人SmithD1抗原在原核细胞中的表达与纯化及临床应用

Prokaryotic expression and purification of human Smith D1 antigen

目的获得人 Smith D1(Sm D1)抗原基因,并进行原核表达及建立斑点免疫金渗虑(dot immunogold filtration assay,DIGFA)检测方法。方法采用基因工程技术,通过 PCR 从人白血病HL-60细胞 cDNA 文库中扩增人 Sm D1抗原基因,构建 pGEX-ST-Sm D1重组表达载体,在大肠杆菌BE21中通过异丙基硫代半乳糖苷酶(IPTG)诱导表达 Sm D1蛋白。利用初步纯化的 Sm D1抗原建立DIGFA。结果重组质粒测序和酶切结果显示 Sm D1抗原基因正确插入 pGEX-5T,重组蛋白、复性及纯化产物经12%十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE),在相对分子质量为39 000处有一明显的蛋白表达条带,免疫印迹法分析表明,重组蛋白具有人 Sm D1抗原的抗原性。DIGFA 与色疫印迹法检测结果的差异无统计学意义(P>0.05),二者的符合率为91.7%。DIGFA 敏感度为100%,特异度为83.3%结论通过基因工程技术制备获得初步纯化的谷胱苷肽转移酶(GST)-Sm D1融合蛋白,用该抗原建立的 DIGFA 与免疫印迹法相似,且具有快速、简便和可靠等优点。

Objective To obtain recombinant human Smith D1 （Sm D1 ） antigen and establish detecting assay. Methods Human Smith D1 antigen was synthesized by PCR using human Leukemic cDNA. The prokaryotic expression vector pGEX-ST-Sm D1 was constructed and transformed into E. coli. BL21 cell. Protein expressed under the induction of IPTG. We established DIGFA for detecting anti-Sm D1 antibodies with purified Sm D1 antigens. Results Sequence and restriction analysis revealed Sm D1 gene was cloned in frame into pGEX-ST ,SDS-PAGE profile showed a clear protein band with a relative molecular weight of 39 000 and western blotting indicated that the expressed product specifically reacted to polyclonal anti-human Sm D1 genes. There was no significant difference between DIGFA and IB. The agreement between DIGFA and IB was 91.7% as calculated by Kappa statistical method. The sensitivity and specificity of DIGFA were 100% and 83.3% repectively. Conclusions Human Sm D1 gene is successfully cloned, expressed and purification. The DIGFA, using purified Sm D1 antigens, is as good as IB, rather simpler, more rapid and reliable assay.