Please wait a minute...

中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2010, Vol. 30 Issue (03): 1-8    
研究报告     
用无选择标记的转基因烟草表达胸腺素α1（Tα1）
芦丽亚,宋维,游晓慧,江婷,赵凌侠**
上海交通大学农业与生物学院 植物生物技术研究中心 上海 200240
Expression of Bioactive Thymosin α1 Using Marker Free Transgenic Tobacco
Plant Biotechnology Research Center, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China
 全文: PDF(1101 KB)   HTML
摘要：

胸腺素α1（thymosin α1，Tα1）作为免疫调节剂在T细胞成熟、分化和功能发挥方面扮演着重要角色，临床主要被用于免疫缺陷、病毒感染和自身免疫性疾病（HBV、HCV、HIV和癌症等）的治疗。由于组织提取Tα1原料限制、化学合成价格昂贵和传统表达系统（原核或转基因动物）存在安全隐患，使Tα1临床应用受限。用烟草表达Tα1，首先按植物偏爱密码子设计合成tα1基因（124bp）并重组串联成4×tα1，构建植物双元表达载体p35s∷4×tα1（含twin T-DNAs），用农杆菌（Agrobacterium tumefaciens）介导法转化烟草。PCR和Southern blot结果证实获得了14转基因烟草，并发现靶基因4×tα1与筛选基因npt II在转基因烟草T0代基因组中发生了分别整合。对177株T1代转基因烟草检测，获得了2株仅整合4×tα1而无筛选标记npt II的植株。ELISA结果显示，4×Tα1在转基因烟草叶片中的表达量介于180.46（81#）~756.87 ng/g·fw（86#），Western blot证实植物源4×Tα1具有免疫活性。MTT实验结果显示，植物源的4×Tα1蛋白具有促进BALB/c鼠脾淋巴细胞增殖的功能，为用安全植物表达系统生产包括Tα1在内的药用蛋白提供重要参考。
关键词： 胸腺素α1;  转基因烟草;  生物活性;  无选择标记    
Abstract:

Thymosin α1 (Tα1), an immune booster, plays critical roles in the maturation, differentiation and function of T-cells. Tα1 mainly was used to cure various diseases of immunodeficiency and virus infection in clinic, such as hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), cancers and so on. Several reasons confine application of the Tα1 in clinic, which include the available thymus of calf or porcine for extraction of Tα1, higher cost to yield Tα1 by chemical synthesis method, and existing safety problems by traditional expression system such as Escherichia coli and transgenic animal using genetic engineering way, because of contamination of E.coil-derived endotoxin and some zoonotic pathogens. In order to meet clinical demand for Tα1, the plant-derived Tα1 was tentatively expressed in transgenic tobacco. The tα1 gene (124 bp) was designed and synthesized according to the plant codon usage bias and created a novel 4×tα1 concatemer (four copies of the tα1 gene arranged end-to-end in tandem, designated 4×tα1). Subsequently, a plant binary expression vector, p35s∷4×tα1 with twin T-DNAs was constructed and introduced into tobacco via Agrobacterium tumefaciens-mediated transformation. Fourteen independent transgenic tobacco plants were confirmed by PCR and Southern blot analysis, and target 4×tα1 gene and selective nptⅡ gene were integrated into tobacco genome at different copies or sites in T0 generation, respectively. In order to analyze the separation of 4×tα1 and nptⅡ, 177 transgenic tobacco plants was assayed with PCR in T1-generation, two transgenic tobacco plats which only carried 4×tα1 was obtained but without selective gene nptII. The ELISA results showed that content of the 4×Tα1 in transgenic tobacco plats ranged from 180.46 ng/g·fw (line 81) to 756.87ng/g·fw (line 86), and Western blot result confirmed that tobacco-derived 4×Tα1 possessed immunocompetence. The MTT ［3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide-experiment showed the 4×Tα1 protein derived from transgenic tobacco exhibited bioactivity that can stimulate the proliferation of mice splenic lymphocytes in vitro. The experimental data will provide significant reference to produce recombinant therapeutic proteins including Tα1 using safe plant expression system.
Key words: Thymosin α1    Transgenic tobacco    Bioactive    Marker free
收稿日期: 2009-10-30 出版日期: 2010-03-25
基金资助:

国家“863”计划(2007AA100503)、国家自然科学基金（30871722）、上海市科技攻关重大专项（03DZ19310， 073158202）资助项目
通讯作者: 赵凌侠     E-mail: lxzhao@sjtu.edu.cn
服务　　
把本文推荐给朋友
加入引用管理器
E-mail Alert
RSS
作者相关文章　　
芦丽亚
宋维
游晓慧
江婷
赵凌侠

引用本文:

芦丽亚 宋维 游晓慧 江婷 赵凌侠. 用无选择标记的转基因烟草表达胸腺素α1（Tα1）[J]. 中国生物工程杂志, 2010, 30(03): 1-8.

HU Li-E, SONG Wei, LIU Xiao-Hui, JIANG Ting, DIAO Ling-Xia. Expression of Bioactive Thymosin α1 Using Marker Free Transgenic Tobacco. China Biotechnology, 2010, 30(03): 1-8.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/    或    https://manu60.magtech.com.cn/biotech/CN/Y2010/V30/I03/1

［1］ Goldstein A L, Slater F D, White A. Preparation, assay, and partial purification of a thymic lymphocytopoietic factor (thymosin). Proc Natl Acad Sci USA, 1966, 56(3): 10101017. 
［2］ Goldstein A L. History of the discovery of the thymosins. Ann N Y Acad Sci, 2007, 1112(1): 113. 
［3］ Goldstein A L, Low T L, McAdoo M, et al. Thymosin alpha1: isolation and sequence analysis of an immunologically active thymic polypeptide. Proc Natl Acad Sci USA, 1977, 74(2): 725729. 
［4］ Good R A, Dalmasso A P, Martinez C, et al. The role of the thymus in development of immunologic capacity in rabbits and mice. J Exp Med, 1962, 116(5): 773796. 
［5］ Wada S, Kinoshita Y, Kamizuru M, et al. A study of antitumor effects of thymosin on rat and mouse urinary bladder carcinoma induced by NbutylN(4hydroxybutyl)nitrosamine. Oncol Res, 1996, 8(3): 139144. 
［6］ Kullavanuaya P, Treeprasertsuk S, ThongNgam D, et al. The combined treatment of interferon alpha2a and thymosin alpha 1 for chronic hepatitis C: the 48 weeks end of treatment results. J Med Assoc Thai, 2001, 84(suppl1): 462469. 
［7］ Amarapurkar D, Das H S. Thymosin alpha in the treatment of chronic hepatitis B: an uncontrolled openlabel trial. Indian J Gastroenterol, 2002, 21(2): 5961. 
［8］ Goldstein A, Badamchian M. Thymosins: chemistry and biological properties in health and disease. Expert Opin Biol They, 2004, 4(4): 559573. 
［9］ Chen C, Li M, Yang H, et al. Roles of thymosins in cancers and other organ systems. World J Surg, 2005, 29(3): 264270. 
［10］ Billich A. Thymosin alpha1. SciClone Pharmaceuticals. Curr Opin Investig Drugs, 2002, 3(5): 698707. 
［11］ Lico C, Chen Q, Santi L. Viral vectors for production of recombinant proteins in plants. J Cell Physiol, 2008, 216(2): 366377. 
［12］ Lie′ nard D, Sourrouille C, Gomord V, et al. Pharming and transgenic plants. Biotechnol Annu Rev, 2007, 13: 115147. 
［13］ Schillberg S, Fischer R, Emans N. Molecular farming of antibodies in plants. Naturwissenschaften, 2003, 90(4): 145155. 
［14］ Streatfield S J. Approaches to achieve highlevel heterologous protein production in plants. Plant Biotechnol J, 2007, 5(1): 215. 
［15］ 曹慧颖，张锐，郭三堆. 串联的人胸腺素α1基因在番茄中的高效表达. 中国农业科学, 2009, 42(7): 22912296. Chao Y H, Zhang R, Guo S W. Scientia Agricultura Sinica, 2009, 42(7): 22912296. 
［16］ 牛颜冰，史正文，王德富，等. 重组马铃薯X 病毒注射番茄果实高效表达胸腺素α1. 生物工程学报, 2009, 25(4): 537541. Niu Y B, Shi Z W, Wang D F , et al. Chin J Biotech, 2009, 25(4): 537541. 
［17］ Chen Y H, Wang A X, Zhao L X, et al. Expression of thymosin α1 concatemer in transgenic tomato (Solanum lycopersicum) fruits. Biotechnol Appl Biochem, 2009, 52: 303312. 
［18］ Murray E E, Lotzer J, Eberle M. Codon usage in plant genes. Nucleic Acids Res, 1989, 17(2): 477498. 
［19］ Stewart C N Jr,Via L E. A rapid CTAB DNA isolation technique useful for RAPD fingerprinting and other PCR applications. Bio techniques, 1993, 14(5): 748749. 
［20］ Bradford M M. ARapid and Sensitive Method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding. Anal Biochem, 1976, 72(5): 248254. 
［21］ Schneider H, Muhle C, Douar A M, et al. Sustained delivery of therapeutic concentrations of human clotting factor IX  a comparison of adenoviral and AAV vectors administered in utero. J Gene Med, 2002, 4(1): 4653. 
［22］ Mosmann T. Rapid colorimetric assay for cell growth and survival:Application to proliferation and crytotoxicity assays. J Immunol Methods, 1983, 65(12): 5563. 
［23］ Chen Y H, Zhao L X, Shen G A, et al. Expression and Analysis of thymosin alpha 1 concatemer in Escherichia coli. Biotechnol Appl Biochem, 2008, 49(1): 5156. 
［24］ Maxfield L F, Fraize C D, Coffin J M. Relationship between retroviral DNA integration site selection and host cell transcription. Proc Natl Acad Sci USA, 2005, 102(5): 14361441. 
［25］ Wang S W, Stevenson A L, Kearsey S E, et al. Global role for polyadenylationassisted nuclear RNA degradation in posttranscriptional gene silencing. Molecul Cell Biol, 2008, 28(2): 656665. 
［26］ Pujol M, Gavilondo J, Ayala M, et al. Fighting cancer with plantexpressed pharmaceuticals. Trends Biotechnol, 2007, 25(10): 455459. 
［27］ Fischer R, Emans N. Molecular farming of pharmaceutical proteins. Transgenic Res, 2000, 9(45): 279299. 
［28］ Ma J K, Drake P M, Christou P. The production of recombinant pharmaceutical proteins in plants. Nat Rev Genet, 2003, 4(10): 794805. 
［29］ Mchughen A. The limited value of measuring gene flow via errant pollen from GM plants. Environ Biosafety Res, 2006, 5(1): 12. 
［30］ Viard F, Arnaud J F, Delescluse M, et al. Tracing back seed and pollen flow within the cropwild Beta vulgaris complex: genetic distinctiveness vs. hot spots of hybridization over a regional scale. Mol Ecol, 2004, 13(6): 13571364. 
［31］ Azeez G. Ampicillin threat leads to wider transgene concern. Nature, 2005, 435(7041): 561. 
［32］ Williams C G. The fit between organic and pharma crops in North Carolina. Nat Biotechnol, 2007, 25(2): 166167. 
［33］ de Vetten N, Wolters A M, Raemakers K, et al. A transformation method for obtaining markerfree plants of a crosspollinating and vegetatively propagated crop. Nat Biotechnol, 2003, 21(4): 439442. 
［34］ Ram N, Ayala M, Lorenzo D, et al. Expression of a singlechain Fv antibody fragment specific for the hepatitis B surface antigen in transgenic tobacco plants. Transgenic Res, 2002, 11(1): 6164. 
［35］ Galeffi P, Lombardi A, Donato M D, et al. Expression of singlechain antibodies in transgenic plants. Vaccine, 2005, 23(15): 18231827. 
［36］ Santi L, Giritch A, Roy C J, et al. Protection conferred by recombinant Yersinia pestis antigens produced by a rapid and highly scalable plant expression system. Proc Natl Acad Sci USA, 2006, 103(4): 861866. 
［37］ KumagaiSano F, Hayashi T, Sano T, et al. Cell cycle synchronization of tobacco BY2 cell. Nat Protoc, 2006, 1(6): 26212627. 
［38］ Nocarova E, Fischer L. Cloning of transgenic tobacco BY2 cells; an efficient method to analyse and reduce high natural heterogeneity of transgene expression. BMC Plant Biol, 2009, 9(44): 111.
[1] 张虎,刘镇洲,陈家敏,高保燕,张成武. 利用海洋硅藻生产生物活性物质研究进展*[J]. 中国生物工程杂志, 2021, 41(4): 81-90.
[2] 陈春琳,秦松,宋宛霖,刘志丹,刘正一. 褐藻寡糖生物法制备研究进展 *[J]. 中国生物工程杂志, 2020, 40(10): 85-95.
[3] 唐健雪,肖永乐,彭俊杰,赵世纪,万小平,高荣. 融合抗菌肽基因在重组毕赤酵母的表达及体外活性研究 *[J]. 中国生物工程杂志, 2018, 38(6): 9-16.
[4] 姚民,朱淑华,李佛生,张士彦,唐琳. 异源表达AtCYSa基因烟草的耐盐和抗虫特性分析[J]. 中国生物工程杂志, 2018, 38(4): 8-16.
[5] 王冬冬, 张国利, 岳玉环, 吴广谋, 田园, 刘雨玲, 吉元刚, 王金鹏, 李建, 潘荣荣, 马洪圆. 抗A型产气荚膜梭菌α毒素全人源双价单链抗体的构建、表达及其活性的初步研究[J]. 中国生物工程杂志, 2017, 37(4): 18-25.
[6] 韩双, 杨志丽, 陈丽梅. 过量表达拟南芥CAT提高烟草对气体甲醛的吸收和抗性[J]. 中国生物工程杂志, 2015, 35(5): 41-48.
[7] 冯天祥, 王玲, 陈海敏, 盛清, 左锐, 谢文杰. 植物内生放线菌功能及生物活性物质研究进展[J]. 中国生物工程杂志, 2015, 35(4): 98-106.
[8] 孙静, 王斌, 段志青, 胡凝珠, 李建芳, 李彦涵, 胡云章. 重组人LIF融合蛋白表达纯化及其活性鉴定[J]. 中国生物工程杂志, 2013, 33(5): 50-55.
[9] 路庆鹏, 许正宏, 史劲松, 窦文芳. 毕赤酵母STE13基因敲除对GGH表达及其生物活性的影响[J]. 中国生物工程杂志, 2013, 33(4): 28-33.
[10] 刘蕾, 孙振, 宋中邦, 肖素勤, 陈丽梅. 在叶绿体中过量表达AOD1和HPS-PHI创建光合甲醇同化途径增加烟草同化甲醇能力[J]. 中国生物工程杂志, 2013, 33(12): 69-78.
[11] 罗莉, 何勇智, 张勇侠, 王明蓉. 功能性包涵体的研究进展[J]. 中国生物工程杂志, 2013, 33(1): 114-121.
[12] 郭云萍, 孙璐, 张立剑, 王增禄, 高超, 杨强, 刘毅, 张英起, 屈延, 陶凌. 无标签重组人硫氧还蛋白的大规模表达、纯化及活性检测[J]. 中国生物工程杂志, 2012, 32(08): 62-67.
[13] 李娟, 余榕捷, 王静静, 黄霖, 刘晓飞. 重组PAC1-EC1(N)对表达不同PAC1变体的细胞系细胞活性的影响[J]. 中国生物工程杂志, 2011, 31(06): 22-28.
[14] 汤熙翔, 易志伟, 李宁, 马群, 李慧, 秦丹, 肖湘. 深海宏基因组文库克隆子发酵产物的生物活性筛选[J]. 中国生物工程杂志, 2011, 31(06): 58-63.
[15] 妙亮, 徐立华, 冀胥, 边六交. 可溶型三聚体血管生长抑制因子Kringle5的克隆,表达,纯化及活性研究[J]. 中国生物工程杂志, 2011, 31(03): 18-22.
主管：中国科学院 　主办：中国科学院文献情报中心 　中国生物技术发展中心 　中国生物工程学会