| Ⅰ群禽腺病毒Hexon蛋白的原核表达及间接ELISA检测方法的建立与应用 |
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| 引用本文：张 曼，韩 飞，高 睿，何亚鹏.Ⅰ群禽腺病毒Hexon蛋白的原核表达及间接ELISA检测方法的建立与应用[J].西北农业学报,2018,27(5):634~640 |
| DOI:10.7606/j.issn.1004-1389.2018.05.004 |
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| 基金项目:陕西省咸阳市重大科技创新（2016K01-43）；杨凌农业高新技术示范区重大科技项目（2016-XY-16）。 |
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| 中文摘要:旨在原核表达Ⅰ群禽腺病毒（Fowl adenovirus groupⅠ，FAVⅠ）的Hexon蛋白，并以Hexon蛋白为包被抗原建立血清的间接ELISA检测方法。将FAVⅠ群Hexon基因克隆至原核表达载体pET-28a（+）中进行重组Hexon蛋白的诱导表达，对表达产物进行纯化，并进行Westernblot鉴定；纯化后的Hexon蛋白作为包被抗原，优化反应条件，建立血清的间接ELISA检测方法，并进行特异性、灵敏性和重复性试验，最后对临床样品进行检测。结果表明，原核表达获得大小约为37.4 ku的重组Hexon蛋白；Westernblot结果表明重组蛋白有良好的特异性和抗原反应性。FAVⅠ间接ELISA法优化反应条件为：抗原最佳包被质量浓度为5.0 mg/L，封闭条件为37 ℃作用1 h，血清以1∶200倍稀释作用1 h，酶标二抗以1∶5 000倍稀释作用1 h，TMB显色时间为10 min。在该优化条件下，OD450≥0.322为阳性，反之为阴性；特异性试验结果表明，对鸡新城疫、禽流感、沙门菌、金黄色葡萄球菌、多杀性巴氏杆菌和大肠埃希菌阳性血清检测均为阴性；对阳性血清的敏感性可达1∶1 200， 70份阳性血清的阳性率为97.1%；重复性试验结果表明，批内和批间OD450值的变异系数分别为1.2%～3.5%和5.1%~8.3%；用此方法对临床279份鸡血清样品检测的阳性率为51.3%。表明建立的ELISA检测方法可用于FAVⅠ抗体水平的检测。 |
| 中文关键词:Ⅰ群禽腺病毒 Hexon蛋白 原核表达 间接ELISA |
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| Establishment and Application of Prokaryotic Expression of Hexon Protein of Fowl Adenovirus GroupⅠand Indirect ELISA in Escherichia coli |
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| Abstract:Using the Hexon protein as the antigen,prokaryotic expression of fowl adenovirus groupⅠ Hexon protein, the indirect ELISA detection method was established. FAVⅠ serotype A Hexon gene was cloned into Escherichia coli expression vector pET-28a (+) to induce the expression of recombinant Hexon protein. Hexon protein was purified and tested by Western blot. Using the recombinant Hexon protein as the antigen; the indirect ELISA detection method was established to detect the level of FAVⅠantibody in serum. Specificity, sensitivity and reproducibility tests were carried out, and the clinical samples were tested. The recombinant protein with the size of 37.4 ku was obtained. Western blot showed that the purified Hexon protein had good specificity and antigen reactivity. The optimal reaction conditions were established as follows: the optimal mass concentrations of antigen was 5.0 mg/L, closed at 37 ℃ for 1 h; the serum dilution was 1∶200，reacting 1 h ; the second antibody dilution was 1∶5 000, reacting 1 h ; the TMB reacting time was 10 min. Criteria for determining the negative and positive results of serum samples were OD450≥0.322 and OD450<0.322, respectively. Specificity tests showed that the results of newcastle disease, avian influenza, Salmonella spp, Staphylococcus aureus, Pasteurella multocida, E.coli positive sera were negative. The sensitivity of positive serum was up to 1∶1 200. 70 positive sera were tested and the positive rate was 97.1%. The repeatability test showed that the coefficient of variation of OD450 between the same batch and different batch were 1.2%-3.5% and 5.1%-8.3%, respectively. The clinical 279 serum samples were tested by established system and the positive rate was 51.3%. The established method in this test could be used for clinical detection of serum antibody and epidemiological investigation of FAVⅠ. |
| keywords:Fowl adenovirus groupⅠ Hexon protein Prokaryotic expression Indirect ELISA |
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