人心肌肌钙蛋白I的原核表达及其兔抗体的制备

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。

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

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 3x10(-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.