宫颈癌组织HPV58的检测及其E7基因的克隆和表达

Detection of HPV 58 and cloning of its E7 gene in cervical cancer

目的 检测宫颈癌组织中人乳头瘤病毒 5 8型 (HPV5 8)并克隆表达其E7基因。方法采用GP5 +/GP6 +引物系统扩增 5 8例宫颈癌组织 ,将荧光偏振 (FP)检测技术与模板指导的末端延伸反应 (TDI FP)结合 ,检测HPV5 8,确定HPV5 8感染阳性宫颈癌组织。用特异引物从HPV5 8阳性标本中扩增HPV5 8早期表达蛋白E7基因 ,将其连入pGEM TEasy载体 ,获得克隆重组体HPV5 8E7 pGEM T ,并测序验证。将E7基因与pRSET A融合表达载体连接 ,获得E7表达重组体pRSET 5 8E7,转化大肠杆菌BL2 1(DE3) ,并用IPTG诱导表达。结果 5 8例宫颈癌标本中 ,HPV5 8阳性 10例 ,占 5 2例HPV阳性标本的 19.2 %。从其中扩增到了HPV5 8E7基因并构建了其克隆和表达重组体。其表达重组体经IPTG诱导后 ,可表达Mr16× 10 3 的HPV5 8E7His6融合蛋白 ,表达量占菌体蛋白的 30 %。结论 欧美国家少见的高危HPV5 8在中国陕西人宫颈癌组织中并不少见。HPV5 8E7重组表达体可在大肠杆菌中高效表达 ,为HPV5

Objective To detect HPV 58, a common type of human papillomavirus (HPV), clone and express its E7 gene from biopsy specimens of cervical cancer. Methods HPV 58 from 58 biopsy tissues of cervical cancer was detected by GP5+/GP6+ PCR followed by template-directed dye-terminator incorporation assay with fluorescence polarization detection (TDI-FP). HPV 58 E7 gene was amplified from one HPV 58-positive sample, and then cloned into pGEM-T Easy vector. The recombinant plasmid, HPV58E7-pGEM-T was confirmed by sequencing. Subsequently, E7 gene was cloned into prokaryotic expression vector pRSET-A. The constructed pRSET-58E7 plasmids were transfected into BL21(DE3)cells, and induced to express 58 E7 protein by IPTG. Results Among the 58 biopsy tissues of cervical cancer, 10 were HPV 58-positive, accounting for 19.2% of 52 HPV-positive cases. HPV 58 E7 gene was amplified from one HPV 58-positive sample. The constructed plasmids were identified containing HPV58 E7 gene by restriction enzyme analysis and sequencing. SDS-PAGE analysis showed that HPV58 E7 His6 fusion protein of M_r16×10~3 was expressed by pRSET-58E7 after induction by IPTG. The fusion protein accounted for 30% of total bacterial proteins. Conclusion HPV 58 is not uncommon in Chinese women with cervical cancer in Shaanxi province. Constructed HPV58 E7 recombinant plasmids can be effectively expressed in E.coli, which may provide a tool in diagnosis and vaccine design for HPV of HPV58-associated tumors.