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目的：比较不同致病型鸡传染性支气管炎病毒(Infectious bronchitis virus, IBV) IBV与鸡氨肽酶N 体外结合能力的差异。方法：将已构建的pET-32a-chAPN 转入大肠杆菌BL21 中进行诱导表达，SDS-PAGE 分析表达结果。表达的His-chAPN 纯化后进行 western-blotting验证，应用酶促反应检测其活性。以不同稀释度的纯化chAPN 包被ELISA 板，加入经过荧光定量PCR方法定量的含有相同数量病毒粒子的三株不同致病型IBV（呼吸型M41、肾型S-03 和腺胃型A2-2），测定三株与chAPN 的结合能力。结果：pET-32a-chAPN在受体菌内成功表达，主要以包涵体的形式存在。经纯化复性的重组His-chAPN，Western blotting分析显示其具有良好的抗原性，酶促反应显示该重组重组His-chAPN 与天然的具有良好的生物活性。ELISA结果显示chAPN 与三株IBV均能结合，其结合能力为A2-2>S-03>M41，并表现为剂量依赖性。结论：不同致病型IBV与chAPN 的体外结合力有一定差异。

英文摘要:

Objective:To compare the binding capacity on different pathotype infectious bronchitis virus （IBV）strains with cellular protein chicken AminopeptidaseN (chAPN) in vitro.Methods:The pET-32a-chAPN was converted into BL21 cells to induce expression. SDS-PAGE analysis were used to examine the fusion protein. The purified His-chAPN was detected by Western blotting and its activity was determined by enzymatic reaction. ELISA plates were coated by the different dilution His-chAPN, and the equivalent amount of three different pathotype IBV strains (respiratory type IBV strain M41, nephropathogenic IBV strain S-03 and proventriculitis type IBV strain A2-2) quantified by Q-PCR were added into the wells, determined the difference among the binding capacity on three different pathotype IBV strains and chAPN in vitro.Results:It showed that the recombinant pET-32a-chAPN was expressed successfully in an inclusion body form. The purified His-chAPN had expected antigenicity tested by western blotting analysis and had enzymatic activity which was similar to the natural chAPN. ELISA data demonstrated the His-chAPN was able to bind three IBV strains dose-dependently, A2-2>S-03>M41.Conclusion:The binding capacity on different pathotype IBV strains with chAPN in vitro was different to a certain extent.

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