高危型人乳头瘤病毒E6/E7质控品的建立

目的: 建立高危型人乳头瘤病毒(HPV)E6/E7质控品,用于评价高危型HPV E6/E7 mRNA检测试剂盒的性能。方法: 根据高危型HPV 16和HPV 18的E6/E7基因序列设计引物,从感染HPV 16和HPV 18宫颈上皮细胞中提取DNA作为模板,通过PCR扩增获得带有目的基因的产物。采用限制性内切酶对带有目的基因的PCR产物和表达载体分别进行酶切,将目的基因与表达载体连接,然后转化DH5α感受态细胞,筛选出阳性菌落进行测序。然后将阳性菌液进行超声诱导,制备假病毒溶液。采用荧光定量逆转录-聚合酶链反应(RT-PCR)法对假病毒溶液提取的RNA进行鉴定。结果: 通过序列测定和荧光定量RT-PCR法检测,结果表明成功建立了高危型HPV E6/E7假病毒质控品。结论: 本研究建立了HPV E6/E7质控品的方法,可为高危型HPV E6/E7 mRNA检测试剂的质量控制提供质控品。

Objective: To establish high-risk human papillomavirus(HPV)E6/E7 control materials for evaluating the detection kits of high-risk HPV E6/E7 mRNA. Methods: The primers were designed according to E6/E7 gene sequence of HPV 16 and HPV 18.DNA was extracted from the cervical epithelial cells infected with HPV 16 or HPV 18 as the template.The products with the target gene were obtained by PCR method.The target gene and the expression vector were respectively digested with the restriction enzyme.Then the target gene was ligated into the expression vector,further transformed into DH5α,and the positive clones were selected for sequence.The correct clones were induced by ultrasound to prepare the pseudotyped virus solution.The RNA extraction was identified by fluorescence quantitative reverse transcription-polymerase chain reaction(RT-PCR). Results: High-risk HPV E6/E7 control materials were successfully established by sequencing and fluorescence quantitative RT-PCR. Conclusion: The method for HPV E6/E7 control materials was established,which can provide control materials for the quality control of the detection kits.

[1] ZHENG ZM,BAKER CC.Papillomavirus genome structure,expression,and post-transcriptional regulation[J].Front Biosci,2006,11: 2286

[2] HARPER DM,FRANCO EL,WHEELER C,et al.Efficacy of a bivalent L1 virus-like particle vaccine in prevention of infection with human papillomavirus types 16 and 18 in young women: a randomised controlled trial[J].Lancet,2004,364(9447):1757

[3] LIE AK,KRISTENSEN G.Human papillomavirus E6/E7 mRNA testing as a predictive marker for cervical carcinoma[J].Expert Rev Mol Diagn,2008,8(4):405

[4] KRAUS I,MOLDEN T,HOLM R,et al.Presence of E6 and E7 mRNA from human papillomavirus types 16,18,31,33,and 45 in the majority of cervical carcinomas[J].J Clin Microbiol,2006,44(4):1310

[5] CASTLE PE,DOCKTER J,GIACHETTI C,et al.A cross-sectional study of a prototype carcinogenic human papillomavirus E6/E7 messenger RNA assay for detection of cervical precancer and cancer[J].Clin Cancer Res,2007,13(9):2599

[6] CATTANI P,SIDDU A,D'ONGHIA S,MARCHETTI S,et al.RNA(E6 and E7)assays versus DNA(E6 and E7)assays for risk evaluation for women infected with human papillomavirus[J].J Clin Microbiol,2009,47(7):2136

[7] CUSCHIERI K,WENTZENSEN N.Human papillomavirus mRNA and p16 detection as biomarkers for the improved diagnosis of cervical neoplasia[J].Cancer Epidemiol Biomarkers Prev,2008,17(10):2536

[8] STOPPLER MC,CHING K,STOPPLER H,et al.Natural variants of the human papillomavirus type 16 E6 protein differ in their abilities to alter keratinocyte differentiation and to induce p53 degradation[J].J Virol,1996,70(10):6987

[9] HEBNER C,BEGLIN M,LAIMINS LA.Human papillomavirus E6 proteins mediate resistance to interferon-induced growth arrest through inhibition of p53 acetylation[J].J Virol,2007,81(23):12740
[10] KIYONO T,FOSTER SA,KOOP JI,et al.Both Rb/p16INK4a inactivation and telomerase activity are required to immortalize human epithelial cells[J].Nature,1998,396(6706):84
[11] DYSON N,HOWLEY PM,MUNGER K,et al.The human papilloma virus-16 E7 oncoprotein is able to bind to the retinoblastoma gene product[J].Science,1989,243(4893):934
[12] MUNGER K,BASILE JR,DUENSING S,et al.Biological activities and molecular targets of the human papillomavirus E7 oncoprotein[J].Oncogene,2001,20(54):7888
[13] BURK RD,CHEN Z,VAN DOORSLAER K.Human papillomaviruses: genetic basis of carcinogenicity[J].Public Health Genomics,2009,12(5/6):281
[14] MOLDEN T,KRAUS I,KARLSEN F,et al.Human papillomavirus E6/E7 mRNA expression in women younger than 30 years of age[J].Gynecol Oncol,2006,100(1):95