携带16 kD抗原多肽的PP7噬菌体样颗粒的制备及其对结核病诊断的价值评估  

Preparation of PP7 bacteriophage-like particles carrying the polypeptide from 16kD antigen and their value evaluation in the diagnosis of tuberculosis

作　　者：伊正君[1] 孙艳花[2] 赵荣兰[1] 路晓红 李纾[4] 李猛[1] 孙艳丽[1] 

机构地区：[1]潍坊医学院医学检验学系,山东潍坊261053 [2]潍坊市人民医院血液科,山东潍坊261000 [3]潍坊市人民医院检验科,山东潍坊261000 [4]山东省口腔组织再生重点实验室,山东大学口腔医学院,济南250102

出　　处：《临床检验杂志》2018年第1期57-61,共5页Chinese Journal of Clinical Laboratory Science

基　　金：山东省高等学校科技计划项目(J14LK14);山东省自然科学基金(ZR2015HL036);山东省医药卫生科技发展计划项目(2013WS0288);潍坊医学院教师公派教师国内访学项目

摘　　要：目的获得表面展示有16kD抗原多肽(16kD91-110)的PP7噬菌体样颗粒(bacteriophage-like particle,BLPs),并评价其在结核病诊断中的价值。方法用普通PCR技术扩增出含16kD91-110多肽编码基因的PP7衣壳蛋白基因,将其插入到pETDuet-2PP7载体中,获得pETDuet-2PP7-16kD91-110质粒;将上述重组质粒转化大肠埃希菌,诱导表达产物用SDS-PAGE和western blot进行鉴定;将纯化的2PP7-16kD91-110作为刺激抗原,用ELISA间接法检测结核病患者血清中的16kD抗体水平。结果SDS-PAGE及电镜结果显示成功表达2PP7-16kD91-110BLPs,并且展示于PP7 BLPs表面的16kD91-110多肽表位能特异性与抗16kD抗体结合;对活动性肺结核和潜伏性肺结核患者,以2PP7-16kD91-110BLPs为刺激抗原的全血释放γ干扰素试验的敏感性分别为75%和82.9%,与北京万泰结核分枝杆菌相关γ干扰素释放试剂盒相比,差异无统计学意义。结论成功制备表面展示有16kD91-110多肽的PP7 BLPs,为应用γ干扰素释放试验检测结核分枝杆菌提供了一种安全性高、稳定性好、价格低廉的刺激抗原,为结核病的诊断提供一种新的思路。Objective：To obtain the PP7 bacteriophage like particles （BLPs） carrying the polypeptide from 16 kD antigen （16kD91-110） on their surface, and evaluate their diagnostic value in tuberculosis. Methods：First, the PP7 capsid protein gene containing the encoding gene of polypeptide 16kD91-110 was amplified by PCR and inserted into the plasmid pETDuet 2PP7. Then, the obtained recombinant plasmid pETDuet-2PP7-16kD91-110 was transferred into Escherichia coli, and the recombinant protein was induced and identified by SDS-PAGE and western blot. Next, the purified 2PP7-16kD91-110 BLPs were used as stimulating antigen to inject into the blood of patients with tuberculosis, and their serum antibody levels against 16 kD antigen were detected by indirect ELISA. Results：The results of SDS-PAGE and transmission electron microscope showed that the 2PP7-16kD91-110 BLPs were prepared, and that the 16kD91-110 polypeptide epitopes displayed on the surface of PP7 BLPs could bind with the antibodies against 16 kD antigen specifically. After using 2PP7-16kD91-110 BLPs as antigen to stimulate the blood cells from active tuberculosis and latent tuberculosis patients, respectively, the sensitivity of interferon γ release assay was 75.0% and 82.9%, respectively, in the diagnosis of active tuberculosis and latent tuberculosis, which was similar to the results of Wantai TB IGRA （interferon γ release assay） kits from Beijing Wantai Biological Pharmacy Enterprise Co., Ltd.. Conclusion：The PP7 BLPs with the 16kD91-110 polypeptide displayed on their surface are prepared successfully, which provides a kind of safe, stable and low cost stimulating antigen for the detection of interferon γ release, and a new method for the diagnosis of tuberculosis.

关 键 词：PP7噬菌体 16kD抗原 结核病 多肽 

分 类 号：R446.5[医药卫生—诊断学]