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

[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。

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

[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.