鼠抗人肥大细胞类糜蛋白酶N端片段抗体的制备与鉴定

Preparation and characterization of the mouse polyclonal antibody against the N-terminal fragment of human mast cell chymase

目的:在大肠杆菌中表达人肥大细胞类糜蛋白酶(hMCC)N端片段(hMCC-N),并制备其鼠源性多克隆抗体。方法:通过PCR法扩增hMCC-N端基因片段,将其克隆至pMAL-c2x原核表达载体,转化大肠杆菌BL21(DE3),用IPTG诱导重组蛋白的表达,通过Amylose树脂亲和层析纯化目的蛋白,以纯化的重组蛋白免疫小鼠,制备鼠抗hMCC-N端片段多克隆抗体,并以间接ELISA测定抗体效价,以Westernblot鉴定抗体的特异性。结果:PCR扩增得到360bp的hMCC-N端基因片段,克隆入表达载体pMAL-c2x,构建了与标签蛋白麦芽糖结合蛋白基因融合表达载体pMAL-c2x/hMCC-N,融合蛋白在大肠杆菌中得到了稳定表达,并经亲和层析,纯化出可溶性的重组蛋白,用其免疫小鼠,获取了鼠抗hMCC-N端片段的多克隆抗体,ELISA结果显示效价达1∶12800,Westernblot分析表明该抗体能特异结合hMCC。结论:成功地制备了效价高、特异性较强鼠抗hMCC-N端片段抗体,为进一步建立hMCC的ELISA检测方法打下了良好的基础。

Objective To express the N-terminal fragment of human mast cell chymase（hMCC-N） in E.coli and prepare its polyclonal antibody from mouse, Methods The DNA coding for the hMCC-N was amplified by PCR and cloned into prokaryotic expression vector pMAL-c2x. The recombinant plasmid pMAL-c2x/hMCC-N was transformed into E.coli BL21 （DE3） and expressed with IPTG induction. The recombinant protein hMCC-N was purified with amylose affinity chromatograph and used as immunogen to immunize the mouse. The titer and specificity of the generated antibody were analyzed by indirect ELISA and western blot, Results The DNA fragment of hMCC-N was amplified and fused to MalE gene of expressing vector pMAL-c2x successfully. The recombinant protein hMCC-N was successfully expressed in E.coli, and soluble recombinant protein was purified successfully. The antibody against hMCC-N from mouse was obtained, the titer of which was 1 ： 12 800. The result of western blot showed that this antibody could react with hMCC specifically. Conclusion The mouse anti-hMCC-N antibody with high titer and high specificity has been prepared successfully, which provides the basis for further research on the detection of hMCC by ELISA.