用无选择标记的转基因烟草表达胸腺素α1(Tα1)

Expression of Bioactive Thymosin α1 Using Marker Free Transgenic Tobacco

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twinT-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nptII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nptII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#)～756.87ng/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。

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.