兰州地区人乳头瘤病毒16型E6、E7基因的克隆及表达
|
摘要
背景与目的：
人乳头瘤病毒(human papillomavirus, HPV)是一种为无包膜的小型双链环状DNA病毒,目前已鉴定有118型,而其中有约40型可感染生殖道粘膜。根据HPV感染后诱发病变的良恶性不同进一步分为高危型和低危型两大类,90%以上的宫颈癌组织中可检测到高危型HPV16,18,31等型的DNA,以HPV16型为主。HPV16型E6、E7基因在HPV16感染相关的宫颈癌及54%以上中重度宫颈上皮内瘤变(cervical intraepithelial neoplasia, CIN)中持续性表达,因此HPV16型E6、E7蛋白与宫颈癌的发生发展密切相关,而E6、E7蛋白被认为是用于免疫治疗的首选蛋白。本研究主要进行HPV16型E6、E7基因的克隆,并在体外诱导表达E6、E7蛋白,为今后的蛋白疫苗的动物实验、进一步探索HPV16型E6、E7蛋白致宫颈癌分子机制及宫颈癌及癌前病变的免疫治疗奠定基础。
方法：
1.用PCR法从宫颈癌组织中扩增HPV16型E6、E7基因；
2.将HPV16型E6、E7基因片段与克隆载体pMD 19-T Simple vector连接,构建HPV16型E6、E7的克隆质粒pMD 19-T-E6,pMD 19-T-E7；
3.用限制性内切酶EcoRⅠ、HindⅢ将HPV16型E6、E7基因从克隆质粒pMD 19-T-E6, pMD 19-T-E7切下,用T4连接酶与表达载体pET28a(+)连接,构建HPV16型E6、E7的表达质粒pET28a(+)-E6、pET28a(+)-E7;
4.将表达质粒pET28a(+)-E6、pET28a(+)-E7转化至大肠杆菌BL21中,诱导HPV16型E6、E7蛋白表达,运用SDS-PAGE蛋白电泳检测HPV16 E6、E7蛋白的表达。
结果：
1.以宫颈癌组织标本中提取的DNA作为模板,PCR扩增反应之后,得到了大小分别约为490bp、310bp
的基因片段；
2.将重组的克隆质粒pMD 19-T-E6, pMD 19-T-E7和表达质粒pET28a(+)-E6、pET28a(+)-E7测序,测序结果均显示E6基因的253位碱基C253AT突变为T253AT,编码氨基酸由组氨酸突变成酪氨酸,E7基因测序结果与HPV16型E7序列完全一致；
3.构建的表达质粒pET28a(+)-E6、pET28a(+)-E7可在大肠杆菌BL21中稳定表达HPV16型E6、E7蛋白。
结论：
1.成功克隆了HPV16型E6、E7基因；
2.成功构建了HPV16型E6、E7蛋白原核表达系统；
3.成功诱导HPV16型E6、E7蛋白的原核表达。
Background and objective:
HPVs are small, non-enveloped viruses that contain circular double-stranded DNA.118 different genotypes of HPVs have been identified, of which about 40 infect the genital mucosa. These viruses can be classified into high-risk or low-risk groups according to the propensity for malignant progression of the associated lesions which they cause. High-risk HPVs, such as HPV16,18 and 31, are associated with more than 90% of cervical cancers, and of which HPV16 is the most. Two early oncogenes E6 and E7 are invariably expressed in cervical cancer and more than 54% of cervical intraepithelial neoplasia (CIN)Ⅱ-Ⅲ, so E6 and E7 proteins are closely related to the occurrence and development of cervical cancer. This study investigates on the cloning of HPV type 16 E6 and E7 gene, and expression of E6 and E7 proteins in E.coli. This is a basic study for the protein vaccine on animal, the molecular mechanism of cervical cancer induced by E6 and E7 oncogenes and immunologic therapy of cervical cancers and precancerous lesions in future.
Methods:
1. PCR technique was used to amplify the HPV16 E6 and E7 genes fragment from the DNA of cervical cancer tissue;
2. HPV 16 E6 and E7 gene fragments were cloned into pMD 19-T Simple vector to construct cloning plasmids pMD 19-T-E6, pMD 19-T-E7;
3. The cloning plasmids pMD 19-T-E6, pMD 19-T-E7 were digested with EcoR I and HindⅢ, then the HPV16 E6 and E7 genes were cloned into the expression vector pET28a(+) to construct expressing plasmids pET28a(+)-E6 and pET28a(+)-E7, using the T4 DNA ligase;
4. The recombinant expressing plasmids pET28a(+)-E6 and pET28a(+)-E7 transformed into E. coli BL21were induced by IPTG to express the E6 and E7 proteins. We used the SDS-PAGE to test the E6 and E7 proteins.
Results:
1. Using PCR technique to amplify extracted DNA of cervical cancer tissue sample as template, we obtained approximately 490bp and 310bp gene fragments;
2. DNA sequencing results of the recombinant cloning plasmids pMD 19-T-E6 and pMD 19-T-E7 and expressing plasmids pET28a(+)-E6 and pET28a(+)-E7 showed that E6 gene mutated at the 253 base C253AT to T253AT, amino acid histidine changed into tyrosine, and E7 gene sequencing results showed the right of HPV16 E7 sequence;
3. HPV16 E6, E7 protein were expressed by the plasmid pET28a (+)-E6, pET28a (+)-E7 in E.coli BL21.
Conclusions:
1. Successfully amplified the HPV16 E6 and E7 genes;
2. Successfully constructed the prokaryotic expression plasmid of HPV16 E6 and E7 genes;
3. Successfully induced the prokaryotic expression of HPV16 E6 and E7 proteins.
人乳头瘤病毒(human papillomavirus, HPV)是一种为无包膜的小型双链环状DNA病毒,目前已鉴定有118型,而其中有约40型可感染生殖道粘膜。根据HPV感染后诱发病变的良恶性不同进一步分为高危型和低危型两大类,90%以上的宫颈癌组织中可检测到高危型HPV16,18,31等型的DNA,以HPV16型为主。HPV16型E6、E7基因在HPV16感染相关的宫颈癌及54%以上中重度宫颈上皮内瘤变(cervical intraepithelial neoplasia, CIN)中持续性表达,因此HPV16型E6、E7蛋白与宫颈癌的发生发展密切相关,而E6、E7蛋白被认为是用于免疫治疗的首选蛋白。本研究主要进行HPV16型E6、E7基因的克隆,并在体外诱导表达E6、E7蛋白,为今后的蛋白疫苗的动物实验、进一步探索HPV16型E6、E7蛋白致宫颈癌分子机制及宫颈癌及癌前病变的免疫治疗奠定基础。
方法：
1.用PCR法从宫颈癌组织中扩增HPV16型E6、E7基因；
2.将HPV16型E6、E7基因片段与克隆载体pMD 19-T Simple vector连接,构建HPV16型E6、E7的克隆质粒pMD 19-T-E6,pMD 19-T-E7；
3.用限制性内切酶EcoRⅠ、HindⅢ将HPV16型E6、E7基因从克隆质粒pMD 19-T-E6, pMD 19-T-E7切下,用T4连接酶与表达载体pET28a(+)连接,构建HPV16型E6、E7的表达质粒pET28a(+)-E6、pET28a(+)-E7;
4.将表达质粒pET28a(+)-E6、pET28a(+)-E7转化至大肠杆菌BL21中,诱导HPV16型E6、E7蛋白表达,运用SDS-PAGE蛋白电泳检测HPV16 E6、E7蛋白的表达。
结果：
1.以宫颈癌组织标本中提取的DNA作为模板,PCR扩增反应之后,得到了大小分别约为490bp、310bp
的基因片段；
2.将重组的克隆质粒pMD 19-T-E6, pMD 19-T-E7和表达质粒pET28a(+)-E6、pET28a(+)-E7测序,测序结果均显示E6基因的253位碱基C253AT突变为T253AT,编码氨基酸由组氨酸突变成酪氨酸,E7基因测序结果与HPV16型E7序列完全一致；
3.构建的表达质粒pET28a(+)-E6、pET28a(+)-E7可在大肠杆菌BL21中稳定表达HPV16型E6、E7蛋白。
结论：
1.成功克隆了HPV16型E6、E7基因；
2.成功构建了HPV16型E6、E7蛋白原核表达系统；
3.成功诱导HPV16型E6、E7蛋白的原核表达。
Background and objective:
HPVs are small, non-enveloped viruses that contain circular double-stranded DNA.118 different genotypes of HPVs have been identified, of which about 40 infect the genital mucosa. These viruses can be classified into high-risk or low-risk groups according to the propensity for malignant progression of the associated lesions which they cause. High-risk HPVs, such as HPV16,18 and 31, are associated with more than 90% of cervical cancers, and of which HPV16 is the most. Two early oncogenes E6 and E7 are invariably expressed in cervical cancer and more than 54% of cervical intraepithelial neoplasia (CIN)Ⅱ-Ⅲ, so E6 and E7 proteins are closely related to the occurrence and development of cervical cancer. This study investigates on the cloning of HPV type 16 E6 and E7 gene, and expression of E6 and E7 proteins in E.coli. This is a basic study for the protein vaccine on animal, the molecular mechanism of cervical cancer induced by E6 and E7 oncogenes and immunologic therapy of cervical cancers and precancerous lesions in future.
Methods:
1. PCR technique was used to amplify the HPV16 E6 and E7 genes fragment from the DNA of cervical cancer tissue;
2. HPV 16 E6 and E7 gene fragments were cloned into pMD 19-T Simple vector to construct cloning plasmids pMD 19-T-E6, pMD 19-T-E7;
3. The cloning plasmids pMD 19-T-E6, pMD 19-T-E7 were digested with EcoR I and HindⅢ, then the HPV16 E6 and E7 genes were cloned into the expression vector pET28a(+) to construct expressing plasmids pET28a(+)-E6 and pET28a(+)-E7, using the T4 DNA ligase;
4. The recombinant expressing plasmids pET28a(+)-E6 and pET28a(+)-E7 transformed into E. coli BL21were induced by IPTG to express the E6 and E7 proteins. We used the SDS-PAGE to test the E6 and E7 proteins.
Results:
1. Using PCR technique to amplify extracted DNA of cervical cancer tissue sample as template, we obtained approximately 490bp and 310bp gene fragments;
2. DNA sequencing results of the recombinant cloning plasmids pMD 19-T-E6 and pMD 19-T-E7 and expressing plasmids pET28a(+)-E6 and pET28a(+)-E7 showed that E6 gene mutated at the 253 base C253AT to T253AT, amino acid histidine changed into tyrosine, and E7 gene sequencing results showed the right of HPV16 E7 sequence;
3. HPV16 E6, E7 protein were expressed by the plasmid pET28a (+)-E6, pET28a (+)-E7 in E.coli BL21.
Conclusions:
1. Successfully amplified the HPV16 E6 and E7 genes;
2. Successfully constructed the prokaryotic expression plasmid of HPV16 E6 and E7 genes;
3. Successfully induced the prokaryotic expression of HPV16 E6 and E7 proteins.
引文
[1]Takashi Yugawa, Tohru Kiyono. Molecular mechanisms of cervical carinogenesis by high-risk human papillamaviruses:novel functions of E6 and E7 oncoproteins [J]. Rev Med Virol,2009,19:97-113.
[2]曹泽毅.中华妇产科学(第六版)[M].人民卫生出版社,2005：2010-2012.
[3]许雪梅.人乳头瘤病毒及宫颈癌疫苗的研究-解读2008年诺贝尔生理学或医学奖[J].生物化学与生物物理进展,2008,35(10)：1095-1103.
[4]Crum CP, Mclachin CM, Tate JE, et al. Pathobiology of vulvar squamous neoplasia [J]. Curr Opin Obstet Gynecol,1997,9:63-69.
[5]Steenbergen RD, de Wilde J, Wilting SM, et al. HPV-mediated transformation of the anogenital tract [J]. J Clin Virol 2005,32(Suppl 1):S25-S33.
[6]Kayes O, Ahmed HU, Arya M, et al. Molecular and genetic pathways in penile cancer [J]. Lancet Oncol, 2007,8:420-429.
[7]Christy M. Hebner, Laimonis A. Laimins. Human papillomaviruses:basic mechanisms of pathogenesis and oncogenicity [J]. Rev Med Virol,2006,16:83-97.
[8]Khan MJ, Castle PE, Lorincz AT, et al. The elevated 10-year risk of cervical precancer and cancer in women with human Papillomavirus (HPV) type 16 or 18 and the possible utility of type-specific HPV testing in clinical practice [J]. J Natl Cancer Inst,2005,97 (14):1072-1079.
[9]Gaelle Boulet, Caroline Horvath, Davy Vanden Broeck et al. Human Paillomarivus E6 and E7 Oncogenes [J]. Int J Biochem Cell Biol,2007:2006-2011.
[10]戴悦,柯杨.HPVE6蛋白的相互作用蛋白研究进展[J].国外医学：肿瘤学分册,2004,31(5)：349-352.
[11]Massimo Tommasino, Rosita Accardi, Sandra Caldeira, et al. The role of TP53 in cervical carcinogenesis [J]. Hum Mutat,2003,21:307-312.
[12]药立波.医学分子生物学(第二版)[M].人民卫生出版社,2005：160-163.
[13]Shai A, Brake T, Somoza C, et al. The human Papillomavirus E6 oncogene dysregulates the cell cycle and contributes to cervical carcinogenesis through two independent activities [J].Cancer Res,2007,67 (4):1626-1635.
[14]Brooks L A, Sullivan A, O'Nions J, et al. E7 proteins from oncogenic human papillomavirus types transactivate p73:role in cervical intraepithelial neoplasia[J]. Br J Cancer,2002,86(2):263-268.
[15]修晓新,张淑兰.HPVE6、E7与端粒酶激活研究进展[J].国外医学：计划生育.生殖健康分册,2006,25(3)：164-167.
[16]S. Renaud, D. Loukinov, Z. Abdullaev, e t al. Dual role of DNA methylation inside and outside of CTCF-binding regions in the transcriptional regulation of the telomerase hTERT gene [J]. Nucleic Acids Res, 2007,35(4):1245-1256.
[17]Veldman T, Horikawa I, Barrett JC, et al. Transcriptional activation of the telomerase hTERT gene by human papillomarirus type 16 E6 oncoprotein [J]. J Virol 2001,75(9):4467-4472.
[18]Baege AC, Berger A, Schlegel R, et al. Cervical epithelial cells transduced with the papillomavirus E6/E7 oncogenes maintain stable levesls of oncoprotein expression but exhibit progressive, major increases in hTERT gene expression and telomerase activity [J]. Am J Pathol,2002,160(4):1251-1257.
[19]赵思成,骆丹.HPV E6蛋白及其在致癌过程中作用的靶蛋白[J].中国生物化学与分子生物学报.2009,25(3)：225-228.
[20]Veldman T, Liu X. Yuan H, et al. Human papillomavirus E6 and Myc proteins associate in vivo and bind to and cooperatively activate the telomerase reverse transcriptase promoter [J]. Proc Natl Acad Sci USA.2003, 14:8211-8216.
[21]Xu M, Luo W, Elzi DJ, et al. NFX1 interacts with mSin3A/histone deacetylase to repress hTERT transcription in keratinocytes [J]. Mol Cell Bio.2008,28:4819-4828.
[22]Gewin L, Myers H, Kiyono T, et al. Identification of a novel telomerase repressor that interacts with the human papillomarirus type-16 E6/E6-AP complex [J]. Genes Dev,2004,18(18):2269-2282.
[23]James M A, Lee J H, Klingelhuts A J. Human papillomavirus type 16 E6 activates NF-kappaB, induces cIAP-2 expression, and protects against apoptosis in a PDZ bingding motif-dependent manner [J]. Virol, 2006,80(11):5301-5307.
[24], Sanclemente G, Gill DK. Human papillomavirus molecular biology and pathogenesis [J]. Eue Acad Dermatol Venereol.2002,16(3):231-240.
[25]Munger K, Baldwin A, Edwards, KM, et al. Mechanisms of human papillomavirus-induced oncogenesis[J]. J Virol,2004,78:11451-11460.
[26]Wang J, Sampath A, Raychaudhuri P, et al. Both Rb and E7 are regulated by the ubiquitin proteasome pathway in HPV-containing cervical tumor cells [J]. Oncogene,2001,20(34):4940-4749.
[27]Sun Gwan Hwang, Daeyoup Lee, Jiyun Kim, et.al. Human papillomavirus type 16 E7 binds to E2F1 and activates E2F1-driven transcription in a retinoblastoma protein-independent manner [J]. J Biol Chem,2002, 277(4):2923-2930.
[28]Heck DV, Yee CL, Howley PM, et al. Efficiency of binding the retinoblastoma protein correlates with the transforming capacity of the E7 oncoproteins of the humam papillomaviruses [J]. Proc Natl Acad Sci USA, 1992,89(10):4442-4446.
[29]张洪文,宋丽君.HPV对细胞周期调控影响的研究进展[J].国外医学：生理病理科学与临床分册.2004,24(4)：325-327.
[30]葛静,鲁永鲜.HPV与宫颈癌关系及疫苗研究进展[J].国外医学：病毒学分册,2005,12(5)：129-132.
[31]Luism AS, Sharonp W, Robera B, et al. Polymorphism of the HPV16 E6 gene of cervical carcinomas [J]. Int J oncol,1995,7:261-266.
[32]Gostout BS, Zanetta GM, Maleemonkol S, et al. Differential distribution of sequence variatins in HPV-16 E6 [J]. Gynecol Oncol,2000,79:11-17.
[33]Zenbe I, Wilander E, Delius H, et al. Human papillomavirus 16 E6 variants are more prevalent in invasive cervical carcinoma than the prototype [J]. Cancer Res,1998,58:829-833
[2]曹泽毅.中华妇产科学(第六版)[M].人民卫生出版社,2005：2010-2012.
[3]许雪梅.人乳头瘤病毒及宫颈癌疫苗的研究-解读2008年诺贝尔生理学或医学奖[J].生物化学与生物物理进展,2008,35(10)：1095-1103.
[4]Crum CP, Mclachin CM, Tate JE, et al. Pathobiology of vulvar squamous neoplasia [J]. Curr Opin Obstet Gynecol,1997,9:63-69.
[5]Steenbergen RD, de Wilde J, Wilting SM, et al. HPV-mediated transformation of the anogenital tract [J]. J Clin Virol 2005,32(Suppl 1):S25-S33.
[6]Kayes O, Ahmed HU, Arya M, et al. Molecular and genetic pathways in penile cancer [J]. Lancet Oncol, 2007,8:420-429.
[7]Christy M. Hebner, Laimonis A. Laimins. Human papillomaviruses:basic mechanisms of pathogenesis and oncogenicity [J]. Rev Med Virol,2006,16:83-97.
[8]Khan MJ, Castle PE, Lorincz AT, et al. The elevated 10-year risk of cervical precancer and cancer in women with human Papillomavirus (HPV) type 16 or 18 and the possible utility of type-specific HPV testing in clinical practice [J]. J Natl Cancer Inst,2005,97 (14):1072-1079.
[9]Gaelle Boulet, Caroline Horvath, Davy Vanden Broeck et al. Human Paillomarivus E6 and E7 Oncogenes [J]. Int J Biochem Cell Biol,2007:2006-2011.
[10]戴悦,柯杨.HPVE6蛋白的相互作用蛋白研究进展[J].国外医学：肿瘤学分册,2004,31(5)：349-352.
[11]Massimo Tommasino, Rosita Accardi, Sandra Caldeira, et al. The role of TP53 in cervical carcinogenesis [J]. Hum Mutat,2003,21:307-312.
[12]药立波.医学分子生物学(第二版)[M].人民卫生出版社,2005：160-163.
[13]Shai A, Brake T, Somoza C, et al. The human Papillomavirus E6 oncogene dysregulates the cell cycle and contributes to cervical carcinogenesis through two independent activities [J].Cancer Res,2007,67 (4):1626-1635.
[14]Brooks L A, Sullivan A, O'Nions J, et al. E7 proteins from oncogenic human papillomavirus types transactivate p73:role in cervical intraepithelial neoplasia[J]. Br J Cancer,2002,86(2):263-268.
[15]修晓新,张淑兰.HPVE6、E7与端粒酶激活研究进展[J].国外医学：计划生育.生殖健康分册,2006,25(3)：164-167.
[16]S. Renaud, D. Loukinov, Z. Abdullaev, e t al. Dual role of DNA methylation inside and outside of CTCF-binding regions in the transcriptional regulation of the telomerase hTERT gene [J]. Nucleic Acids Res, 2007,35(4):1245-1256.
[17]Veldman T, Horikawa I, Barrett JC, et al. Transcriptional activation of the telomerase hTERT gene by human papillomarirus type 16 E6 oncoprotein [J]. J Virol 2001,75(9):4467-4472.
[18]Baege AC, Berger A, Schlegel R, et al. Cervical epithelial cells transduced with the papillomavirus E6/E7 oncogenes maintain stable levesls of oncoprotein expression but exhibit progressive, major increases in hTERT gene expression and telomerase activity [J]. Am J Pathol,2002,160(4):1251-1257.
[19]赵思成,骆丹.HPV E6蛋白及其在致癌过程中作用的靶蛋白[J].中国生物化学与分子生物学报.2009,25(3)：225-228.
[20]Veldman T, Liu X. Yuan H, et al. Human papillomavirus E6 and Myc proteins associate in vivo and bind to and cooperatively activate the telomerase reverse transcriptase promoter [J]. Proc Natl Acad Sci USA.2003, 14:8211-8216.
[21]Xu M, Luo W, Elzi DJ, et al. NFX1 interacts with mSin3A/histone deacetylase to repress hTERT transcription in keratinocytes [J]. Mol Cell Bio.2008,28:4819-4828.
[22]Gewin L, Myers H, Kiyono T, et al. Identification of a novel telomerase repressor that interacts with the human papillomarirus type-16 E6/E6-AP complex [J]. Genes Dev,2004,18(18):2269-2282.
[23]James M A, Lee J H, Klingelhuts A J. Human papillomavirus type 16 E6 activates NF-kappaB, induces cIAP-2 expression, and protects against apoptosis in a PDZ bingding motif-dependent manner [J]. Virol, 2006,80(11):5301-5307.
[24], Sanclemente G, Gill DK. Human papillomavirus molecular biology and pathogenesis [J]. Eue Acad Dermatol Venereol.2002,16(3):231-240.
[25]Munger K, Baldwin A, Edwards, KM, et al. Mechanisms of human papillomavirus-induced oncogenesis[J]. J Virol,2004,78:11451-11460.
[26]Wang J, Sampath A, Raychaudhuri P, et al. Both Rb and E7 are regulated by the ubiquitin proteasome pathway in HPV-containing cervical tumor cells [J]. Oncogene,2001,20(34):4940-4749.
[27]Sun Gwan Hwang, Daeyoup Lee, Jiyun Kim, et.al. Human papillomavirus type 16 E7 binds to E2F1 and activates E2F1-driven transcription in a retinoblastoma protein-independent manner [J]. J Biol Chem,2002, 277(4):2923-2930.
[28]Heck DV, Yee CL, Howley PM, et al. Efficiency of binding the retinoblastoma protein correlates with the transforming capacity of the E7 oncoproteins of the humam papillomaviruses [J]. Proc Natl Acad Sci USA, 1992,89(10):4442-4446.
[29]张洪文,宋丽君.HPV对细胞周期调控影响的研究进展[J].国外医学：生理病理科学与临床分册.2004,24(4)：325-327.
[30]葛静,鲁永鲜.HPV与宫颈癌关系及疫苗研究进展[J].国外医学：病毒学分册,2005,12(5)：129-132.
[31]Luism AS, Sharonp W, Robera B, et al. Polymorphism of the HPV16 E6 gene of cervical carcinomas [J]. Int J oncol,1995,7:261-266.
[32]Gostout BS, Zanetta GM, Maleemonkol S, et al. Differential distribution of sequence variatins in HPV-16 E6 [J]. Gynecol Oncol,2000,79:11-17.
[33]Zenbe I, Wilander E, Delius H, et al. Human papillomavirus 16 E6 variants are more prevalent in invasive cervical carcinoma than the prototype [J]. Cancer Res,1998,58:829-833