的DAS-ELISA方法。通过棋盘法对该方法的反应条件进行优化,建立标准曲线,并对建立的DAS-ELISA方法进行性能评价及初步应用。结果本实验制备的多克隆抗体亲和力可达1×10^(-8),使用该高亲和力抗体建立的DAS-ELISA方法灵敏度为78 ng/mL,定量范围为78~312 ng/mL;与创伤弧菌溶细胞毒素(Vibrio vulnificus hemolysin,VVH)、产气荚膜梭菌α毒素(Clostridium perfringensαtoxin,CPA)以及产气荚膜梭菌ε毒素(epsilon toxin,ETX)均无交叉反应;利用扇贝、花蛤和魁蚶制备海鲜模拟样本,在上述3种模拟样本内添加重组蛋白构建标准曲线,评价本方法复杂基质中检出能力时发现灵敏度均并未发生改变;同时在上述3种模拟样本中添加菌液上清评价本方法检测天然毒素能力,检出率为100%。结论成功构建了一种用于副溶血弧菌耐热直接溶血素检测的DAS-ELISA方法,该方法特异性强、灵敏度高,适用于复杂基质检测,有望开发成副溶血弧菌耐热直接溶血素快速诊断试剂盒,具有良好的应用前景。

This study aimed to establish a double antibody sandwich ELISA(DAS-ELISA)for the detection of thermostable direct hemolysin in Vibrio parahaemolyticus.The gene sequence of thermostable direct hemolysin of Vibrio parahaemolyticus was amplified by PCR and cloned into the pET-28a vector for prokaryotic expression,and the expressed protein was purified and identified.New Zealand White rabbits were immunized with the high purity recombinant protein to prepare polyclonal antibodies.The affinity of the antibodies was determined on the basis of molecular interactions to develop a DAS-ELISA method for the detection of thermostable direct hemolysin in Vibrio parahaemolyticus.The reaction conditions were optimized through the tessellation method,a standard curve was established,and the performance of the established DAS-ELISA method was evaluated.A polyclonal antibody with an affinity of 1×10^(-8)was prepared,and the sensitivity of the DAS-ELISA method with this high affinity antibody was 78 ng/mL,with a quantitative range of 78-312 ng/mL.No cross-reactivity with Vibrio vulnificus hemolysin,Clostridium perfringens alpha toxin and Clostridium perfringens epsilon toxin was observed.The sensitivity of the method was not altered in the detection in complex matrices,evaluated by addition of recombinant proteins to the standard curves of the three mock samples prepared from scallops,clams and arks.The detection rate was 100%when the method was evaluated by addition of bacterial supernatant to the three mock samples.Thus,a DAS-ELISA method for the detection of thermostable direct hemolysin of V.parahaemolyticus was successfully constructed.This method is specific,sensitive and suitable for the detection of complex matrices.It is expected to be developed into a rapid diagnostic kit for thermostable direct hemolysin of V.thus indicating its good application prospects.