基因串联原核高效表达胸腺肽α1

High Level Prokaryotic Expression of Thymosin α1 by Gene Repeats

人工合成含有SD序列、胸腺肽α1(Tα1)基因、纯化标签和酶切位点的DNA序列作为构建元件,利用同尾酶将其依次克隆到pET-32a(+)中,得到含有1～8个不同重复数目的含SD序列基因的串联表达载体,利用PCR初步鉴定和进一步的测序证实序列完全正确。进而利用lacZ基因作为指示基因,将带有SD序列的lacZ基因克隆到不同Tα1基因串数载体末尾,利用蓝白斑实验验证启动子后不同SD序列的效率,证实串联载体中的各个SD序列均能有效启动翻译。在此基础上对各串表达载体进行摇瓶发酵,SDS-PAGE电泳检测证实各串均可正确表达Tα1,且为可溶性表达,为小肽原核高效表达提出了新方法,有望成为小肽高效表达的新途径。

A new thymosin α1 （ Tα1 ） gene [ SD -His ·tag-EK - Tα1] named SD-Tα1 was synthesized, consecutively consisting of SD sequence, sequence encoding His tag, enterokinase recognition site and Tα1 cDNA. This gene monomer was multiplied up to 8-tandems using Isocaudamer XbaⅠ and SpeⅠ. These different repeats of SD-Tα1 were inserted into the plasmind pET-32a（ ＋ ） respectively and then transformed into E. coli BLR（ DE3 ） to achieve a series of genetic bacteria E. coli BLR（ DE3 ） containing 1 - 8 copies of SD-Tα1. PCR analysis and the sequencing showed that 1 - 8 copies of SD-Tα1 were all correctly cloned into the pET-32a（ ＋ ） vector. Furthermore, the reporter gene lacZ with its own SD sequence （SD-lacZ） was added as a tail of SD-Tα1 tandems to verify the translation efficiency of the fusion gene through blue-white clone screening. The expected blue clones were obtained such as genetic E. coli BLR（ DE3 ） strain containing pET32a-1Tα1 -lacZ, pET32a-3Tα1 -tacZ, pET32a-STα1-lacZ and pET32a-8Tα1-lacZ. The results implied that each SD-Tα1 repeat can be authentically transcribed and submitted for translation. The fermentation and SDS-PAGE analysis indicated that Tα1 peptide was expressed in a soluble form in all the genetic bacterial strains containing 1 -8 repeats of SD- Tα1. In conclusion, a series of multiple repeats of Tα1 gene had successfully been constructed and expressed in E. coli with a new construction method. Also, a platform of high prokaryotie expression of small peptides was established.