HPV18 E7基因的原核表达及其在宫颈癌患者血清抗体中的检测

The Expression of the Full-length Gene of Human Papillomavirus Type 18 E7 in Prokaryotic Cells and Its Serum Antibody in Patients with Cervical Cancer

目的研究人类乳头瘤病毒18型E7全长基因(HPV18 E7)在原核表达系统中的表达,及其作为特异性抗原在宫颈癌血清抗体检测中的意义,为HPV感染与肿瘤相关的血清学诊断研究奠定基础。方法PCR法扩增HPV18 E7全长基因,构建重组质粒pET32a(+)/HPV18 E7,转化表达HPV18 E7融合蛋白后经纯化、SDS-PAGE和Western blot分析鉴定。再以该融合蛋白作为诊断抗原,间接ELISA法检测宫颈癌、尖锐湿疣及健康对照三组血清特异性IgG抗体水平。结果HPV18 E7融合蛋白表达量约占菌体总蛋白的35.00%;宫颈癌、尖锐湿疣及健康对照组的血清特异性抗体均值分别为0.604±0.328,0.287±0.202和0.342±0.225,三组间差异有显著性意义(F=32.771,P<0.01);宫颈癌组与尖锐湿疣组及健康对照组的HPV18E7均值比较,差异均有显著性意义(P均<0.01),而尖锐湿疣组与健康对照组抗体均值比较,差异无统计学意义(P>0.05)。结论HPV18 E7融合蛋白具较强的抗原性,对宫颈癌诊断试剂的研究具有应用价值。

Objective To study the expression of full-length oncoproteins of human papillomavirus type 18 E7 in prokaryotic expression system, and to screen the serum antibody in the patients with cervical cancer by antigen of the recombinant HPV 18 E7 protein, try to establish the method for the serum diagnosis of HPV infection and the HPV associated disease. Methods HPV18 E7 full-length oncoproteins was amplified by PCR and then cloned into the vector pET32a（ ± ） to construct the recombinant plasmid pET32a（ ± ）/ HPV 18E7, then the recombinant plasmid was transformed into E. coli BL21. The pET32a（ ± ）/ HPV18 E7 fusion protein was purified and analysised by SDS-PAGE and Western blot. Then it was used as diagnosis antigen to screen the levels of serum IgG from the patients with cervical cancer, condyloma acuminatum and healthy control subjects by ELISA. Results The recombinant HPV18 E7 protein was about 35.00% of the total bacterial protein. The HPV18 E7 fusion protein was also used to screen the antibody in human serum. The mean values of the antibody of the subjects with cervical cancer, condyloma acuminatum and healthy control were 0. 605 ± 0. 328,0.287 ± 0.202 and 0. 342 ± 0. 225 respectively. Differences between the three groups were significant（ F = 32. 771 ,P 〈 0.01 ）. Intercomparison analysis by Tamhane method showed that the mean values between cervical cancer and condyloma acuminatum, cervical cancer and healthy control had significant difference （all P 〈 0.01 ）, while condyloma acuminatum and healthy control showed no significant difference （P〉0.05）. Conclusion HPV18 E7 fusion protein has good antigenicity, and should be as the serodiagnosis reagent of cervical cancer.