摘要
[目的]研究Asial型口蹄疫病毒P1基因原核表达及其抗血清的制备。[方法]利用基因克隆技术获得Asia1型口蹄疫病毒(FM-DV)的P1基因,然后将P1基因重组到pET-32a(+)质粒中;转化大肠杆菌BL21感受态细胞,经IPTG诱导及蛋白纯化后,进行SDS-PAGE分析;将重组菌BL21培养物用超声波裂解,对该融合蛋白进行分离纯化后免疫新西兰兔,制备P1蛋白抗血清。[结果]获得了重组性阳性克隆;SDS-PAGE结果表明在105 kD处出现了目的条带;Western-blot分析表明,抗血清可与原核表达的P1蛋白特异性结合,ELISA方法检测抗血清的效价可达到1∶5 120,[结论]为建立FMDV的血清学诊断方法及其基因工程疫苗的研究奠定了基础。
[Objective] The aim was to study on the prokaryotic expression of P1 gene of type Asia1 foot and mouth disease virus（FMDV） as well as the preparation of its antiserum.[Method] The P1 gene of type Asia1 FMDV was obtained by gene cloning techniques,and then the P1 gene was cloned into pET-32a（＋） plasmid;subsequently transformed the recombinant plasmid into E.coli BL21（DE3）,after the IPTG induction and protein purification,SDS-PAGE was carried out;the ultrasonic wave was use to lyse the cultivated recombinant strain,and after the isolation and purification,this fusion protein was utilized to immunize New Zealand rabbits so as to prepare P1 protein antiserum.[Result] The positive clones were obtained;SDS-PAGE result showed that the target band was appeared at 105 kD;Western blot analysis showed that the antisera could bind to the expressed P1 fusion protein specifically;the ELISA titer of the rabbit anti-FMDV-P1 sera was approximately 1∶5 120.[Conclusion] This study had provided foundations for FMDV serological diagnostic methods and genetically engineered vaccine.
出处
《安徽农业科学》
CAS
北大核心
2011年第5期2804-2806,共3页
Journal of Anhui Agricultural Sciences
基金
国家转基因重大专项项目(2009ZX08007-006B)
国家自然科学基金项目(31072160)
山东省科技攻关项目(2009GG20002032)
山东省自然基金项目(Y2008D20)
兽医生物技术国家重点实验室开放课题基金项目(SKLVBF200806)
