Please wait a minute...

中国生物工程杂志

China Biotechnology
China Biotechnology  2010, Vol. 30 Issue (03): 1-8    DOI:
    
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
Download: HTML   PDF(1101KB) HTML
Export: BibTeX | EndNote (RIS)      

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 wordsThymosin α1      Transgenic tobacco      Bioactive      Marker free     
Received: 30 October 2009      Published: 25 March 2010
Fund:  

China National ‘863’ High-Tech Program; the National Natural Science Foundation of China

Corresponding Authors: Ling-xia ZHAO     E-mail: lxzhao@sjtu.edu.cn
Cite this article:

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.

URL:

https://manu60.magtech.com.cn/biotech/     OR     https://manu60.magtech.com.cn/biotech/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] ZHANG Hu,LIU Zhen-zhou,CHEN Jia-min,GAO Bao-yan,ZHANG Cheng-wu. Research Progress on the Production of Bioactive Compounds from Marine Diatoms[J]. China Biotechnology, 2021, 41(4): 81-90.
[2] Min YAO,Shu-hua ZHU,Fo-sheng LI,Shi-yan ZHANG,Lin TANG. Analysis of Salt Tolerance and Insect Resistance of Transgenic Tobacco Expressing AtCYSa from Arabidopsis[J]. China Biotechnology, 2018, 38(4): 8-16.
[3] HAN Shuang, YANG Zhi-li, CHEN Li-mei. Over Expression of Arabidopsis CAT Improved the Absorption and Tolerance of Gas HCHO in Tobacco[J]. China Biotechnology, 2015, 35(5): 41-48.
[4] LIU Lei, SUN Zhen, SONG Zhong-bang, XIAO Su-qin, CHEN Li-mei. Simultaneous Over-expressions of AOD1 and HPS-PSI in Chloroplasts Creates a Novel Photosynthetic CH3OH-assimilation Pathway and Enhances Its Ability to Assimilate CH3OH[J]. China Biotechnology, 2013, 33(12): 69-78.
[5] SONG Feng, SUN Min, LUO Ke-Meng. A Simple and Rapid PCR-based Method for Identifying Transgenic Tobacco Plants Carrying a Single Copy of the Integrated Gene[J]. China Biotechnology, 2010, 30(04): 83-88.
[6] . Advance on the Biotransformation of Bioactive Natural Leading Compounds[J]. China Biotechnology, 2007, 27(9): 110-115.
[7] . Transgenic tobacco expressing the crocus CsZCD gene showed the production of crocetin[J]. China Biotechnology, 2007, 27(6): 51-55.
[8] . Expression of a kind of curcin induced from Jatropha curcas (curicin 2) in tobacco[J]. China Biotechnology, 2007, 27(4): 94-98.
[9] . Purification and Characterization of α-AE Protein Expressed in E.coli[J]. China Biotechnology, 2007, 27(11): 6-10.