【作者】 张卉；

【导师】 陈心秋； 田厚文；

【作者基本信息】 广西医科大学 ， 肿瘤学， 2007， 博士

【摘要】 根据各种流行病学资料,肛门生殖器疣（GW）是世界上一个不容忽视的公众健康问题,每年用于GW的诊断治疗费用给人们带来了不小的经济负担。传统的治疗方法存在一定的缺陷,而且复发率高,于是人们在寻找更有效的治疗或辅助治疗方法。GW与人乳头瘤病毒（Human papillomavirus,HPV）6型和11型的密切关系10年前就已经明确。GW病灶会由一个逐渐扩增成多个,常常是旧的和新的疣体并存。可以解释为当上皮细胞局部受损伤的时候,上皮细胞的完整性遭到破坏,旧病灶脱落释放的HPV通过表皮的微小创伤进入组织,感染新的上皮基底层细胞。所以被HPV感染的细胞受感染的程度不一:处于非整合状态的部位,HPV的早晚期基因E6、E7、L1、E2都有表达;与宿主细胞DNA整合的仅能检测到E6、E7的表达;对于这种纷乱混杂的感染状态,就应该既要预防HPV的扩散感染,又要清除已被感染的细胞和相关病灶。鉴于上述原因,人们一直在研制HPV疫苗,通过预防和治疗HPV感染的策略,达到预防和治疗GW的目的。预防性疫苗免疫策略主要集中在如何诱发出针对HPV相关表位的高滴度中和抗体来预防感染。以HPV衣壳蛋白L1,L2为靶抗原制备的病毒样颗粒（VLP）是预防性疫苗的主流,目前Merk公司的16、18、6、11型四价VLP疫苗已经上市。治疗性疫苗的关键是诱发针对病毒蛋白的细胞免疫,清除病灶和感染细胞,由于HPV-6,11型的两个早期蛋白E6,E7在疣体中持续表达,并且与疣复发密切相关,它们是治疗性疫苗理想的靶抗原。由英国和比利时分别研制的HPV6b L2E7融合蛋白疫苗都已经进行了Ⅱ期临床实验。本研究在实验室已构建的HPV6bL2E7、HPV16L2E7治疗性疫苗基础上,构建HPV11L2E7融合蛋白原核表达系统,得到纯化的L2E7蛋白,并在小鼠体内评价其免疫原性。首先我们从尖锐湿疣组织经PCR扩增获得人乳头瘤病毒11型L2、E7编码区基因,其序列与发表的序列比对（序列号M14119）,L2有两处突变,因未引起氨基酸的改变,为同义突变;E7有一处碱基由“G”变为“T”,相应位置的氨基酸由“A”变为“S”。构建pMD18T HPV11L2E7质粒,测序证实获得了L2E7融合基因。将融合的L2E7基因连接至缺失His的原核表达载体pETga,构建pET9a HPV11L2E7原核表达重组质粒。在大肠杆菌宿主菌BL21（DE3+）中经IPTG诱导高效表达了L2E7融合蛋白（553个氨基酸）,经SDS-PAGE电泳和Western Blotting鉴定,表明成功表达了L2E7蛋白。诱导表达L2E7蛋白,收获、洗涤包涵体溶解于8M尿素（PH 8.3）进行CM离子交换介质纯化,纯化的蛋白通过Gel-Pro 3.1软件分析其纯度为83.8%。纯化的L2E7蛋白与佐剂CpG-ODN和氢氧化铝免疫6～8周龄的BALB/c小鼠,进行细胞、体液免疫指标的检测。酶联免疫斑点实验（Enzyme linkedimmunospot,ELISPOT）显示,经该蛋白两次免疫后,小鼠脾单个核细胞（SMNC）体外HPV11E7肽库特异性IFN-γ分泌的斑点数为42.5±6.473/8×10⁵脾细胞,与对照组（1.8±0.7528/8×10⁵脾细胞）相比较,差别具有统计学意义（Q=16.3600,P<0.01）。细胞因子染色表明,实验组CD4/IFN-γ双阳性的细胞比例为1.8±0.1%,CD8/IFN-γ双阳性的细胞比例为0.3±0.1%,都高于对照组。酶联免疫吸附实验（ELISA）检测小鼠血清中L2和E7的特异性抗体,L2_100-120和E7的IgG都达到了1∶8000。综上所述,原核表达纯化的HPV11L2E7融合蛋白不仅可以在小鼠体内引起HPV11E7特异性的细胞免疫反应,并且也能诱发针对HPV11L2和E7的特异性体液免疫反应,能作为尖锐湿疣免疫治疗候选疫苗。更多还原

【Abstract】 Genital wart（GW）is a significant public health problem across the world according to the data of epidemiology.Annually the cost of GW’s diagnosis and therapy is a great burdern to people.Due to the defects of traditionary therapies and the high recurrence of GW,people began to pursue the effective therapeutic or aid-therapeutic methods.It was cleared that the close association between GW and Human papillomavirus （HPV）type 11 and 6 10 years ago.Once genital wart exists in one place then it usually spreads to other places and becomes polyinfection.HPV released from past lesions would infect the new basal cell layer when epithelial cells were injured and the integrity of epithelia was destroyed.Therefore the infections of cells’ were at different levels such as the early gene E6,E7 and late gene L1,L2 were expressed in non-integrated cells howerver in integrated cells only E6,E7 protein were detected.So we need to treat the disordered infections by preventing the diffusion of HPV infection and clearing the infected cells and lesions.Thus people study vaccines to prevent and treat HPV infection with HPV vaccine so that to prevent and treat GW.Prophylactic vaccination strategies have focused on eliciting high titers of neutralizing antibodies to epitopes displayed on papillomaviruses and preventing the primary infection by HPV.In this respect,the virus-like particles（VLPs）targeting the capsid protein L1,L2 of HPV become the main currency.The quadrivalent VLPs HPV-16/18/6/11 produced by Merk went on the market in 2006.As to therapeutic vaccine,it is the vital point to induce the cell-mediated immune response to viral protein,then to eliminate the lesion and infected cells.Two early proteins of HPV-6,-11 E6 and E7 are expressed at GW continuously and are required for the maintenance and recurrence of GW.They represent good targets for developing therapeutic vaccines.Two kinds of fusion proteins HPV6bL2E7 produced by Britain and Belgium respectively had been carried on phaseⅡclinical trials.In our study,we constructed the Escherichia coli（E.coli）prokaryotic expression system pET9aHPV11L2E7 on the basis of two therapeutic vaccines HPV6bL2E7 and HPV16L2E7 constructed by us.Purified the fusion protein L2E7 and studied the immunnogenicity in mice.At first,we amplified HPV11 L2,E7 coding region from condyloma acuminata tissues by PCR.Then compared the sequences with published sequences on genebank（serial numobr:M14119）.We found that there were two mutations in L2 gene and which were same-sense mutations.In E7 gene there was one mutation: the G changed to T which caused the corresponding amino acids changed from A to S.Thereafter we constructed the plasmid pMD18T HPV11L2E7 then sequenced and which showed that we got the fusion gene L2E7.Subsequently we connected fusion gene L2E7 to His depletion plasmid pET9a,established the recombinant plazmid pET9aHPV11L2E7 and sequenced.Fusion protein L2E7（553 amino acids） was expressed in host strain BL21（DE3⁺）by IPTG inducing and identified by using SDS-PAGE and Western Blotting which indicated we had expressed L2E7 succesfully.We expressed L2E7 protein in host strain BL21（DE3⁺）and harvested the inclusion then washed it twice with 1M Urea.At last we lysised the inclusion in 8M Urea（PH8.3）and purified with CM column.The purity of L2E7 analysed with software Gel-Pro 3.1 was 83.8%.Purified L2E7 mixed with adjuvant CpG-ODN and AL（OH）₃ inoculated to BALB/c mice and its cell-mediated and humoral immunnogenicity was assessed. IFN-γ,enzyme-linked immunospot（ELISPOT）showed after primed/boost the frequency of HPV11 E7 peptides pool specific IFN-γsecreting counts are 42.5±6.473/8×10⁵ splenocyte mononuclearcytes（SMNC）.Compared with the control group（1.8±0.7528/8×10⁵SMNC）the difference between experiment group and control group were statistic significant.Cytokine staining manifested that CD4/IFN-γpositive cells（1.8±0.1%）and CD8/IFN-γ,positive cells（0.3±0.1%）were higher than those in control group.Enzyme-linked immunosorbent assay（ELISA）were used to evaluate the HPV11 L2 and E7-specific antibody in the serum of mice.The titers of anti-L2_100-120,E7 IgG both reached 1:8000.In summery the mice in vivo experiment indicated that the purified protein L2E7 could induce HPVllE7 specific cell-mediated immune responses and humoral immune responses.It can be used as a candidate of genital wart immune therapeutic vaccine.更多还原