高危HPV16 E4基因的表达纯化及临床应用  

Expression purification and clinical application of high-risk HPV16 E4 gene

作　　者：高艳娥[1] 惠慧[1] 张菊[2] 樊江波[1] 阎小君[2] 

机构地区：[1]西安交通大学医学院第二附属医院妇产科,西安710004 [2]第四军医大学全军基因诊断技术研究所,西安710032

出　　处：《中南大学学报（医学版）》2008年第8期676-681,共6页Journal of Central South University ：Medical Science

基　　金：陕西省科技计划项目[2007K09-08(2)]~~

摘　　要：目的:构建人乳头瘤病毒(HPV)16 E4重组表达体并表达纯化获得HPV16 E4重组蛋白,用于检测不同人群血清相应抗体,初步探讨本地区HPV16 E4血清抗体反应与宫颈癌的相关性。方法:将HPV16E4基因与pRSET-A融合表达载体连接,获得E4表达重组体,转化大肠杆菌BL21(DE3)并用IPTG诱导表达。表达的包涵体变性后经Ni柱纯化,复性并经活性鉴定后,用以包被酶联免疫吸附实验(ELISA)板,检测正常女性、慢性宫颈炎和宫颈癌患者血清抗体。结果:pRSET-16E4表达重组体的工程菌经IPTG诱导后可表达Mr15×103的HPV16 E4组氨酸融合蛋白,表达量占菌体蛋白的30%。表达形式为包涵体,重组蛋白经Ni-NTA琼脂糖树脂纯化后,纯度达95%以上,其活性经ELISA法证实。80例正常女性、46例慢性宫颈炎和32例宫颈癌患者中,其血清抗体阳性率分别为10.00%,39.13%和28.13%,宫颈癌患者HPV16 E4血清抗体阳性率显著高于正常人,且慢性宫颈炎患者HPV16 E4血清抗体阳性率也显著高于正常人,但宫颈癌组HPV16 E4血清抗体阳性率与慢性宫颈炎组间的差异无统计学意义。结论:在pRSET-A/BL21中表达获得的HPV16 E4融合蛋白,可用于宫颈癌相关HPV的血清学研究;宫颈癌和慢性宫颈炎患者HPV16E4血清抗体阳性率均明显高于正常人。Objective To determine the relationship between serum antibody against HPV16 E4 and cervical cancer, and to construct HPV 16 E4 protein expression vector as an antigen to detect its corresponding serum antibody among different populations. Methods HPV16 E4 early gene was ligated into pRSET-A expression vector. The constructed plasmids were transformed into BL21 （DE3） cells, and induced to express HPV 16 E4 protein by isopropylthio-β-D-galactoside （IPTG）. The expressed E4 inclusions were denatured, purified through Ni-column, and renatured. After the activity was revealed, antibodies against HPV 16 E4 in the sera from healthy women and patients with chronic cervicitis and cervical cancer were respectively determined by enzyme-linked immunosorbent assay （ELISA） using the fusion protein as the antigen. Results HPV 16 E4 fusion protein of Mr 15×10^3 was expressed by pRSET-16 E4 after IPTG induction. The fusion protein accounted for 30% of the total bacterial proteins and expressed as inclusive body. After purification with Ni-NTA agarose resin, the recombinant protein revealed purity of 95% , and activity of the renatured protein was identified by ELISA. The serum antibody-positive rate of HPV 16 E4 was 10.00% , 39. 13% and 28. 13%, respectively in 80 healthy women, 46 chronic cervicitis patients, and 32 cervical cancer patients. The antibody-positive rate in cervical cancer patients and chronic cervicitis patients were significantly higher than that in healthy women （P〈0.01） , while the difference between the antibody-positive rate in cervical cancer patients and chronic cervicitis patients was not significant. Conclusion HPV 16 E4 protein expressed from pRSET-A/BL21 can be used in serological studies on cervical cancer-related HPV infection. Serum antibody against HPV16 E4 is present in a significantly higher percentage in cervical cancer and chronic cervicitis patients than in healthy women.

关 键 词：人乳头瘤病毒16型 E4蛋白 基因表达 血清抗体 宫颈癌 

分 类 号：R737.33[医药卫生—肿瘤]