【摘要】 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。更多还原

【Abstract】 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lacZ,pET32a5Tα1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 prokaryotic expression of small peptides was established.更多还原