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

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 6b E7全长基因,构建重组原核表达质粒pET32a(+)/HPV 6b E7,原核表达产物采用SDS-PAGE和Western blot进行鉴定.进一步经镍螯合亲和层析法(Ni-NTA Agarose)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(40μg/ml).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＜0.05),吸光度(A)均值分别为1.82±0.48、1.36±0.39和1.39±0.27.CA组宫颈分泌物特异性sIgA抗体水平亦显著高于健康对照组(P＜0.05),A值分别为0.63±0.26和0.53±0.06.经PCR检测,CA患者组织HPV 6b E7DNA阳性率为78.6%(44/56).与PCR检测结果比较,HPV6bE7特异性IgG和sIgA抗体用于检测CA患者HPV 6b感染的敏感性分别为68.2%(30/44)、54.6%(24/44),特异性均为100%(12/12).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.

Studies on the establishment of a method to detect HPV 6b E7-specific antibodies of serum and cervical secretion from patients with condyloma acuminatum and its epidemiological significance

Objective To establish a method for detection of the human papi 11 omavirus(HPV)6b E7-specific antibodies in serum and cervical secretion from patients with condyloma acuminatum(CA).Methods A full-length HPV 6b E7 gene was amplified by PCR from the CA tissue to construct the recombinant plasmid pET32a(+)/HPV 6b E7.The expression of prokaryotic protein was analyzed by SDS-PAGE and Western blot,then purified with Ni-NTA Agarose affinity column and used as an diagnostic antigen for establishing indirect ELISA method,to detect specific serum IgG and specific cervical secretion slgA from 56 CA patients,81 healthy control.Sera from 43 cervical cancer was served as control.HPV 6b DNA from 56 CA patients 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(40 μg/ml).Based on HPV 6b E7 fusion protein being used as coating antigen,results from ELISA showed that the absorbance rates(A)of serum IgG from CA,cervix cancer and healthy control groups were 1.82±0.48,1.36 ± 0.39 and 1.39 ± 0.27,respectively.The level of IgG antibody in the serum of CA group was significantly higher than that in cervix cancer group and healthy control(P＜0.05).The 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 of HPV 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 sIgA antibodies by ELISA used to detect the patients infected with HPV 6b infection,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 HPV 6b infection.