摘要
目的利用原核系统可溶性表达结核分枝杆菌PPE17蛋白并进行纯化,研究其免疫学特性,并评价该重组蛋白在结核病血清学诊断方面的价值。方法从结核分枝杆菌H37Rv基因组中利用PCR扩增PPE17核酸序列然后克隆至融合表达载体pET-DsbC中,转入大肠埃希菌BL21(DE3)进行诱导、表达和纯化,用Western Blot和EL ISA方法进行抗原性初步评价。结果融合蛋白Ds-bC-PPE17在原核系统内经IPTG诱导表达后,主要以可溶性形式表达存在,经镍柱层析获得了纯的重组蛋白,纯度达95%以上。Western Blot和EL ISA方法结果证明重组DsbC-PPE17蛋白具有较强的抗原活性。用纯化的PPE17蛋白做抗原,临床诊断结核病人血清,阳性率达60%。结论融合蛋白DsbC-PPE17在大肠埃希菌中以可溶性表达形式存在,高纯度的重组融合蛋白有可能成为结核病的血清学诊断抗原。
Objective To express soluble Mycobacterium tuberculosis（TB） PPE17 protein in prokaryotic system and evaluate its diagnosis value after purification.Methods The PPE17 gene of Mycobacterium tuberculosis was obtained from H37Rv strain by PCR and cloned into pET-DsbC fusion vector.DsbC-PPE17 His-tag fusion protein was expressed in E.coli with IPTG induction and purified by Nickel Cobalt pre-packed column.A panel of serum specimens from 40 TB patients and 20 healthy volunteers were used to evaluate the ELISA assay coated with the purified protein.Results The purity of the expressed protein reached 95% after the column purification.The expressed protein of DsbC-PPE17 showed strong antigenicity by Western Blot and ELISA assays.The sensitivity and specificity of the ELISA were 60%（24/40） and 95%（19/20）,respectively.Conclusion The recombinant expressed DsbC-PPE17 fusion protein can be used for TB clinical diagnosis.
出处
《中国预防医学杂志》
CAS
2011年第11期910-913,共4页
Chinese Preventive Medicine
基金
国家传染病重大专项课题(2008ZX-10003-012)
