重组人脂蛋白相关磷脂酶A2的原核表达及单克隆抗体制备

Prokaryotic expression of recombinant human lipoprotein-associated phospholipase A2 and preparation of monoclonal antibodies

目的构建重组人脂蛋白相关磷脂酶A2(Lp-PLA2)的原核表达质粒,制备重组人Lp-PLA2,并通过免疫诱导获得人Lp-PLA2的单克隆抗体。方法以人cDNA文库为模板,通过PCR的方法获得PLA2G7基因的编码区序列,并与原核表达载体pET32a进行连接,构建原核表达质粒,转化大肠杆菌Rostta(DE3)。通过在30℃0.2mmol/L异丙基硫代半乳糖苷(IPTG)条件下诱导蛋白表达,经过纯化获得重组人Lp-PLA2;免疫Balb/c小鼠,制备单克隆抗体;并通过酶联免疫吸附试验法测定效价,检测抗体特异性。结果通过NotⅠ和SalⅠ双酶切及测序证实原核表达质粒构建成功;经纯化得到47×10^3左右的蛋白,与Lp-PLA2抗体进行蛋白免疫印迹法杂交呈阳性,表明已得到重组人Lp-PLA2。通过对小鼠免疫,最终获得单克隆抗体,效价为1∶2×10^6,与纤维蛋白原、C-反应蛋白无交叉反应。结论成功获得重组人Lp-PLA2,并制备得到了效价较高的单克隆抗体,为后续Lp-PLA2检测试剂盒的开发奠定了基础。

Objective To construct a prokaryotic expression plasmid of human lipoprotein-associated phospholipase A2(Lp-PLA2),prepare recombinant human Lp-PLA2,and obtain monoclonal antibodies against human Lp-PLA2 by immunoinduction.Methods Human cDNA library was used as template,the coding sequence of PLA2 G7 gene was obtained by PCR and linked with prokaryotic expression vector pET32 a.The prokaryotic expression plasmid was constructed and then transformed into Escherichia coli Rostta(DE3).The recombinant human Lp-PLA2 was purified by inducing the expression of the protein under the condition of 0.2 mmol/L isopropylgalactoside(IPTG)at 30℃.The monoclonal antibody was prepared by immunizing Balb/c mice.The titer and the specificity of the antibody were determined by ELISA.Results The plasmid was identified by the double-enzyme digestion(NotⅠand SalⅠ)and sequencing.The purified protein was about 47×10^3 in size.The protein showed a positive result in the Western blot hybridization with the commercialized Lp-PLA2 antibody,indicating that the recombinant Lp-PLA2 has been achieved.By immunizing the mice,the Lp-PLA2 monoclonal antibodies was achieved with a titer at 1∶2×10^6,and no cross-reaction with other biomarkers such as fibrinogen or C-reactive protein.Conclusion Human recombinant Lp-PLA2 was successfully obtained and monoclonal antibodies with high titer were prepared,which laid a foundation for the development of subsequent Lp-PLA2 detection kit.