鸭疫里默氏杆菌外膜蛋白A原核表达及间接ELISA方法的建立和初步应用

Prokaryotic expression of outer membrane protein A of Riemerella anatipestis and establishment and preliminary application of indirect ELISA method

关键词:  鸭疫里默氏杆菌  外膜蛋白A  原核表达  ELISA。

Key words:  Riemerella anatipestifer  Outer membrane protein A  Prokaryotic expression  ELISA.

摘要:

本试验为建立鸭疫里默氏杆菌（Riemerella anatipestifer, RA）间接酶联免疫吸附试验（ELISA）方法，以RA贵州分离株RAG06基因组为模板扩增并克隆鸭疫里默氏杆菌OmpA基因，构建pET32a-OmpA重组原核表达质粒，转化大肠杆菌BL21（DE3）感受态细胞，重组菌经诱导、超声、纯化后，通过SDS-PAGE和Western blotting分析鉴定获得OmpA重组蛋白大小约57 KD；以OmpA重组蛋白为包被抗原初步建立ELISA方法并优化，经多次方阵检测结果显示，OmpA蛋白以2.243 mg/mL的浓度进行包被，血清以1:100稀释，抗原包被条件为4℃过夜、封闭条件为37℃ 120min、血清反应时间为37℃60min、酶标抗体工作浓度为1:1000、酶标抗体反应时间为37℃ 60min、显色时间为15min为最佳条件。确定临界值为0.389。特异性试验表明RA阳性血清有较好反应外，其他血清均无明显反应。批内变异系数2.77%-8.00%，批间变异系数为1.10%-7.80%。当阳性血清稀释比例为1:1600时，仍可判断为阳性，敏感性较高。应用该方法检测贵州省120份鸭血清、65份鹅血清证明该方法能用于水禽源鸭疫里默氏杆菌的检测，应用该方法检测灭活疫苗免疫后的鸭血清和卵黄抗体，结果表明符合灭活疫苗消长规律。本试验建立的ELISA方法，为RA的流行病学调查和RA抗体监测提供了血清学诊断方法。

Abstract:

This trial was conducted to develop an indirect Enzyme Linked Immunosorbent Assay (ELISA) for Riemerella anatipestifer (RA).?The recombinant prokaryotic expression plasmid PET32A-OMPA was constructed and transformed into escherichia coli BL21 (DE3) competent cells. After induction, ultrasound and purification of the recombinant strain, the OmpA gene of RA was amplified and cloned using the RAG06 genome of Guizhou strain RA as template. About 57 KD OmpA recombinant protein was identified by SDS-PAGE and Western blotting analysis.ELISA method was preliminarily established and optimized by using OmpA recombinant protein as coated antigen. The results of multiple square array detection showed that OmpA protein was coated at the concentration of 2.243 mg/mL and the serum was diluted at 1:100.The optimal conditions were as follows: 4℃ overnight for antigen coating, 37℃ for 120min for sealing, 37℃ for 60min for serum reaction time, 37℃ for 60min for elisa concentration 1:1000, 37℃ for 60min for ELISA reaction time, and 15min for chromogenic time. The critical value is determined to be 0.389.??The specificity test showed that RA positive serum had good reaction, but no obvious reaction to other serums. The coefficient of variation within batches was 2.77%-8.00%, and the coefficient of variation between batches was 1.10%-7.80%.When the dilution ratio of positive serum is 1:1600, it can still be judged as positive with high sensitivity.120 duck serum samples and 65 goose serum samples were detected in Guizhou Province, which proved that the method could be used for the detection of RA from waterfowls. The serum and egg yolk antibodies of ducks immunized with inactivated vaccine were detected by this method, and the results showed that it was consistent with the growth and decline rule of inactivated vaccine. The ELISA method established in this study provided a serological diagnosis method for the epidemiological investigation and antibody monitoring of RA.