血清4型禽腺病毒Fiber蛋白的原核表达及其在抗体检测中的应用

Prokaryotic Expression of Fiber Protein in Serotype 4 Avian Adenovirus and Its Application in Antibody Detection

为建立特异的血清4型禽腺病毒(FAdV-4)血清学抗体检测技术,试验将FAdV-4纤突蛋白基因fiber-2克隆至载体pET-28a中,构建原核表达载体pET-28a-Fiber-2,获得纯化的重组蛋白His-Fiber-2,以纯化的His-Fiber-2作为包被抗原建立检测Fiber-2抗体的间接ELISA方法,对该方法进行特异性、重复性、稳定性、灵敏性试验,并与BioChek FAdV商品化ELISA试剂盒检测结果进行比较。结果显示:建立的ELISA仅与抗FAdV-4阳性鸡血清发生反应,与检测的抗其他病原的血清以及SPF鸡血清均无反应性,批间重复性与批内重复性良好,其变异系数分别在4.345%~6.983%和4.326%~9.391%之间,包被酶标板保存12个月后,变异系数为4.4%~10.0%,其检测灵敏度是间接免疫荧光检测技术的8~32倍,是基于包被GST-Fiber-2蛋白ELISA的2倍;建立的ELISA对FAdV-4灭活疫苗免疫的鸡血清检出率高于BioChek试剂盒,两者对临床感染FAdV-4的鸡血清的检测结果一致。研究表明,试验构建的基于重组蛋白His-Fiber-2的检测FAdV-4抗体的间接ELISA方法为临床上FAdV-4感染监测以及疫苗的免疫评价提供了技术,具有良好的应用前景。

To establish a specific serological antibody detection method for serotype 4 fowl adenovirus(FAdV-4),the fiber-2 gene of FAdV-4 was cloned into the vector pET-28a to construct the prokaryotic expression vector pET-28a-Fiber-2 in this study,then the recombinant protein His-Fiber-2 was purified and used as coating antigen,and an indirect ELISA method for de-tecting Fiber-2 antibody was successfully established.The specificity,repeatability,stability,sensitivity of the ELISA method were dtected,and the detect effect were compared with those of BioChek FAdV commercial ELISA kit.The results showed that the established ELISA method only reacted with anti-FAdV-4 positive chicken serum,and did not react with the detected serum against other pathogens and SPF chicken serum,which had a good interbatch/intra batch repeatability,with coefficients of variation ranged from 4.345%to 6.983%and 4.326%to 9.391%,respectively;The coated ELISA plates were stable after stored for 12 months,and the coefficient of variation was from 4.4%to 10.0%,and which was 8 to 32 times more sensitive than indirect immunofluorescence detection technique and twice as sensitive as an ELISA based on coated GST-Fiber-2 protein.In comparison with,the detection rate of chicken serum immunized with inactivated FAdV-4 vaccine by the established ELISA was higher than that of BioChek kit,and the detection results of serum from chicken clinically infected with FAdV-4 were identical.In conclu-sion,the established ELISA method for detection of FAdV-4 antibody based on the recombinant protein His-Fiber-2 provided technology for the surveillance of FAdV-4 infection and evaluation of vaccines,and showed promising application.