人组织型纤溶酶原激活剂(t-PA)(1354-1802)的原核表达及抗血清制备  

Expression of t-PA(1354-1802) and Preparation of Its antiserum

作　　者：王青泉[1,2] 武建明[2] 李秀梅[2] 王立群[1] 何洪彬[2] 王洪梅[2] 刘晓[2] 

机构地区：[1]东北农业大学生命科学学院,黑龙江哈尔滨150030 [2]山东省农业科学院奶牛研究中心,山东济南250100

出　　处：《安徽农业科学》2012年第16期8905-8907,共3页Journal of Anhui Agricultural Sciences

基　　金：国家转基因重大专项(2009ZX08007-006B;2011ZX08007-002);山东省科技攻关项目(2009GG20002032);山东省自然科学基金项目(2010ZRC02039);济南市高校院所自主创新计划项目(201004027)

摘　　要：[目的]制备人组织型纤溶酶原激活剂t-PA(1354-1802)抗血清,并测定其效价,为深入研究t-PA基因功能奠定基础。[方法]利用基因克隆技术获得t-PA(1354-1802)催化域基因片段,然后将其重组到pET-32a(+)质粒中,进行鉴定及序列分析;将测序正确的阳性质粒转化大肠杆菌BL21感受态细胞,IPTG诱导、蛋白纯化及免疫兔试验。[结果]SDS-PAGE结果显示,37 kD处出现特异性条带;对该融合蛋白进行分离纯化后免疫新西兰兔,制备t-PA蛋白抗血清,Western blot分析表明,抗血清可与标准品t-PA蛋白特异性结合,间接ELISA方法检测抗血清的效价可达到1∶12 800。[结论]成功制备了t-PA(1354-1802)抗血清,为t-PA基因功能的深入研究奠定了基础。[Objective]To prepare t-PA（1354-1802） antiserum and measure its titer,so as to lay a foundation for the deep studies on gene function of t-PA.[Method]The 449 bp fragment of t-PA was obtained by gene cloning techniques,and then recombined into pET-32a（＋） vector.The recombinant vector was confirmed by PCR and DNA sequence analysis.The positive recombinant plasmid was transformed into BL21（DE3） and then induced with IPTG for expression.[Result]An expected specific band of about 37 kD was determined by SDS-PAGE.The fusion protein was purified and then used to immunize the New Zealand rabbits,and protein t-PA（1354-1802） antiserum was prepared.Western blot analysis showed that the antiseum could bind to standard of t-PA protein.The ELISA titer of the rabbit i-t-PA（1354-1802） antiserum was 1∶ 128 00 approximately.[Conclusion]t-PA（1354-1802）antiserum is successfully prepared,which lays foundation for the in-depth study of t-PA gene.

关 键 词：T-PA 原核表达 抗血清 

分 类 号：S852.5[农业科学—基础兽医学]