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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。
OBJECTIVE: To construct a prokaryotic expression plasmid of human cardiac troponin I (hcTnI) gene and express in E. coli and prepare rabbit anti-hcTnI antibody. Methods: The hcTnI gene was synthesized by chemical method and inserted into the multiple cloning site of the fusion expression vector pET21a (+) to construct the recombinant expression plasmid pET21a (+) hcTnI. The recombinant plasmid was transformed into E. coli BL21 (DE3) plysS, and the positive recombinant was screened. The expression of the target protein was induced by IPTG. The immunological activity of the expressed product was identified by Western blot. Rabbits were immunized with the expressed hcTnI protein to prepare anti-hcTnI antibodies and purified and characterized. Results: The hcTnI gene was successfully synthesized and sequenced. The correct sequence was subcloned into the expression vector pET21a (+). Positive clones were obtained by PCR screening and restriction enzyme digestion. The sequence analysis showed that the inserted sequence was identical to cTnI gene. The target protein of Mr 24000 was expressed in E. coli, accounting for about 28% of the total bacterial proteins. Western blot analysis showed that the expressed hcTnI protein had good immunoreactivity. Rabbit immunized with purified hcTnI, can effectively stimulate the production of specific antibodies, antiserum titers of 3 × 10-4, and has good specificity. Conclusion: The prokaryotic expression vector pET21a (+) hcTnI of hcTnI gene was successfully constructed and highly expressed in E. coli. Antibodies to rabbit anti-hcTnI were prepared with good titer and specificity, which laid the foundation for further determination of hcTnI by enzyme-linked immunosorbent assay.