脂蛋白相关磷脂酶A2的原核表达及多克隆抗体的制备

Prokaryotic Expression of Lipoprotein-associated Phospholipase A2 and Preparation of Its Polyclonal Antibody

目的构建人脂蛋白相关磷脂酶A2(Lipoprotein-associated phospholipase A2,LP-PLA2)基因重组原核表达质粒,表达重组蛋白,制备人LP-PLA2多克隆抗体。方法从分化的THP-1细胞中提取总RNA,RT-PCR扩增LP-PLA2全长编码基因,插入原核表达载体pCold TF中,构建重组原核表达质粒,转化大肠杆菌BL21(DE3),IPTG低温(15℃)诱导表达,表达的重组蛋白经Ni-Sepharose 6FF亲和层析及DEAE-Sephadex离子交换层析后,免疫新西兰大白兔,制备多克隆抗体,采用间接ELISA法测定抗血清效价,Western blot法检测多抗的反应原性。结果重组表达质粒pCold TF-LP-PLA2经双酶切及测序证实构建正确;TF-LP-PLA2获得可溶性表达,相对分子质量约为93 000;纯化的重组蛋白纯度可达90%,可与市售兔抗人LP-PLA2抗体反应;制备的抗血清效价达1∶5.12×106以上,反应原性良好。结论已成功原核表达了重组LP-PLA2蛋白,并制备了高效价的多克隆抗体,为下一步制备单克隆抗体及建立LP-PLA2免疫检测方法奠定了基础。

Objective To construct a prokaryotic expression vector for lipoprotein-associated phospholipase A2（LP-PLA2） gene,express recombinant protein and prepare the polyclonal antibody against human LP-PLA2.Methods Total RNA was extracted from differentiated THP-1 cells,from which the full-length gene encoding LP-PLA2 was amplified by RT-PCR and inserted into prokaryotic expression vector pCold TF.The constructed recombinant plasmid was transformed to E.coli BL21（DE3） for expression under induction of IPTG at low temperature.The expressed protein was purified by Ni-Sepharose 6FF affinity chromatography and DEAE-Sephadex ion exchange chromatography and immunized into New Zealand rabbits.The prepared polyclonal antibody was determined for titer by indirect ELISA and for reactogenicity by Western blot.Results Restriction analysis and sequencing proved that recombinant plasmid pCold TF-LP-PLA2 was constructed correctly.The expressed TF-LP-PLA2 in a soluble form,with a relative molecular mass of about 93 000,reached a purity of 90% after purification and reacted with commercial antibody against human LP-PLA2.The prepared antiserum reached a titer of more than 1 ∶ 5.12 × 106 and showed good reactogenicity.Conclusion Recombinant LP-PLA2 protein was successfully expressed in prokaryotic cells,and high titer polyclonal antibody was prepared,which laid a foundation of further preparation of monoclonal antibody and development of immunological method for determination of LP-PLA2.