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鸡传染性支气管炎病毒HN99株N基因在大肠杆菌中的表达及产物免疫活性检测 被引量:3

Expression of avian infectious bronchitis virus HN99 strain nucleocapsid protein gene in Eco. li and immunological activity detection of expressed product
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摘要 鸡传染性支气管炎病毒HN99株核蛋白(N)基因PCR产物经BamHⅠ、HindⅢ双酶切,克隆入大肠杆菌原核表达载体pMAL-p2X,构建重组表达质粒pMAL-p2X-N,转化大肠杆菌TB1并进行诱导表达。通过pMALTM融合蛋白纯化系统对表达产物进行非变性纯化,将纯化的N蛋白按常规方法免疫新西兰大白兔,制备兔抗鸡传染性支气管炎病毒N蛋白的多克隆抗体,并用ELISA法检测其生物活性。结果表明,表达的重组核蛋门纯化后出现2条带,相对分子质量分别约为92和82 ku,与Western blot出现的2条蛋白带一致;纯化的重组蛋白经裂解因子作用后,出现2条蛋白带,相对分子质量分别为45和35 ku,与预期结果相符合;制备的多克隆抗体可与不同鸡传染性支气管炎病毒株发生反应,与亲本毒株的反应性略高于与其他毒株的反应性。以上结果表明,原核表达的可溶性N蛋白具有高度的生物活性,有望作为新的基因工程抗原用于鸡传染性支气管炎病毒的群特异性诊断。 PCR product of avian infectious bronchitis virus(IBV) H N99 strain nucleocapsid protein gene was inserted into E. coli pMAL-p2X expression vector and fused to MBP tag at N-terminus. Then the recombinant plasmid pMAL-p2X-N was transformed into E. coli TB1 and induced by IPTG. N protein were purified by affinity chromatography taking advantage of the affinity of MBP for maltose. These purified N proteins were used to raise polyclonal antibody according to classic procedure. SDS-PAGE and Western blot results indicated that MBP-N fusion protein had been successful expressed. Two kinds of protein appeared after expression, with the molecular weight of 92 ku and 82ku respectively. Purification product of MBP-N fusion protein cleavage by Factor Xa, MBP tag were cleaved,and their relative molcular weight was respectively 45 ku and 35 ku. ELLISA confirmed that antiserum to N protein could react with different strains of IBVs, and was more active when it reacted with its relative strains. In conclusion,the non-denaturalized N proteins were active and hopeful as a kind of gene engineering antigen for a group of IBVs diagnosis.
出处 《西北农林科技大学学报(自然科学版)》 CSCD 北大核心 2007年第4期32-36,40,共6页 Journal of Northwest A&F University(Natural Science Edition)
基金 河南省科技攻关项目(0424050015)
关键词 鸡传染性支气管炎病毒 N基因 免疫活性 avian infectious bronchitis virus N gene immunological activity
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参考文献15

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二级参考文献23

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