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目的原核表达耶氏肺孢子菌(Pneumocystis jirovecii,Pj)主要表面糖蛋白(major surface glycoprotein,Msg)氨基端片段,获得可溶性Pj重组Msg抗原(recombinant Msg,rMsg),利用酶联免疫吸附试验(ELISA)检测266例成年人外周血抗Pj rMsg IgG抗体。方法从耶氏肺孢子菌感染者的痰液获得Pj DNA,构建表达载体pGEX-3X-Pj Msg,转化感受态大肠埃希菌DE3,用异丙基-β-D-硫代吡喃半乳糖苷(IPTG)诱导表达。利用谷胱甘肽转移酶(GST)标签进行亲和纯化,对纯化Pj rMsg抗原进行鉴定并建立ELISA方法,检测266例成年人外周血抗Pj rMsg IgG抗体。结果成功构建了表达质粒pGEX-3X-Pj Msg,并诱导表达分子质量单位约39ku的可溶性Pj rMsg蛋白,产量为13mg/500ml菌液,以此为抗原,采用ELISA检测266例成年人外周血抗Pj rMsg IgG抗体,阳性率为42.5%(113/266)。其中男、女性患者阳性率分别为43.3%(58/134)和41.7%(55/132),差异无统计学意义(χ2=0.071,P>0.05);20~29岁、30~39岁、40~50岁组阳性率分别为31.8%(27/85)、48.3(42/87)和46.8%(44/94),差异无统计学意义(χ2=5.911,P>0.05)。结论成功表达了可溶性Pj rMsg抗原,Pj rMsg-ELISA检测结果表明北京地区成人耶氏肺孢子菌感染率较高。
Objective To express the recombinant Msg antigen (rMsg) in prokaryotic Pneumocystis jirovecii (Pj) major surface glycoprotein (Msg) by enzyme-linked immunosorbent assay (ELISA) ) Detected 266 adult peripheral blood anti-Pj rMsg IgG antibodies. Methods Pj DNA was obtained from the sputum of Pneumocystis sp., And the expression vector pGEX-3X-Pj Msg was constructed. The competent cell strain Escherichia coli DE3 was transformed into E. coli DE3. Glycoside (IPTG) induces expression. The purified Pj rMsg antigen was identified by GST tag and the anti-Pj rMsg IgG antibody was detected in 266 adult peripheral blood samples by ELISA. Results The recombinant plasmid pGEX-3X-Pj Msg was successfully constructed and the soluble Pj rMsg protein of about 39ku was expressed. The yield of the product was 13mg / 500ml, which was used as the antigen to detect the peripheral blood anti-266 Pj rMsg IgG antibody, the positive rate was 42.5% (113/266). The positive rates of male and female patients were 43.3% (58/134) and 41.7% (55/132) respectively, with no significant difference (χ2 = 0.071, P> 0.05) The positive rates in the 40-50 age group were 31.8% (27/85), 48.3 (42/87) and 46.8% (44/94) respectively, with no significant difference (χ2 = 5.911, P> 0.05). Conclusion The soluble Pj rMsg antigen was successfully expressed. The results of Pj rMsg-ELISA showed that the infection rate of Pneumocystis sp. In adults was high in Beijing.