摘要
目的 克隆结核分枝杆菌 14 0 0 0、370 0 0、ESAT 6与mtb81抗原基因 ,于原核表达系统进行表达并纯化 ,测定抗原性与特异性。方法 利用聚合酶链反应 (PCR)自结核分枝杆菌基因组DNA扩增 14 0 0 0、380 0 0、ESAT 6与mtb81抗原基因 ,经测序鉴定后克隆于pGEX 4T 1表达载体 ,转化于大肠杆菌BL2 1菌株进行诱导表达 ,利用亲和层析纯化表达产物 ,通过酶联免疫吸附测定 (ELISA)检测其抗原性与特异性。结果 携带重组质粒的菌株经诱导产生高水平的表达产物 ,纯化的表达产物具备较高的纯度、抗原性与特异性。其中 380 0 0、14 0 0 0、ESAT 6及mtb81抗原的阳性检出率分别为 5 4 %、6 0 %、4 4 %及 36 %。 4种抗原联用阳性检出率可达 86 %。结论 14 0 0 0、380 0 0、ESAT 6与mtb81重组抗原具备良好的抗原性与特异性 。
Objective To express the 14 000, 37 000, and 6 000 early secretory antigenic target(ESAT 6) and mtb81 antigen genes in bacteria, and to purify the product and determine their activity Methods The 14 000, 37 000, ESAT 6, and mtb81 antigen genes were amplified from Mycobacterium tuberculosis genomic DNA by polymerase chain reactions and cloned into pGEX 4T 1 expression vector BL21 strain of Escherichia coli was transformed with the recombinant vectors and induced to express recombinant proteins The proteins were purified by affinity chromatography The biological activity of purified proteins were estimated by enzyme linked immunoabsorbant assay(ELISA) Results The BL21 strains of Escherichia coli with recombinant vectors showed high level of 14 000, 37 000, ESAT 6, and mtb81 gene expressions after induction The products were purified successfully and showed high antigenicity and specificity The sensitivity of 38 000,14 000,ESAT 6, and mtb81 were 54%, 60%, 44%, and 36%, respectively Conclusion The expressions and purifications of recombinant 14 000, 37 000, ESAT 6, and mtb81 antigens with natural activity facilitate their research and application
出处
《中华结核和呼吸杂志》
CAS
CSCD
北大核心
2004年第3期188-190,共3页
Chinese Journal of Tuberculosis and Respiratory Diseases
