无标签NT-proBNP重组蛋白的获得及多克隆抗体的制备

Preparation of tag-free NT-proBNP recombinant protein and its polyclonal antibody

目的通过原核表达,获得不带标签的NT-proBNP重组蛋白,并用其免疫新西兰大白兔,制备兔抗NT-proBNP抗体。方法从重组菌pET-32a(+)-NT-proBNP提取重组质粒,通过PCR扩增出目的 DNA片段,插入pCold TF载体,转化入大肠埃希菌BL21(DE3),经IPTG诱导表达融合蛋白。经镍柱亲和纯化、HRV 3C蛋白酶酶切、镍柱亲和纯化,制备不带标签的纯蛋白质,并用临床商品化试剂盒验证其抗原性。用该蛋白质免疫新西兰大白兔制备抗体,western blot鉴定其特异性。结果成功获得高纯度的不带标签的NT-proBNP蛋白,该蛋白质能被商品化的试剂盒识别。用该蛋白质免疫新西兰大白兔成功制备出高效价高特异的抗体。结论成功制备高纯度的无标签NT-proBNP蛋白及其多克隆抗体,为NT-proBNP在临床上的广泛应用奠定了基础。

Objective To express tag-free N-terminal pro-B-type natriuretic peptide （NT-proBNP） by prokaryotic expression system and prepare polycolonal antibodies against the recombinant NT-proBNP. Methods NT-proBNP gene sequence was obtained from pET- 32a （ ＋ ）-NT-proBNP and inserted into pCold TF vector. The recombinant pCold TF-NT-proBNP was transferred into E. coli BI21 （DE3） and the recombinant NT-proBNP was expressed by IPTG induction. Subsequently, the recombinant NT-proBNP was purified by Nickel affinity column, and further digested with HRV 3C protease. After purification with Nickel affinity column again, tag-free recombinant NT-proBNP was obtained and its antigenicity was analyzed with the commercial kit employed in clinical test. Polyclonal antibodies against the recombinant tag-freeNT-proBNP were prepared by immunizing rabbits and the specificity of antibodies was tested by Western blot. Results Highly purified tag-free NT-proBNP protein was prepared and could be detectable by Roche NT-proBNP test kit. The polyclonal antibodies against recombinant tag-free NT-proBNP with high titer and specificity were also prepared. Conclusion Highly purified tag-free recombinant NT-proBNP protein and its specific polyclonal antibodies were successfully prepared for clinical research of NT-proBNP and widespread application in diagnosis.