【摘要】 目的:构建EB病毒(epstein-barr virus,EBV)EBNA1基因原核表达载体,并探讨其在鼻咽癌血清学诊断中的应用。方法:收集2003-02-01-2005-08-30中山大学附属肿瘤医院(216例)和江苏省肿瘤医院(84例)300例鼻咽癌(nasopharyngeal carcinoma,NPC)患者的血清样本,同时收集2003-07-01-2005-07-30中山大学附属第三医院500名健康体检者标本作为对照组。以EBV DNA为模板,采用PCR法扩增目的基因EBNA1,定向克隆到原核表达载体pGEX-5X-1中,构建pGEX-5X-EBNA1重组质粒,转化大肠埃希菌BL21(DE3),IPTG诱导表达GST/EBNA1融合蛋白。经SDS-PAGE、蛋白质印迹法鉴定表达产物后,纯化目的蛋白作为包被抗原,制备ELISA试剂检测鼻咽癌患者和正常人群EBNA1-IgA抗体。结果:在大肠埃希菌中成功表达了GST/EBNA1融合蛋白,相对分子质量为54×103,ELISA法检测结果显示,鼻咽癌患者EBNA1-IgA抗体阳性检出率为80.7%(242/300),对照组阴性检出率为91.0%(455/500);EBV-IgA检测鼻咽癌患者抗体阳性检出率为82.3%(247/300),对照组阴性检出率为93.0%(465/500),2种检测方法的阳性检出率(χ2=1.77,P=0.19)和阴性检出率(χ2=1.36,P=0.25)比较,差异均无统计学意义。结论:初步评估了EB病毒GST/EBNA1重组融合蛋白在鼻咽癌血清筛选中的诊断价值,获得了在鼻咽癌筛选中有一定应用价值的pGEX-5X-EBNA1大肠埃希菌工程菌株。更多还原

【Abstract】 OBJECTIVE:To establish prokaryotic expression of EBNA1 gene in E.coli and analyze of the application of recombinant EBNA1 protein in serological diagnosis of nasopharyngeal carcinoma(NPC).METHODS:The serum specimen from Affiliated Cancer Hospital of San Yet-Sen University(n=216)and Jiangsu Cancer Hospital(n=84)were collected from 2003-02-01 to 2005-08-30.Totally 500 serum specimen of normal persons from Third Affiliated Hospital of San Yet-Sen University from 2003-07-01 to 2005-07-30 were collected as control group.DNA extracted from the B95-8cells was used as the templates in a polymerase chain reaction(PCR).EBNA1 gene 759bp was amplified and cloned to pGEX-5X-1vector.The recombinant plasmid pGEX-5X-EBNA1 was transformed into E.coli BL21(DE3).The expression of GST/EBNA1 fusion protein was induced by IPTG,identified by both SDS-PAGE and Westem blot,and then purified with glutathione-sepharose beads.The purified recombinant protein was coated to microplate for detection of EBNA1-IgA antibody in NPC patients by ELISA.RESULTS:We have successfully expressed the GST/EBNA1 fusion protein in the E.coli.The molecular weight of product was approximately 54×103.The recombinant fusion protein GST/EBNA1 showed good immunoreactivity by Westernblot.A novel ELISA was established using GST/EBNA1 protein.Serum samples were collected from 300 patients with NPC and 500 healthy controls and tested by this ELISA.The sensitivity and the specificity of GST/EBNA1 tests in the NPC and healthy controls sera were 80.7%(242/300)and 91.0%(455/500)separately.Meanwhile we tested the same samples using EBV-IgA kits from hongkong shennong company.The sensitivity and the specificity in the NPC sera were 82.3%(247/300)and 93.0%(465/500)separately.There was no significant difference between two reagents(sensitivity,χ2=1.77,P=0.19;specificity,χ2=1.36,P=0.25).CONCLUSIONS:The recombinant protein GST/EBNA1 is expressed in E.coli.,the diagnostic value of the recombinant protein in screening for NPC patients is primary evaluated and the valuable pGEX-5X-EBNA1 strain is obtained.更多还原