共查询到19条相似文献,搜索用时 109 毫秒
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目的 表达人乳头瘤病毒16型(HPV16) E2蛋白,并制备小鼠抗HPV16 E2血清.方法 采用PCR技术扩增HPV16E2基因,构建入pET21b载体,重组表达载体pET21 b-HPV16E2经鉴定后转化大肠埃希菌BL21(DE3),诱导表达并鉴定表达产物.经纯化、变性和复性方法,制备可溶性HPV16 E2蛋白.免疫BALB/c小鼠制备抗血清,检测小鼠IFN-γ、CD4+T细胞、CD8+T细胞、CD4/CD8比值和抗血清滴度变化.结果 酶切和测序结果表明pET21b-HPV16 E2构建成功.表达蛋白相对分子质量(Mr)为42 000,Western blot法证明具有较高特异性.小鼠抗血清效价升高,CD4+T细胞数量和CD4/CD8比值升高,小鼠IFN-γ无升高.结论 成功制备可溶性HPV16 E2蛋白和小鼠抗HPV16 E2高效价的抗血清. 相似文献
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目的 表达人乳头瘤病毒16型(HPV16) E4蛋白,并制备小鼠抗HPV16 E4血清.方法 将HPV16 E4基因克隆入pQE30,重组质粒pQE30-HPV 16E4经鉴定后转化大肠埃希菌M15(PREP4),诱导表达并鉴定表达产物.因纯化、变性和复性方法,制备可溶性HPV16 E4蛋白.免疫Bal B/C小鼠制备抗血清,检测小鼠IFN-γ水平、CD4/CD8比值和抗血清滴度变化.结果 酶切和测序结果表明pQE30-HPV16F4构建成功.表达分子相对分子量(Mr)为10 000,Western blot法证明具有较高特异性.小鼠抗血清效价升高,CD4/CD8无升高,小鼠γ干扰素(IFN-γ)无升高.结论 成功制备可溶性HPV16 E4蛋白和小鼠抗HPV16 E4高效价的抗血清. 相似文献
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目的 克隆HPVl6 E5基因,构建原核重组工程菌并诱导表达,对表达产物HPVl6E5蛋白进行鉴定.方法 提取宫颈癌组织DNA作为模板,用PCR方法扩增HPVl6 E5基因,经BamH I和HindⅢ双酶切后,插入相同酶切的pET21b载体质粒,转化DH5α,筛选阳性克隆.经酶切和测序鉴定后转化大肠埃希菌BL21(DE3),建屯重组工程菌株pET21b-HPVl6E5/BL21(DE3).经IPTG诱导表达,SDS-PAGE和Western印迹检测表达产物.结果 HPVl6 E5基因扩增片段0.27kb.测定序列与HPVl6原型株E5基因比较,出现4处核苷酸变异,分别为3979、4042、4077和4089位,引起144L和165V氨基酸改变.重组质粒经酶切和序列测定证实构建正确.SDS-PAGE分析重组菌在16 kDa处出现蛋白条带.该蛋白条带可被组氨酸标签单克隆抗体特异性识别.结论 成功克隆HPVl6E5基因,并构建原核表达质粒,E5 蛋白在BL21(DE3)中表达.本实验为进一步研究E5生物学活性、转化活性和肿瘤杀伤免疫作用奠定了实验基础. 相似文献
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利用突变修饰后消除转化活性并保留抗原性的中国山东地方株人乳头瘤病毒16型(human papillomavirus type 16,HPVl6)E6E7融合基因(fmE6E7),研制治疗HPVl6相关疾病的DNA疫苗。用PCR扩增fmE6E7基因后,插人真核表达质粒获得pVRl012-fmE6E7,瞬时转染Cos-7细胞,免疫荧光法检测证实其表达后,在C57BL/6小鼠后腿肌肉进行裸DNA免疫,5lCr释放法体外分析免疫鼠的细胞毒性T淋巴细胞活性Cytotoxic T lymphocyte,CTL),间接ELISA法检测免疫鼠血清中E7特异性抗体。研究表明修饰后的中国地方株E6E7融合基因可诱导机体产生特异的抗体反应和CTL反应,与单独野生型E7基因免疫相比,E6E7融和基因可更好的活化CTT反应。表明修饰后消除转化活性的中国地方株E6E7融合基因可作为HPVl6治疗性DNA疫苗的靶基因。 相似文献
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运用杂交瘤技术,我们成功地建立了两株能稳定分泌小鼠抗人乳头瘤病毒16E6蛋白单克隆抗体的杂交瘤细胞,经鉴定单克隆抗体属IgG1k。试验结果表明,所得单克隆抗体仅1与PV16E6融合蛋白反应。不与HPV16E7、L1、L2融合蛋白以及L1-L2真核表达蛋白反尖,也只与Caski细胞反应,不与Hela细胞反应,初步结果说明,该抗体是HPV16E16蛋白特异性的McAb。 相似文献
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构建了含人乳头瘤病毒16型(HPV16)-E6E7ORFs(nt83-855)片段和HPV16长控制区(LCR)加E6E7ORFs片段(nt7007-7904/0-879)的逆转录病毒载体pH21和pH18质粒,利用Lipofectin分别将它们导入病毒包装细胞pA317中,经过筛选获得G418抗性的病毒包装细胞,产生的重组病毒H21和H18感染的NIH3T3细胞都具有恶性细胞的形态学特征,并能在裸鼠体内形成肿瘤。Southern杂交结果证明,上述两基因片段都整合到细胞基因组中。本实验结果说明HPV16-E6E7基因片段是HPV16转化NIH3T3细胞的关键早期区,其自身LCR区在该转化过程中没有显示出重要作用。 相似文献
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运用杂交瘤技术,我们成功地建立了两株能稳定分泌小鼠抗人乳头瘤病毒16E6蛋白单克隆抗体的杂交瘤细胞。经鉴定单克隆抗体均属IgG1k。试验结果表明,所得单克隆抗体仅与HPV16E6融合蛋白反应,不与HPV16E7、L1、L2融合蛋白以及L1-L2真核表达蛋白反应,也只与Caski细胞反应,不与Hela细胞反应。初步结果说明,该抗体是HPV16E16蛋白特异性的McAb。 相似文献
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高危型乳头瘤病毒E6蛋白能结合并降解p5 3蛋白 ,导致细胞发生转化 ,是其致癌的关键环节。但最近大量研究表明p5 3并非E6致癌的唯一靶位 ,E6蛋白的致癌机制还远没有阐明 ,探索E6蛋白与其新的靶分子的相互作用对肿瘤的基因治疗和全面揭示其致癌机制具有重要意义。 1997年 ,Kaghad等发现了一个在结构和功能上都和p5 3极为相似的新基因 ,命名为p73。p73基因虽然发现时间不长 ,却已成为研究的热点。p73无论在结构上还是在功能上均与p5 3极为相似。p73蛋白具有与p5 3同样的四个主要功能区 ,其中DNA结合结构域两者的相同序列… 相似文献
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人乳头瘤病毒58型E6蛋白对p53的作用研究 总被引:3,自引:0,他引:3
目的 研究HPV58 E6蛋白与p53蛋白的作用关系,以初步探讨其致癌机理。方法 先通过GST沉降试验和体外降解试验,体外研究HPV58 E6结合降解p53蛋白的作用,进而将表达质粒导人SAOS-2细胞,在细胞内研究HPV58 E6对p53诱导细胞凋亡活性的影响。结果 HPV58 E6能够有效结合p53蛋白并进一步诱导其降解,而且在细胞内,HPV58 E6具有抑制p53蛋白的诱导凋亡的功能。结论 p53蛋白的降解影响其细胞周期调控和诱导凋亡的功能,导致细胞发生恶性转化引发子宫颈癌等肿瘤。 相似文献
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Asadurian Y Kurilin H Lichtig H Jackman A Gonen P Tommasino M Zehbe I Sherman L 《Journal of medical virology》2007,79(11):1751-1760
Genetic variations in the E6 oncogene have been associated with different risk for cancer progression. In the present study, the functional significance of human papillomavirus (HPV) polymorphism in the E6 oncogene was investigated. Ten HPV16 E6 variants containing amino acid substitutions in the N-terminal region of E6 were evaluated for different biological and biochemical activities in human keratinocytes, the target cells for HPV infection. Western blot analyses of primary foreskin human keratinocytes or immortalized human keratinocytes, stably transduced with the E6 variants, revealed reduced p53 and Bax levels in all E6 expressing cultures. The reduction induced by most E6 proteins was at similar levels and comparable to the reduction induced by the E6 prototype. The ability of the proteins to induce serum/calcium-differentiation resistant colonies in primary keratinocytes was more variable. Overall activities of the variants ranged between 0.24- and 2.18-fold of the E6 prototype activity. The I27R/L83V variant showed the lowest activity whereas the R8Q variant showed the highest activity. The L83V polymorphism previously associated with risk for cancer progression in some populations, showed significant activity, comparable to that of the E6 prototype, in reducing p53 and Bax levels. Furthermore, this variant showed enhancement in the ability to induce colonies resistant to serum/calcium-triggered differentiation, however, the difference from the prototype was not statistically significant. This, and augmentation of other described functions might result in differences in L83V pathogenicity. 相似文献
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Valeria Cento Massimo Ciccozzi Luigi Ronga Carlo Federico Perno Marco Ciotti 《Journal of medical virology》2009,81(9):1627-1634
High‐risk human papillomaviruses (HPVs) are risk factors for the development of cervical cancer. HPV 16 is the most common type, being present in about 60% of cervical cancers worldwide. Previous studies have reported upon the association between HPV 16 E6 variants and increased risk of cervical intraepithelial neoplasia and invasive cervical cancer. In this study, the presence of HPV 16 polymorphisms in the E6, E7, and L1 genes was investigated in relation to the presence of high‐grade lesions. Sequencing of the E6 gene revealed the presence of nucleotide mutations resulting in 15 amino acid changes. Of these, the G134D and C136R fall within the CXXC zinger finger domain important for p53 binding. In the E7 gene, four nucleotide variations were identified with two leading to the amino acid substitutions L15V and S31R. The L1 gene showed 13 nucleotide changes leading to 11 amino acid substitutions. Among these, the R364C and N367D are located at the base of the HI‐loop of the L1 protein, considered to be the immunodominant epitope of HPV 16. No significant relationship between HPV 16 variants and high‐grade lesions was found. Phylogenetic analysis showed that all the HPV 16 variants identified belonged to the European lineage, except one which was of the Asian‐American lineage. The European‐350G variant was detected most frequently (22 of 34, 64.7%). The study provides some new data on the genetic diversity of HPV 16 which may help to understand the oncogenic potential of the virus and to improve management of patients. J. Med. Virol. 81:1627–1634, 2009. © 2009 Wiley‐Liss, Inc. 相似文献
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目的研究人乳头瘤病毒(HPV)相关肿瘤患者的HPV16E6抗体水平及其流行病学意义;探讨用杆状病毒-昆虫细胞表达载体系统表达的HPV16E6蛋白的抗原性。方法用PCR从HPV16基因组中扩增出HPV16E6完整基因,克隆至转移载体pVL1393中,重组质粒DNA与线性杆状病毒DNA共转染昆虫细胞Sf-9,经噬斑筛选获得带有编码E6基因的重组杆状病毒株,并在昆虫细胞中表达。结果Westernblot和高效液相色谱法检测HPV16E6表达蛋白,其分子量约为18000。免疫印迹检测显示其能与兔抗HPV16E6多抗特异性结合。酶联免疫吸附试验表明此重组蛋白能被人HPV16阳性血清所识别。结论昆虫细胞表达的HPV16E6蛋白,具有良好的抗原性,可用于检测HPV16E6特异性免疫球蛋白IgG和IgM抗体 相似文献
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目的检测HPV16/18和HPV16E6/E7 DNA在宫颈癌组织中的表达,探讨其在宫颈癌发病中的作用.方法应用PCR和琼脂糖凝胶电泳方法检测46例宫颈癌组织中HPV16/18和HPV16E6/E7DNA.结果 46例宫颈癌中56.5%(26/46)扩增HPV16/18 DNA,其中宫颈鳞癌25例,宫颈腺癌1例.正常对照组20例HPV16/18DNA均为阴性,与宫颈癌组相比差异有显著性(P<0.01).HPV16/18 DNA阳性拷贝对数值为4.32±2.45.HPV16E6,E7DNA分别有53.8%(14/26)、46.2%(12/26)扩增.结论 HPV16/18和HPV16E6/E7 DNA与宫颈癌的发生密切相关,是宫颈癌恶性转化的关键之一,预示着宫颈癌有较强的增殖能力和转移能力. 相似文献
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The high risk forms of human papillomavirus (HPV) (primarily types 16 and 18) are the leading cause of cervical cancer worldwide. Infection results in expression of three oncoproteins, E5, E6, and E7, the latter two being of predominant importance in maintaining a transformed state of the host epithelial cell. While little is known about the role(s) of the HPV E5, the bovine papillomavirus type 1 (BPV1) E5 protein has been well characterized. A study of HPV16 E5 was performed, focusing on the protein's ability to self-interact, its ability to bind to the 16-kDa subunit of the vacuolar H(+)-ATPase (16K), and its cellular localization. As has been previously shown for BPV1 E5, we found that HPV16 E5 is also capable of self-interaction and binding to 16K. Further, we examined which portions of the HPV16 E5 protein were involved in these interactions using progressive deletions of putative transmembrane helices of the protein. All of the E5 deletion mutants tested bound to full-length E5 as well as to 16K, suggesting that these protein-protein interactions are based on hydrophobic interactions. The majority of E5 expressed in HEK 293-T7 cells was perinuclear but did not appear to localize to the cis/medial-Golgi, in contrast to previous reports for both HPV16 E5 and BPV1 E5. 相似文献
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目的 用基因重组技术构建pcDNA-E6E7真核表达载体.方法 经限制性内切酶和序列分析,用脂质体转染技术将其转入B16细胞,G418稳定筛选后IFA法检测其表达,RT-PCK法检测HPV16E6E7mRNA的生成,并将转染细胞接种小鼠皮下,观察成瘤情况.结果 酶切鉴定证实重组质粒中插入的目的基因片段及载体大小、方向和插入住点均正确,在转染的B16细胞中可见绿色荧光并检测到HPV16E6E7mRNA的生成,接种的转染细胞在小鼠皮下100%成瘤.结论 提示B16细胞转染E6E7后其致瘤性与转染空载体组和野生型B16细胞组无明显差异. 相似文献