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1.
An optimized recombinant HPV16 E6E7 fusion gene (HPV16 ofE6E7) was constructed according to codon usage for mammalian cell expression, and a mutant of HPV16 ofE6E7 fusion gene (HPV16 omfE6E7) was generated by site-directed mutagenesis at L57G, C113R for the E6 protein and C24G, E26G for the E7 protein for HPV16 ofE6E7 [patent pending (CN 101100672)]. The HPV16 omfE6E7 gene constructed in this work not only lost the transformation capability to NIH 3T3 cells and tumorigenicity in SCID mice, but also maintain... 相似文献
2.
Ayslan Castro Brant Vladimir Majerciak Miguel Angelo Martins Moreira Zhi-Ming Zheng 《中国病毒学》2019,34(2):211-221
Human papillomavirus 18(HPV18) E6 and E7 oncogenes are transcribed as a single bicistronic E6 E7 pre-mRNA. The E6 ORF region in the bicistronic E6 E7 pre-mRNA contains an intron. Splicing of this intron disrupts the E6 ORF integrity and produces a spliced E6~*I RNA for efficient E7 translation. Here we report that the E6 intron has two overlapped branch point sequences(BPS) upstream of its 30 splice site, with an identical heptamer AACUA■C, for E6~*I splicing. One heptamer has a branch site adenosine(underlined) at nt 384 and the other at nt 388. E6~*I splicing efficiency correlates to the expression level of E6 and E7 proteins and depends on the selection of which branch site. In general, E6~*I splicing prefers the 30 ss-proximal branch site at nt 388 over the distal branch site at nt 384. Inactivation of the nt 388 branch site was found to activate a cryptic acceptor site at nt 636 for aberrant RNA splicing. Together, these data suggest that HPV18 modulates its production ratio of E6 and E7 proteins by alternative selection of the two mapped branch sites for the E6~*I splicing, which could be beneficial in its productive or oncogenic infection according to the host cell environment. 相似文献
3.
Kim SH Kim KS Lee EJ Kim MO Park JH Cho KI Imakawa K Hyun BH Chang KT Lee HT Ryoo ZY 《Life sciences》2004,75(25):3035-3042
Human papillomavirus type 16 (HPV16) has been known as a major causative factor for the development of uterine cervical carcinomas. To investigate the in vivo activity of HPV16 expressed in squamous epithelia, transgenic mice harboring HPV16 E6/E7 with human keratin 14 (hK14) promoter were generated. Grossly, hK14 driven HPV16 E6/E7 transgenic mice exhibited multiple phenotypes, including wrinkled skin that was apparent prior to the appearance of hair in neonates, thickened ears, and loss of hair in adults. Transgenic mice with phenotype exhibiting severe wrinkled skin and a lack of hair growth died at the age of 3-4 weeks. Histological analysis revealed that in transgenic mice survived beyond the initial 3-4 weeks, HPV16 E6/E7 causes epidermal hyperplasia in multiple transgenic lineages with high incidence of transgene penetration. This epithelial hyperplasia was characterized by an expansion of the proliferating compartment and keratinocytes, and was associated with hyperkeratosis. Such activities were significantly higher in the skin of transgenic mice than that of the normal mice. Thus, these transgenic mice appeared to be useful for the expression of HPV16 E6/E7 gene and subsequent analysis on hyperkeratosis. 相似文献
4.
HPV16型为主的多种HPV病毒可诱发机体发生宫颈癌等疾病,以重叠PCR法人工合成HPV16 E6、E7致癌基因的融合基因,以之为目的基因构建了无选择标记基因(Marker-Free)双元载体,期望转化番茄开发新型宫颈癌治疗性疫苗-转基因植物口服疫苗.通过生物信息学分析HPV16 E6、E7基因,设计并合成密码子优化的靶基因E6-E7融合基因;并在目的基因的上游引入分子佐荆LTB基因,与Kozak序列等表达元件相偶联,以提高目的基因在植物表达系统的表达水平、增强其诱导黏膜免疫的免疫原性.目前已构建pX6-LTB-E7和pX6-LTB-E7-E6两个番茄转化双元载体.采用番茄Marker-Free系统转化和表达HPV16 E6、E7目的基因可以在转化后代中剔除标记基因,从而消除由标记基因可能引起的转基因植物口服疫苗的安全性问题,为HPV转基因植物口服疫苗应用奠定基础. 相似文献
5.
Induction of senescence-like state and suppression of telomerase activity through inhibition of HPV E6/E7 gene expression in cells immortalized by HPV16 DNA 总被引:16,自引:0,他引:16
The E6 and E7 oncoproteins of human papillomavirus (HPV) play a major role in the development of cervical carcinoma. In this study, a recombinant adenovirus that expresses the bovine papillomavirus (BPV) E2, which has been shown to inhibit HPV early gene expression, was delivered to two HPV-immortalized cell lines as well as CaSki, a cervical carcinoma cell line. We tested whether the carcinoma and the immortal cells were equally affected by the expression of BPV E2. In all cell lines, BPV E2-mediated inhibition of HPV E6/E7 expression caused a dramatic suppression of cell growth, being preceded by the activation of the p53-Rb growth-inhibitory pathway, and a decrease in hTERT mRNA expression and telomerase activity. This suggests that the HPV E6 and E7 proteins are required not only for induction of the proliferative phenotype and telomerase activity, but also for their maintenance. In both the carcinoma and the immortal lines, the number of cells with enlarged cytoplasm and senescence-associated beta-galactosidase activity, which are markers for cellular senescence, was significantly increased. These results suggest that a senescence program exists in cells immortalized by HPV DNA as well as in cervical carcinoma cells. 相似文献
6.
人乳头瘤病毒(Human papillomavirus,HPV)16型(HPV-16)是引起宫颈癌的一种主要高危型病毒,其2个致癌基因E6和E7的核酸序列变异可能会影响其对宿主细胞的致癌性,已有研究表明其序列突变呈现地域差异性。因此,研究不同地域HPV-16这2个基因的变化情况是宫颈癌流行病学调研的主要内容,也可为研究E6和E7的致癌性积累数据。研究以NCBI登录号为NC_001526.2的HPV-16型病毒的序列为参照,采用Neighbor-joining方法对云南地区74例HPV-16样本的E6、E7的DNA序列构建进化树,结果显示:只有亚洲和欧洲变异亚型,而没有发现非洲1、非洲2、亚-美洲和北美洲这4种变异亚型。DNA序列分析显示:E6的碱基突变以T178G(D25E,59.46%)和T350G(L83V,8.11%)为主,E7的碱基突变主要以A647G(N29S,59.46%)和T846C(同义突变,60.81%)为主。发现E6的新突变有A95G(同义突变,1.35%)和A135G(K11R,1.35%);E7的新突变有C625T(L22F,1.35%)、C627T(同义突变,12.16%)、G689A(G43E,1.35%)、T748G(S63A,1.35%)。此外还发现有一个共突变现象:T178G(D25E,59.46%)-A647G(N29S,59.46%)-T843C(同义突变,21.62%)-T846C(同义突变,60.81%)。 相似文献
7.
人乳头瘤病毒16型E7变异株免疫原性研究 总被引:1,自引:0,他引:1
采用分子克隆技术,构建E7变异株重组质粒[pcDNA3.1-(by)E7]和E7标准株重组质粒[pcDNA3.1-(ys)-E7],并将两种质粒分别皮下免疫Balb/c小鼠,免疫后于不同时间提取小鼠血清和制备脾淋巴细胞悬液,分别用ELISA法和MTT比色法检测特异性抗体和特异性淋巴细胞增殖反应.基因免疫后,ELISA法显示,HPV16 E7变异株和标准株均能诱导特异性抗E7抗体;MTT比色法显示,E7标准株免疫组脾淋巴细胞在体外受到变异株E7蛋白的再次刺激后出现特异性淋巴细胞增殖反应,变异株E7免疫组脾淋巴细胞经过同样处理后,出现非特异性淋巴细胞增殖反应.结果表明HPV16E7变异株能诱导特异性体液免疫应答而不能诱导特异性细胞免疫应答,HPV16 E7变异株无论在结构还是免疫原性上均与标准株有差异.由此推测,HPV16E7变异可能导致其逃逸机体自然感染或疫苗诱导的免疫应答.用基因免疫方法研究E7变异株免疫原性也为其它不能或难以进行体外培养的病毒变异研究提供借鉴. 相似文献
8.
采用分子克隆技术,构建E7变异株重组质粒[pcDNA3.1-(by)E7]和E7标准株重组质粒【pcDNA3.1.(ys)-E7】,并将两种质粒分别皮下免疫Balb/c小鼠,免疫后于不同时间提取小鼠血清和制备脾淋巴细胞悬液,分别用ELISA法和MTT比色法检测特异性抗体和特异性淋巴细胞增殖反应。基因免疫后,ELISA法显示,HPVl6E7变异株和标准株均能诱导特异性抗E7抗体;MTTT比色法显示,E7标准株免疫组脾淋巴细胞在体外受到变异株E7蛋白的再次刺激后出现特异性淋巴细胞增殖反应,变异株E7免疫组脾淋巴细胞经过同样处理后,出现非特异性淋巴细胞增殖反应。结果表明HPV16E7变异株能诱导特异性体液免疫应答而不能诱导特异性细胞免疫应答,HPVl6E7变异株无论在结构还是免疫原性上均与标准株有差异。由此推测,HPV16E7变异可能导致其逃逸机体自然感染或疫苗诱导的免疫应答。用基因免疫方法研究E7变异株免疫原性也为其它不能或难以进行体外培养的病毒变异研究提供借鉴。 相似文献
9.
I. Caroline Le Poole Frank M. van den Berg René M. J. G. J. van den Wijngaard Denise A. Galloway Peter J. van Amstel Anita A. M. Buffing Henk L. Smits Wiete Westerhof Pranab K. Das 《In vitro cellular & developmental biology. Animal》1997,33(1):42-49
Summary Availability of a standard human melanocyte cell line with unlimited growth potential and otherwise normal melanocytic properties
will greatly facilitate research in melanocyte biology and in vitro studies on the etiology of pigmentary disorders and melanoma. Using a retroviral vector, E6 and E7 open reading frames of
human papilloma virus type 16 (HPV 16) have been introduced into cultured normal human melanocytes. Cells selected by increased
resistance to geneticin conveyed by the vector and expressing E6E7 mRNA have been cloned to ensure genetic homogeneity. Since
their establishment as primary cells, cloned PIG1 cells have undergone more than twice the amount of population doublings
of senescent parental cells. Moreover, in passage numbers when parental cells had become senescent, proliferation of clonal
cells was retained at levels exceeding those of normal human melanocytes in third passage by 100%. Further characterization
has revealed that the cells remain dependent on tetradecanoyl phorbol 13-acetate (TPA) for growth and do not proliferate in
soft agar nor form tumors in nude mice. The antigenic profile of the cells was slightly altered as compared to parental cells,
but was incomparable to that of M14 melanoma cells. Importantly, PIG1 cells contain more melanin pigment than parental cells. 相似文献
10.
Lin CT Tsai YC He L Calizo R Chou HH Chang TC Soong YK Hung CF Lai CH 《Journal of biomedical science》2006,13(4):481-488
Summary The HPV oncoproteins E6 and E7 are consistently expressed in HPV-associated cancer cells and are responsible for their malignant transformation. Therefore, HPV E6 and E7 are ideal target antigens for developing vaccines and immunotherapeutic strategies against HPV-associated neoplasms. Recently, it has been demonstrated that codon optimization of the HPV-16 E7 gene resulted in highly efficient translation of E7 and increased the immunogenicity of E7-specific DNA vaccines. Since vaccines targeting E6 also represent an important strategy for controlling HPV-associated lesions, we developed a codon-optimized HPV-16 E6 DNA vaccine (pNGVL4a-E6/opt) and characterized the E6-specific CD8+ T cell immune responses as well as the protective and therapeutic anti-tumor effects in vaccinated C57BL/6 mice. Our data indicated that transfection of human embryonic kidney cells (293 cells) with pNGVL4a-E6/opt resulted in highly efficient translation of E6. In addition, vaccination with pNGVL4a-E6/opt significantly enhanced E6-specific CD8+ T cell immune responses in C57BL/6 mice. Mice vaccinated with pNGVL4a-E6/opt are able to generate potent protective and therapeutic antitumor effects against challenge with E6-expressing tumor cell line, TC-1. Thus, DNA vaccines encoding a codon-optimized HPV-16 E6 may be a promising strategy for improving the potency of prophylactic and therapeutic HPV vaccines with potential clinical implications. 相似文献
11.
Human papillomavirus: E6 and E7 oncogenes 总被引:4,自引:0,他引:4
Boulet G Horvath C Vanden Broeck D Sahebali S Bogers J 《The international journal of biochemistry & cell biology》2007,39(11):2006-2011
12.
Caberg JH Hubert P Herman L Herfs M Roncarati P Boniver J Delvenne P 《Cancer immunology, immunotherapy : CII》2009,58(1):39-47
Human papillomavirus (HPV) infection, particularly type 16, is causally associated with cancer of the uterine cervix. The
persistence or progression of cervical lesions suggests that viral antigens are not adequately presented to the immune system.
This hypothesis is reinforced by the observation that most squamous intraepithelial lesions (SILs) show quantitative and functional
alterations of Langerhans cells (LC). The infiltration of immature LC in the squamous epithelium is mainly controlled by Macrophage
Inflammatory Protein 3α/CCL20. After having shown that CCL20 production is altered in HPV-transformed keratinocytes (KC),
the possible role of HPV16 E6 and E7 viral oncoproteins in the reduced CCL20 levels observed in SILs was investigated by silencing
HPV16 E6 and E7 oncogenes by RNA interference (siRNA). This treatment not only increased CCL20 secretion but also resulted
in the modulation of NF-κB p50, p52 and p65 precursor localization. Moreover, silencing of E6 and E7 oncogenes in HPV16-transformed
KC induced a significantly higher migratory capacity of LC in a Boyden chamber assay and in an in vitro formed (pre)neoplastic
epithelium reminiscent of high-grade SILs. Anti-CCL20 neutralizing antibody experiments showed that the increased migration
of LC is due to the re-expression of CCL20 in E6 and E7 siRNA transfected KC. These data suggest that HPV16 E6/E7-induced
down-regulation of CCL20 observed during the cervical carcinogenesis may contribute to a diminished capacity of the immune
system to control HPV infection.
P. Hubert and J. H. Caberg contributed equally to this work. 相似文献
13.
分别以卡介苗(BCG)基因组DNA及宫颈癌组织提取DNA为模板,通过PCR扩增得到19kD抗原的胞壁区及其上游调控元件(19ss)基因序列和HPV16型E7基因序列。先将19ss基因与大肠杆菌-分枝杆菌穿梭质粒pMV261重组,得到重组质粒pMCW。再将E7基因克隆至pMCW,得到重组质粒pMCW-E7。最后用PCR引物定点诱变法突变E7基因与转化有关的位点,得到重组质粒pMCW-mE7。对重组质粒pMCW-mE7用PCR扩增、双酶切及测序鉴定证实,19ss基因及突变的E7基因正确插入穿梭质粒pMV261。成功构建了重组穿梭质粒pMCW-mE7。 相似文献
14.
进行人乳头瘤病毒6b型(Human papillomavirus type 6b,HPV6b)E7蛋白原核表达并制备其多克隆抗体。用已构建的pGEX-4T-2/HPV6bE7原核表达载体诱导表达大量可溶性融合蛋白GST-HPV6bE7,用Glutathione-Sepharose 4B亲和柱和凝血酶纯化获取HPV6b型E7蛋白。将纯化的E7蛋白免疫新西兰兔并纯化为多克隆抗体IgG。采用Western-Blot及免疫荧光法分析该抗体的效价及特异性。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析显示,异丙基-β-D-硫代半乳糖苷(IPTG)诱导3~6h后pGEX-4T-2/HPV6bE7表达载体在大肠杆菌中高水平表达可溶性融合蛋白。纯化的E7蛋白免疫新西兰兔后可获得兔多克隆抗体IgG。经Western-Blot及免疫荧光鉴定,兔抗IgG具有高效价性和抗HPV6bE7蛋白特异性。获取纯化的HPV6b型E7蛋白具有较好的免疫原性,其免疫兔产生的多克隆抗体IgG效价高,特异性好,有望进一步用于HPV6b型的生物学功能研究和免疫学效应研究。 相似文献
15.
Wang X Santin AD Bellone S Gupta S Nakagawa M 《Cancer immunology, immunotherapy : CII》2009,58(2):309-308
Previously, safety and immunogenicity of human papillomavirus type 16 (HPV16) or 18 E7-pulsed dendritic cells (DC) vaccinations
were demonstrated in a dose-escalation Phase I clinical trial which enrolled ten patients diagnosed with stage IB or IIA cervical
cancer (nine HPV 16-positive, one HPV 18-positive). The goal of the study was to define the T-cell epitopes of HPV 16 or 18
E7 protein in these patients in order to develop new strategies for treating HPV-associated malignancies. This was accomplished
through establishing T-cell lines by stimulating peripheral blood mononuclear cells with autologous mature DC pulsed with
the HPV 16 or 18 E7 protein, examining the T-cell responses using ELISPOT assays, and isolating E7-specific T-cell clones
based on IFN-γ secretion. Then, the epitope was characterized in terms of its core sequence and the restriction element. Twelve
T-cell lines from eight subjects (seven HPV 16-positive, one HPV 18-positive) were evaluated. Positive T-cell responses were
demonstrated in four subjects (all HPV 16-positive). All four were positive for the HPV 16 E7 46-70 (EPDRAHYNIVTFCCKCDSTLRLCVQ)
region. T-cell clones specific for the E7 47–70 region were isolated from one of the subjects. Further analyses revealed a
novel, naturally processed, CD4 T-cell epitope, E7 58–68 (CCKCDSTLRLC), restricted by the HLA-DR17 molecule.
This work was supported by the National Institutes of Health (R21CA094507).
An erratum to this article can be found at 相似文献
16.
Smahel M Tejklova P Smahelova J Polakova I Mackova J 《Cancer immunology, immunotherapy : CII》2008,57(6):823-831
Infection with high-risk types of human papillomavirus (HPV) can cause the development of malignant tumors. To study mechanisms
responsible for immune escape of tumor cells infected with HPV16, we previously used mouse oncogenic TC-1 cells producing
HPV16 E6 and E7 oncoproteins to derive TC-1 clones resistant to immunization against E7. We have found immunoresistance of
the clones to correlate with the point mutation in the E7 oncogene, which resulted in the N53S substitution in the immunodominant
epitope RAHYNIVTF (aa 49–57). Here, we have shown that this mutation reduced stabilization of H-2Db molecules on RMA-S cells and eliminated immunogenicity of E7. The resistance of TC-1 clones was E7-specific as immunization
against E6 inhibited tumor growth. Transduction of the TC-1/F9 clone carrying the mutated epitope with the wild-type E7 gene
restored susceptibility to immunization against E7. Our results suggest that mutagenesis of tumor antigens can lead to the
escape of malignant cells and should be considered in the development and evaluation of cancer immunotherapy. 相似文献
17.
Characterization of HPV-16 E6 DNA vaccines employing intracellular targeting and intercellular spreading strategies 总被引:1,自引:1,他引:0
Peng S Trimble C Ji H He L Tsai YC Macaes B Hung CF Wu TC 《Journal of biomedical science》2005,12(5):689-700
Summary Human papillomavirus (HPV) E6 and E7 are consistently expressed and are responsible for the malignant transformation of HPV-associated
lesions. Thus, E6 and E7 represent ideal targets for therapeutic HPV vaccine development. We have previously used the gene
gun approach to test several intracellular targeting and intercellular spreading strategies targeting HPV-16 E7. These strategies
include the use of the sorting signal of lysosome-associated membrane protein (LAMP-1), Mycobacterium tuberculosis heat shock protein 70 (HSP70), calreticulin (CRT) and herpes simplex virus type 1 (HSV-1) VP22 proteins. All of these strategies
have been shown to be capable of enhancing E7-DNA vaccine potency. In the current study, we have characterized DNA vaccines
employing these intracellular targeting or intercellular spreading strategies targeting HPV-16 E6 for their ability to generate
E6-specific CD8+ T cell immune responses and antitumor effects against an E6-expressing tumor cell line, TC-1, in C57BL/6 mice. We found that
all the intracellular targeting strategies (CRT, LAMP-1, HSP70) as well as the intercellular spreading strategy (VP22) were
able to enhance E6 DNA vaccine potency, although the orientation of HSP70 linked to E6 antigen in the E6 DNA vaccine appears
to be important for the HSP70 strategy to work. The enhanced E6-specific CD8+ T cell immune response in vaccinated mice also translated into potent antitumor effects against TC-1 tumor cells. Our data
indicate that all of the intracellular targeting and intercellular spreading strategies that have been shown to enhance E7
DNA vaccine potency were also able to enhance E6 DNA vaccine potency. 相似文献
18.
19.
目的:构建呈递HPV16 E7 CTLs抗原表位的病毒样颗粒,并初步评价病毒样颗粒作为治疗性疫苗载体的潜能。方法:根据文献选择有效的HPV16 E7 CTLs表位,合成其正负链寡核苷酸序列,并通过退火形成双链DNA片段。将片段克隆于表达乙肝核心抗原的重组质粒p Thio His AHBc Ag,使抗原肽得以呈现于病毒样颗粒。重组菌经IPTG诱导后经SDS-PAGE鉴定目的蛋白表达。菌体破碎后经硫酸铵盐析法和蔗糖密度梯度离心进行纯化,并经高效液相凝胶过滤色谱和电镜鉴定病毒样颗粒的存在。制备的病毒样颗粒免疫接种了TC-1细胞的肿瘤模型小鼠,检测小鼠肿瘤大小。此外,在体外以抗原肽刺激脾细胞,以ELISA检测IFN-γ表达水平。结果:构建的三个重组表达质粒经酶切鉴定及测序分析证实构建正确。表达的重组蛋白大小与预期相符,并形成了病毒样颗粒。免疫小鼠后显示了抑制肿瘤增长的一定作用趋势。此外,抗原肽体外刺激促进了疫苗免疫小鼠脾细胞IFN-γ的表达,显示疫苗引起机体产生E7特异性的细胞免疫应答。结论:HBc Ag VLPs是有潜能的治疗性疫苗载体。 相似文献
20.
Shaochun Chen Chaowei Liao Yiukay Lai Yan Fan Gang Lu Hua Wang Xiaoai Zhang Marie C.M. Lin Shuilong Leng Hsiang-Fu Kung 《Acta biochimica et biophysica Sinica》2014,(1):6-14
In order to develop more effective therapeutic vaccines against cancers with high-risk human papillomavirus (HPV) infection, it is crucial to enhance the immunogenicity, eliminate the oncogenicity of oncoproteins, and take a combination of ET- and E6-containing vaccines. It has been shown recently that PE(AIII)-E7-KDEL3 (E7), a fusion protein containing the HPVI6 oncoprotein E7 and the trans- location domain of Pseudomonas aeruginosa exotoxin A, is effective against TC-1 tumor cells inoculated in mice, there- fore, we engineered PE(AIII)-E6-CRL-KDEL3 (E6), the deoncogenic versions of the E7 and E6 fusion proteins [i.e. PE(AIII)-E7(d)-KDEL3, E7(d), and PE(AIII)-E6(d)-CRL- KDEL3, E6(d)] and tested the immunoefficacies of these fusion proteins as mono- and bivalent vaccines. Results indicated that the E7(d) get higher immunogenicity than its wild type and the E6 fusion proteins augmented the im- munogenicity and antitumor effects of their E7 counterparts. Furthermore, the bivalent vaccine system E7(d) plus E6(d), in the presence of cisplatin, showed the best tumori- static and tumoricidal effects against established tumors in vivo. Therefore, it can be concluded that this novel therapeutic vaccine system, upon further optimization, may shed new light on clinical management of HPV-related carcinomas. 相似文献