CAJ | 학술논문

目的研究人乳头瘤病毒(HPV)次要衣壳蛋白(L2)在临床常见HPV感染型别中的同源性及其交叉反应特性.方法采用生物信息学方法对临床常见HPV感染型别中的L2氨基酸序列进行比对,发现其氨基端1～200序列具有高度同源性.采用PCR法从宫颈癌患者组织DNA中扩增HPV16 L2(1～200)肽段的碱基序列,将其克隆至原核表达载体PGEX-4T-1得到重组质粒PGEX-4T-1-HPV16 L2(1～200).将测序鉴定正确的重组质粒转入E.coli BL21(DF3)中,经异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,SDS-PAGE和Western blot鉴定;进一步通过Western blot检测HPV16L2(1～200)融合蛋白与HPV6、11、16和18型DNA检测阳性临床患者血清的特异性结合能力;以镍螯合亲和层析柱(Ni-NTA Agarose)纯化的HPV16 L2(1～200)融合蛋白作为包被抗原,采用间接ELISA法对98例尖锐湿疣患者、135例宫颈癌患者及96例健康对照者血清进行特异性血清IgG抗体检测.结果在HPV6、11、18、35等14个临床常见型别中,与HPV16 L2的1～200间的氨基酸序列比较,同源性达到52.7%～74.3%;成功构建了含有HPV16 L2(1～200)的重组质粒PGEX-4T-1-HPV16 L2(1～200),含有HPV16 L2(1～200)的融合蛋白在原核表达体系中呈高效表达,表达量占总蛋白的22.6%;表达产物的相对分子质量(Mr)约49×103,与预期Mr相符;以HPV6、11、16和18 DNA阳性患者血清为一抗进行Western blot分析,结果显示,在Mr约49×103处出现特异性条带.ELISA结果显示,尖锐湿疣组、宫颈癌组患者血清及健康对照者血清抗体均值分别为0.753±0.262、0.756±0.274和0.178±0.157,阳性率分别为89.8%、88.9%和9.4%.三组间血清抗体均值及阳性率比较差异均具有统计学意义(P均＜0.001),而尖锐湿疣组和宫颈癌组间的血清抗体均值比较差异无统计学意义(P＞0.05).结论 HPV L2 N端1～200氨基酸序列具有高度同源性,并在HPV6、11、16和18型别间具有交叉反应.
목적 연구인유두류병독(HPV)차요의각단백(L2)재림상상견HPV감염형별중적동원성급기교차반응특성.방법 채용생물신식학방법대림상상견HPV감염형별중적L2안기산서렬진행비대,발현기안기단1～200서렬구유고도동원성.채용PCR법종궁경암환자조직DNA중확증HPV16 L2(1～200)태단적감기서렬,장기극륭지원핵표체재체PGEX-4T-1득도중조질립PGEX-4T-1-HPV16 L2(1～200).장측서감정정학적중조질립전입E.coli BL21(DF3)중,경이병기-β-D-류대반유당감(IPTG)유도표체,SDS-PAGE화Western blot감정;진일보통과Western blot검측HPV16L2(1～200)융합단백여HPV6、11、16화18형DNA검측양성림상환자혈청적특이성결합능력;이얼오합친화층석주(Ni-NTA Agarose)순화적HPV16 L2(1～200)융합단백작위포피항원,채용간접ELISA법대98례첨예습우환자、135례궁경암환자급96례건강대조자혈청진행특이성혈청IgG항체검측.결과 재HPV6、11、18、35등14개림상상견형별중,여HPV16 L2적1～200간적안기산서렬비교,동원성체도52.7%～74.3%;성공구건료함유HPV16 L2(1～200)적중조질립PGEX-4T-1-HPV16 L2(1～200),함유HPV16 L2(1～200)적융합단백재원핵표체체계중정고효표체,표체량점총단백적22.6%;표체산물적상대분자질량(Mr)약49×103,여예기Mr상부;이HPV6、11、16화18 DNA양성환자혈청위일항진행Western blot분석,결과현시,재Mr약49×103처출현특이성조대.ELISA결과현시,첨예습우조、궁경암조환자혈청급건강대조자혈청항체균치분별위0.753±0.262、0.756±0.274화0.178±0.157,양성솔분별위89.8%、88.9%화9.4%.삼조간혈청항체균치급양성솔비교차이균구유통계학의의(P균＜0.001),이첨예습우조화궁경암조간적혈청항체균치비교차이무통계학의의(P＞0.05).결론 HPV L2 N단1～200안기산서렬구유고도동원성,병재HPV6、11、16화18형별간구유교차반응.
Objective To research the homology and cross reaction characteristics of human papillomavirus(HPV)16 type L2 N-terminal(1-200)protein in clinical common HPV infection types.Methods The amino acid sequences of the common HPV infection types(6,11,16,18 ,etc.)were blasted and it was found that 1-200 N-terminal sequence of L2 protein was highly homologous.The gene of HPV16 L2(1-200)was amplificated from tissue sample of cervical cancer patient and inserted into the prokaryotic expression vector PGEX-4T-1 to construct the recombinant plasmid PGEX-4T-1-HPV16 L2(1-200).After sequencing identification,the recombinant plasmid was tranformed into E.coli BL21(DE3).Induced by IPTG,the fusion protein containing HPV16 L2(1-200)was expressed and analyzed by both SDS-PAGE and Western blot.Furthermore,the specific binding capacity of the fusion protein to the HPV 6,11,16 and 18 DNA positive patient serums were analyzed by Western blot.The fusion protein was purified with Ni-NTA Agarose Kit and coated with ELISA reaction plates.The specific serum IgG of 98 condyloma acuminatum patients,135 cervix cancer patients and 96 healthy control subjects were detected respectively by indirect ELISA.Results After comparing the amino acid sequences of the common HPV infection types(HPV6,11,16,18,etc.),We found that the homology of HPV L2(1-200)reached 52.7%-74.3%.The recombinant plasmid PGEX-4T-1-HPV 16 L2(1-200)was constructed successfully.Highly expressed HPV16 L2(1-200)fusion protein was obtained and the expression level was account for up to about 22.6% of total bacterial protein.The relative molecular mass(Mr)of the fusion protein is about 49×103,which matches up to the expected Mr Meanwhile,the serums of HPV 6,11,16,18 DNA positive patients were used as the first antibody and the specific band was detected respectively at about 49 × 103 by Western blot.Indirect ELISA showed that the A490 values of the specific IgG of condyloma acuminatum group,cervical cancer group and healthy control subjects were 0.753 ± 0.262,0.756 ± 0.274 and 0.178 ± 0.157 with the positive rate were 89.8%,88.9% and 9.4% respectively.There was no significance of the specific IgG between condyloma acuminatum group and cervical cancer group(P＞0.05),but it was significant among the three groups(P＜0.001).Conclusion The N-terminal 1-200 amino acids of HPV L2 has high homology and there exits cross reaction among the most common HPV infection types.