| 钟晓芝,陈韶,朱珊丽,薛向阳,李文姝,王乐丹,张丽芳.尖锐湿疣患者血清及宫颈分泌物HPV 6bE7抗体检测方法的建立及流行病学意义[J].Chinese journal of Epidemiology,2010,31(2):199-203 |
| 尖锐湿疣患者血清及宫颈分泌物HPV 6bE7抗体检测方法的建立及流行病学意义 |
| Studies on the establishment of a method to detect HPV 6b E7一specific antibodies of serum and cervical secretion from patients with condyloma acuminatum and its epidemiological significance |
| Received:June 03, 2009 |
| DOI： |
| KeyWord: 尖锐湿疣 人乳头瘤病毒 血清 宫颈分泌物 6b E7抗体 |
| English Key Word: Condylomata acuminata Human papil lomavirus Serum Cervical secretion 6b E7-specific antibodies |
| FundProject:国家自然科学基会(30671882)；浙江省医药卫牛蓐点科技基金(2005ZD011)；温州市科技计划项日(Y20060081) |
| Author Name | Affiliation | E-mail | | ZHONG Xiao-zhi | Department of Microbiology and Immunology, Institute of Molecular Virology and Immunology, Wenzhou Medical College, Wenzhou 325035, China | | | CHEN Shao | Department of Microbiology and Immunology, Institute of Molecular Virology and Immunology, Wenzhou Medical College, Wenzhou 325035, China | | | ZHU Shan-1i | Department of Microbiology and Immunology, Institute of Molecular Virology and Immunology, Wenzhou Medical College, Wenzhou 325035, China | | | XUE Xiang-yang | Department of Microbiology and Immunology, Institute of Molecular Virology and Immunology, Wenzhou Medical College, Wenzhou 325035, China | | | LI Wen-shu | Department of Microbiology and Immunology, Institute of Molecular Virology and Immunology, Wenzhou Medical College, Wenzhou 325035, China | | | WANG Le-dan | Department of Obstetrics and Gynecology, the Second Afiliated Hospital of Wenzhou Medical College | | | ZHANG Li-fang | Department of Microbiology and Immunology, Institute of Molecular Virology and Immunology, Wenzhou Medical College, Wenzhou 325035, China | wenzhouzlf@126.com |
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| Abstract: |
| 目的建立尖锐湿疣(cA)患者血清和宫颈分泌物人乳头瘤病毒(HPV)6b型E7特异性抗体检测方法。方法利用PCR扩增HPV 6bE7全长基因，构建重组原核表达质粒pET32a(+)/HPV 6bE7。原核表达产物采用SDS.PAGE和Westernblot进行鉴定。进一步经镍螯合亲和层析法(Ni-NTAAgarose)纯化后作为包被抗原，以间接ELISA方法分别对56例CA患者、81名健康对照者的血清和宫颈分泌物标本中的特异性抗体进行检测，另取43例宫颈癌患者的血清标本作为对照；同时用PCR方法对56例CA组织标本进行HPV6bDNA的检测。结果从CA组织标本中扩增的HPV 6b E7全长基因与标准序列(GenBank登录号:NC001355)同源性为99.5％。HPV 6b E7融合蛋白在原核表达系统中得到了较高效的表达(40 I.tg/m1)。经间接ELISA检测，CA组血清特异性IgG抗体水平明显高于宫颈癌组和健康对照组(P结论CA患者血清和宫颈分泌物HPV 6b E7特异性抗体，用于诊断HPV 6b感染具有一定的敏感性和较高的特异性，可用于HPV 6b感染的流行病学调查。 |
| English Abstract: |
| Objective To establish a method for detection of the human papi ll omavirus (HPV)6b E7.specific antibodies in serBnl and cervical secretion from patients with eonay7loma acuminatum(CA). Methods A full.1ength HPV 6b E7 gene was amplified by PCR from the CA tissue to construct the recombinant plasmid pET32a(+)/uPV 6b E7.The expression of prokaryotic vrotein Was analyzed by SDS.PAGE and Westem blot，then purified wit}l Ni-NTA Agarose affinity column and used as all diagnostic antigen for establishing indirect ELISA method。to detect specific serlml IgG and specific cervical secretion slgA from 56 CA patients，81 healthy contr01.Sera from 43cervical cancer was served as contr01.HPV 6b DNA from 56 CA pafienB Was identified by PCR. Results Data showed that the nucleotide homology of cloned sequence was 99.5％.compared to the standard sequences of HPV 6b E7 gene(GenBank accession number:NC001355).A high level expression of E7 fusion protein Was obtained in prokaryotic expression system(40μg/ml).Based on HPV 6b E7 fusion protein being used as coating antigen，results from ELlSA showed that the absorbance rates(A)of serunl lgG from CA，cervix cancer and healthy control groups were 1.82±
O.48.1.36±O.39 and 1.39±O-27，respectively.The level of lgG antibody in the serum of CA group was significantly higlIel"than that in cervix cancer gro叩and healthy control(P<0.05).111e A values of cervical secretion sIgA in CA and healthy control groups were 0.63±0.26 and 0.53±0.06.respectively.while the level of sIgA antibody in the cervical secretion of CA group was also significantly higher than that in healthy controls(P<0.05).The positive rate ofHPV 6b E7 DNA in CA tissue was 78.6％(44/56)by PCR method.When compared the results measured by PCR，the HPV 6b E7.specific IgG and slgA antibodies bv ELISA used to detect the patients infected with HPV 6b iIlfection。showed that the sensitivity rates were 68.2％(30/44)and 54.6％(24/44)respectively。and the specificity were all 100％(12/12). Conclusion Based on the serum and cervical secretion specific HPV 6b E7 antibodies from patients with CA to diagnose HPV 6b infection，results showed medium sensitivity and high specificity，and could further be used to investigate the epidemiological characteristics of HPV6b infection. |
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