人心肌肌钙蛋白I的原核表达及其兔抗体的制备  

Prokaryotic expression of human cTnI and preparation of rabbit anti-hcTnI antibody

作　　者：徐霞[1] 杨能[1] 涂洪斌[2] 

机构地区：[1]广州医学院检验系,广东广州510182 [2]广州医学院实验医学研究中心,广东广州510182

出　　处：《细胞与分子免疫学杂志》2005年第4期463-465,469,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：广东省医学科学技术研究基金资助项目(No.A2000265)

摘　　要：目的:构建人心肌肌钙蛋白I(hcTnI)基因的原核表达质粒,在大肠杆菌中表达后,并制备兔抗hcTnI抗体。方法:以化学方法合成hcTnI基因并插入融合表达载体pET21a(+)的多克隆位点,构建重组表达质粒pET21a(+)hcTnI。以重组质粒转化大肠杆菌BL21(DE3)plysS,筛选阳性重组子,经IPTG诱导目的蛋白的表达,表达产物的免疫学活性用Westernblot进行鉴定。以表达的hcTnI蛋白免疫家兔,制备抗hcTnI的抗体并进行纯化及特性鉴定。结果:成功地合成hcTnI基因,测序证实序列正确后亚克隆于表达载体pET21a(+)中,经PCR筛选和酶切鉴定获得阳性克隆,序列分析表明其中的插入序列与cTnI基因完全一致。在大肠杆菌中表达出相对分子质量(Mr)为24000的目的蛋白,约占菌体总蛋白的28%,Westernblot分析显示,表达的hcTnI蛋白具有良好的免疫反应性。以纯化的hcTnI免疫家兔后,能有效地刺激特异性抗体的产生,抗血清的效价为3×10-4,且具有良好的特异性。结论:成功地构建hcTnI基因的原核表达载体pET21a(+)hcTnI,并在大肠杆菌中获得高效表达。制备出兔抗hcTnI的抗体,且效价及特异性均较良好,为进一步建立酶联免疫吸附法检测hcTnI奠定了基础。AIM: To construct the prokaryotic expression vector pET-21a(+)-hcTnI and prepare the rabbit anti-hcTnI antibody using hcTnI expressed in E.coli as immunogen. METHODS: The full-length gene encoding human cardiac troponin I (hcTnI) was synthesized chemically and inserted into expression plasmid pET-21a(+)to construct recombinant plasmid pET-21a(+)-hcTnI. The recombinant plasmid was transformed into E.coli BL21 (DE3)plysS which then expressed hcTnI under IPTG induction. The immunological activity of the expressed hcTnI was analyzed by Western blot. A rabbit was immunized with purified hcTnI to prepare anti-hcTnI antibody and the Ab’s properties were identified. RESULTS: Human cTnI gene was synthesized and confirmed by DNA sequencing. Positive recombinant clones were identified by restriction enzyme digestion analysis and DNA sequencing. After induction with IPTG, hcTnI with M_r being 24 000 was expressed in E.coli BL21 (DE3)plysS, and the expressed hcTnI accounted for 28% of total bacterial protein. Western blot analysis showed that the hcTnI protein could be recognized by an anti-hcTnI antibody. Rabbit polyclonal antibody with a good specificity was obtained. The titer of the polyclonal antibody was 3×10 -4 . CONCLUSION: The recombinant expression plasmid of hcTnI was constructed successfully and expressed in E.coli. The prepared rabbit anti-hcTnI antibody had a high titer and specificity.

关 键 词：肌钙蛋白I 原核表达 抗体 兔 

分 类 号：Q51[生物学—生物化学]