耶氏肺孢子菌主要表面糖蛋白合成及人群IgG抗体调查  

A recombinant Pneumocystis jirovecii major surface glycoprotein antigen and a study of IgG antibody levels in human populations

作　　者：宋营改[1] 乌姗娜[1] 徐安健[1] 靳庆娥[1] 赵高潮[1] 任翊[1] 

机构地区：[1]首都医科大学附属北京友谊医院、北京热带医学研究所,北京100050

出　　处：《中国病原生物学杂志》2014年第4期318-321,326,共5页Journal of Pathogen Biology

基　　金：北京热带病防治研究北京市重点实验室项目(No.2013RDBF03)

摘　　要：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。Objectives To express an N-terminal fragment of the major surface glycoprotein（Msg）of Pneumocystis jirovecii and to obtain a soluble recombinant P.jirovecii Msg（Msg）antigen.An enzyme-linked immunosorbent assay（ELISA）was used to test for IgG antibodies against the recombinant P.jirovecii Msg in 266samples of peripheral blood from random adult patients at a Beijing hospital. Methods P.jirovecii DNA was extracted from sputum from patients clinically diagnosed with pneumocystis pneumonia（PCP）.The prokaryotic expression vector pGEX-3X-Pj Msg was constructed after PCR amplification of the target 5end of the Msg gene.The vector was transformed into Escherichia coli DE3and expression was induced with isopropylβ-D-1-thiogalactopyranoside（IPTG）.Recombinant P.jirovecii Msg was purified using aglutathione S-transferase（GST）affinity column.ELISA was used to detect IgG antibodies against the recombinant P.jirovecii Msg.Samples of peripheral blood were collected from 266random patients ages 20to 50at a Beijing hospital to study the rate at which IgG antibodies against the recombinant P.jirovecii Msg were detected. Results The expression vector pGEX-3X-Pj Msg was successfully constructed.Soluble recombinant P.jirovecii Msg protein was purified and it had a molecular weight of about 39ku.Production efficiency was 13mg of recombinant P.jirovecii Msg per 500ml of bacterial culture.IgG antibodies against the recombinant P.jirovecii Msg were detected in adults in Beijing at a rate of 42.5%（113/266）.IgG antibodies against the recombinant P.jirovecii Msg were detected in women at a rate of 41.7%（55/132）and in men at a rate of 43.3%（58/134）.There were no statistically significant differences in the rate of detection for men and women（χ2=0.071,P=0.790）.Individuals ages 20-29tested positive for IgG at a rate of 31.8%（27/85）,individuals ages 30-29tested positive at a rate of 48.3%（42/87）,and individuals ages 40-50tested positive at a rate of 46.8%（44/94）.There were no statistically sign

关 键 词：耶氏肺孢子菌 主要表面糖蛋白 重组抗原 抗体 

分 类 号：R379.9[医药卫生—病原生物学]