【作者】 廖娟；

【导师】 王红宁；

【作者基本信息】 四川农业大学 ， 预防兽医学， 2007， 硕士

【摘要】 禽传染性支气管炎(Avian Infectious Bronchitis，IB)是由禽传染性支气管炎病毒(IBV)引起的鸡的一种急性、高度接触性的传染病。IBV的变异大，血清型多，是IB免疫预防的一大难题。IBVM蛋白比较保守，在病毒粒子中所占的比例最大，同时具有膜蛋白的典型特点，是重要的免疫原基因。M蛋白的这些特性为M蛋白作为IB的诊断抗原提供了重要的分子生物学及免疫学依据。本研究根据本课题组在GenBank中注册的禽传染性支气管炎病毒中国分离株SAIBK株的M基因序列(注册号AY302742)及毕赤酵母分泌型表达载体pPIC9K的序列设计引物，通过鸡胚培养收获禽传染性支气管炎病毒，提取病毒RNA，用RT-PCR方法扩增出IBV M基因，构建了克隆载体pMD18-M，通过菌落PCR和酶切对克隆载体进行了鉴定。将克隆载体pMD18-M和酵母表达载体pPIC9K分别用限制性内切酶EcoR I和Not I进行双酶切，回收目的片段，用T4 DNA连接酶将两者连接后转化大肠杆菌，筛选得到酵母表达载体，经酶切鉴定M基因已经正确地连接到pPIC9K上。用限制性内切酶Sac I将表达质粒pPIC9K-M线性化，然后用电转化的方法导入毕赤酵母GS115中。将在MD平板上生长的转化子经过PCR鉴定、表型筛选和不同G418抗性浓度筛选后，获得整合型阳性重组菌株，命名为GS115／pPIC9K-MHis~+Mut~+。将重组菌株在1％的甲醇中进行诱导分泌表达，通过收集不同时间段的表达产物进行SDS-PAGE分析表明，在诱导72小时后目的蛋白表达量最大，表达蛋白占上清中总蛋白量的13.3％，表达蛋白的分子量约为33KD。通过Western-blot检测显示，表达蛋白能与IBV阳性血清特异性结合。对表达蛋白进行初步纯化后，以M蛋白为包被抗原的ELISA检测结果显示表达蛋白与特异性抗体反应良好，表明表达的M蛋白生物学活性良好。目前国内外还没有利用毕赤酵母分泌型表达载体pPIC9K来表达IBV M蛋白的报道。本研究表达的IBV M蛋白可作为制备IB诊断抗原的基础材料，对IB的防治有重要理论和实用价值。更多还原

【Abstract】 IBV is a fast and highly—touched infectious disease which has been variating frequently.It has much serological type and was difficult to prevent. IBV M protein was ratherconservative, concerned with replication of viruses it was an important immunogen gene.In addition, M gene holds a largest proportion in IBV particle and has typical feature ofmembrane protein. The characteristics of M protein provided molecular biology andimmunology base for M protein becaming IB diagnostic antigen.A pair of M gene expression fragment PCR primers were designed according toisolated IBV’s M gene sequence which was accepted by Genbank and the sequence ofPichia pastoris expression vector pPIC9K’s Multiple Cloning Site(MCS). Virus wereobtained by chicken embryo cultivating and the RNA of IBV virus was extracted. M genewas amplified by RT-PCR and cloned into the vector pMD18.Then the cloned vectorpMD18-M was identified by colony-PCR and enzyme digestion. The M fragment,producing from pMD18-M by EcoR I and Not I, was ligated with the yeast expressingvector pPIC9K which was also digested by EcoR I and Not I,then the ligation wastransformed to E.coli. The results of PCR and enzyme digestion of plasmid proved thatrecombination vector was obtained (named pPIC9K-M). The expressing plasmidpPIC9K-M was linearized with Sac I, then it was transformed into Pichia pastoris GS115by electroporation. After the transformants were selected by PCR、Methanol-utilization-plus and different G418 concentrations YPD medium, the positiverecombination yeast was obtained(named GS115/ pPIC9K-M His~+Mut~+). GS115/pPIC9K-M His~+Mut~+was induced with 1% methanol at 28℃for target protein expression. Expressed M protein of different time section was analysised by SDS-PAGE,the resultsshowed that the target protein was up to the highest yield after 3 days induced, and theproduct was about 33KD. The amount of target protein shares about 13.3% of the totalprotein which consist in the supematant fluid of yeast culture medium.Western blotting assay showed that the expressed protein could act with the positiveserum of IBV. The protein was initially purifed and acted as the envelope antigen of ELISAdetecting, the results proved expressed protein acted well with the specific antibody andthe expressed M protein has powerful biologic activity.Now there had no report about expressing M protein with Pichia pastoris. The expressedM protein may act as diagnostic antigen meterial of IB.The result has important theoreticaland practical value to prevention and cure of IB.更多还原