EB病毒EBNA1基因原核表达载体构建及其对鼻咽癌筛选价值研究

发布时间：2019-05-05 06:07

【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。
[Abstract]:Aim: to construct prokaryotic expression vector of EB virus (epstein-barr virus,EBV) EBNA1 gene and to explore its application in serological diagnosis of nasopharyngeal carcinoma (NPC). Methods: serum samples of 300 patients with nasopharyngeal carcinoma (nasopharyngeal carcinoma,NPC) were collected from the affiliated tumor Hospital of Sun Yat-sen University (216 cases) and Jiangsu Cancer Hospital (84 cases). At the same time, 500 healthy specimens were collected from the third affiliated Hospital of Sun Yat-sen University in 2003 / 07 / 01 / 2005 / 07 / 30 as the control group. The target gene EBNA1, was amplified by PCR using EBV DNA as template and cloned into prokaryotic expression vector pGEX-5X-1. The recombinant plasmid pGEX-5X-EBNA1 was constructed and transformed into E. coli BL21 (DE3), IPTG-induced expression of GST/EBNA1 fusion protein). The recombinant plasmid was transformed into E. coli BL21 (DE3), IPTG-induced expression of GST/EBNA1 fusion protein). The expressed product was identified by SDS-PAGE, Western blot, and the target protein was purified as the coated antigen. The ELISA reagent was prepared to detect the EBNA1-IgA antibody in NPC patients and normal population. Results: GST/EBNA1 fusion protein was successfully expressed in Escherichia coli with a molecular weight of 54 脳 103. Elisa showed that the positive rate of EBNA1-IgA antibody in NPC patients was 80.7% (242 脳 300). The negative detection rate in the control group was 91.0% (455,500). The positive rate of antibody detected by EBV-IgA was 82.3% (247 / 300) in nasopharyngeal carcinoma patients and 93.0% (465 / 500) in control group, and the positive rate of two methods (蠂 ~ 2 / 1.77, P < 0.01) was higher than that of control group (93.0%). There was no significant difference between the negative detection rate (蠂 ~ 2 = 1.36, P < 0.05) and the negative rate (P = 0.19, 蠂 ~ 2 = 1.36, P = 0.25). Conclusion: the diagnostic value of recombinant GST/EBNA1 fusion protein of EB virus in serum screening of nasopharyngeal carcinoma (NPC) was evaluated, and the engineering strains of pGEX-5X-EBNA1 Escherichia coli with certain application value in NPC screening were obtained.
【作者单位】：中山大学附属第三医院检验科;中山大学附属肿瘤医院检验科;江苏省肿瘤医院检验科;
【基金】：广东省科技计划(2006B36001010) 教育部第44批留学回国人员科研启动基金〔教外司留(2012)940〕
【分类号】：R739.63

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