建鲤重组Cystatin蛋白的原核表达、鉴定及多克隆抗体制备

目的:对建鲤半胱氨酸蛋白酶抑制剂(CPIs)Cystatin进行原核表达和纯化鉴定,并以重组Cystatin为抗原制备多克隆抗体。方法:用异丙基硫代半乳糖苷(IPTG)诱导转入pET-30a-Cystatin的E.coli BL21(DE3)表达重组蛋白,经过Ni 2+-NTA镍离子亲和层析纯化重组Cystatin,高效液相色谱鉴定重组Cystatin的纯度,Azocasein法测定其对木瓜蛋白酶的抑制作用。利用QuickAntibody-Mouse5W快速佐剂和纯化的重组Cystatin免疫Balb/C小鼠获得抗血清,采用夹心ELISA法鉴定抗血清的效价,Western blotting和免疫组织化学法鉴定抗血清的抗原识别特异性。结果:原核表达的目的蛋白相对分子质量约20 000;镍亲和层析纯化后重组Cystain在SDS-PAGE上显示单一条带,纯度>95%;Azocasein法检测,0.014μg重组Cystain抑制木瓜蛋白酶活性达50%以上;ELISA夹心法检测获得的Cystatin抗血清效价达到1∶512 000;Western blotting检测,转入pET-30a空质粒的全菌蛋白中未检测到Cystatin存在,而在转入pET-30a-Cystatin的全菌蛋白及纯化各步样品中均检测到Cystatin信号,且均为单一条带;免疫组织化学检测,建鲤各组织呈现不同强度的Cystatin阳性染色,抗血清中的多克隆抗体与原核和真核表达的Cystatin均特异性结合。结论:成功表达和纯化了建鲤Cystatin;获得含高效价多克隆抗体的Cystatin抗血清,具有高特异性和灵敏性。

Prokaryotic expression and identification of recombinant Cystatin of Cyprinus carpio var.Jian and preparation of its polyclonal antibody

Objective To perform the prokaryotic expression,purification and identification of Cystatin of Cyprinus carpio var.Jian,and to further prepare a polyclonal antibody targeting Cystatin as the antigen.Methods The E.coli BL21(DE3),in which the pET-30a-Cystatin plasmid had been transferred,was induced by IPTG toexpress the recombinant protein,which was purified with Ni 2+-NTA affinity chromatography.Then the purity of recombinant Cystatin and inhibitory activity was identified using high performance liquid and Azocasein respectively. The QuickAntibody-Mouse5Wfast adjuvant and purified recombinant Cystatin were used to immunize Balb/C mice to get antiserum,whose titer was identified with sandwich ELISA and whose specificity to recognize antigens was assessed with Western blotting and immunohistochemistry.Results The molecular weight of interest protein of prokaryotic expression was approximately 20 000.After Ni 2+-NTA affinity chromatography,the recombinant Cystatin was shown as a single band on the SDS-PAGE and its purity was above 95%.Azocasein assay showed that 50%activity of papain was inhibited by the recombinant Cystatin of 0.014μg.ELISA showed Cystatin antiserum with titer of 1∶512 000was obtained.The results of Western blotting showed the Cystatin was absence in the total protein of E.coli BL21(DE3)with transferred plasmid pET-30a,in contrast with transferred plasmid pET-30aCystatin and the sample in every process of purifying,the Cystatin was presence and showed a single band,and immunohistochemistry indicated the different tissues of Cyprinus carpio var.Jian showed different positive staining, the antiserum could specifically bind to the Cystatin expressed in both prokaryotic and eukaryotic systems. Conclusion The recombinant Cystatin of Cyprinus carpio var.Jian is expressed and purified successfully.And finally,the antiserum with high titer polyclonal antibody recognizing Cystatin of Cyprinus carpio var.Jian is obtained,which owns the high specificity and sensitivity.