摘要
背景与目的:人乳头瘤病毒16型(humanpapillomavirustype16,HPV16)是宫颈癌组织中最常见的高危HPV,其相应蛋白的血清抗体与宫颈癌的发生发展相关。本研究构建HPV16E6重组表达载体并表达纯化获得HPV16E6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HPV16E6血清抗体反应与宫颈癌的相关性。方法:将HPV16E6基因与pRSET-A融合表达载体连接,获得E6表达重组体,转化大肠杆菌BL21(DE3)并用异丙基硫代-$-D-半乳糖苷(isopropylthio-$-D-galactoside,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ELISA板,检测正常女性、慢性宫颈炎患者和宫颈癌患者血清抗体。同时采用荧光偏振方法分型检测宫颈癌组织HPVDNA。结果:pRSET-16E6表达重组体的工程菌经IPTG诱导后可表达Mr24×103的HPV16E6组氨酸融合蛋白,表达量占菌体蛋白的22.3%。表达形式为包涵体,重组蛋白纯度达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例宫颈癌患者血清抗体阳性率分别为5.0%、6.5%和31.2%,宫颈癌患者HPV16E6血清抗体阳性率显著高于正常人(P<0.002)及慢性宫颈炎患者(P<0.01),而正常人与慢性宫颈炎患者间的差异无显著性。32例宫颈癌患者癌组织中,HPVDNA阳性率90.6%,HPV16DNA阳性率46.9%。HPV16DNA阳性组血清HPV16E6抗体阳性率(46.7%)高于阴性组(17.6%),但两组间的差异无显著性(P>0.05)。结论:在pRSET-A/BL21中表达获得的HPV16E6融合蛋白,可用于宫颈癌相关HPV的血清学研究;宫颈癌患者HPV16E6血清抗体阳性率明显高于正常人和慢性宫颈炎患者。
BACKGROUND ＆ OBJECTIVE. Human papillomavirus type 16 （HPV16） is the predominant high-risk type of HPV in cervical cancer tissues. Serum antibody responsed to HPV16-related proteins is associated with the development of cervical cancer. This study was to construct and purify recombinant HPV16 E6 protein, detect its corresponding serum antibody among different populations, and explore the correlation of HPV16 E6 serum antibody reaction to cervical cancer. METHODS： HPV16 E6 early gene was constructed into pRSET-A expression vector. The plasmids were transfected into BL21 （DE3） cells, which were induced to express HPV16 E6 protein by isopropylthio-β-D-galactoside （IPTG）. E6 inclusions were denatured, purified through Ni column, and renatured. After the activity of HPV16 E6 protein being identified, the antibodies against HPV16 E6 in serum samples from 80 healthy women, 46 chronic cervicitis patients, and 32 cervical cancer patients were determined by ELISA using the fusion protein as antigen. HPV DNA genotype was estimated in cervical cancer tissues by fluorescence polarization. RESULTS. HPV16 E6 fusion protein of Mr 24×10^3 was expressed in pRSET-16E6 after induction of IPTG. The fusion protein was accounted for 22.3% of total bacterial proteins, and expressed as inclusive body. After purification with Ni-NTA agarose resin, the purity of the recombinant protein was over 95%, and its activity was identified by ELISA. The antibody-positive rate was significantly higher in cervical cancer patients than in healthy women and chronic cervicitis patients （31.2% vs. 5.0% and 6.5%, P〈0.01）. In the 32 cervical cancer patients, the positive rates of HPVs DNA and HPV16 DNA in cancer antibody-positive rate of HPV16 E6 tissues were 90.61% and 46.88%. The was higher in HPV16 DNA-positive cervical cancer patients than in HPV16 DNA-negative patients （46.7% vs. 17.6%）, but the difference was not significant. CONCLUSIONS. HPV16 E6 fusion protein obtained from pRSET-A/BL21 can be used in serologic studies on cervical cancer-related HPV infection. Serum antibody against HPV16 E6 is more common in cervical cancer patients than in healthy women and chronic cervicitis patients.
出处
《癌症》
SCIE
CAS
CSCD
北大核心
2006年第11期1374-1379,共6页
Chinese Journal of Cancer
基金
陕西省科技计划项目(No.2004K13-G2)~~
