| 摘要: |
| 采用固相亚磷酸三酯法人工化学合成了人胰岛素样生长因子-Ⅰ(huIGF-1)基因的4条寡核苷酸片段，再通过片段1，2与3，4末端互补配对和Klenow酶促补平及酶切连接成为完整的huIFG-I DNA片段，用EcoRⅠ/PstⅠ酶切后克隆于pGEM T-Easy Vector，经测序证明所得DNA序列与设计的序列完全一致，选用植物偏爱密码子校正了启始密码子ATG下游的6个密码子，并在ATG处增加Kozak序列后，插入pBI121的BamHⅠ/SacⅠ位点，构建了以35S为启动子的植物表达载体，以HindⅡ/EcoRⅠ切下35S－Kozak-IGF-Ⅰ-NOS(ter)。插入pCAMBIA2300之Hind Ⅲ/EcoRⅠ位点，构建成huIGF-1植物表达载体，通过CaCl2直接转化法将表达载体导入农杆菌EHA105（Rif.r)，并以菌落原位杂交技术和DNA dot blot对重组农杆菌进行了筛选，所获得的重组农杆菌菌 培养huIGF-I药用植物奠定地基础。 |
| 关键词: 胰岛素样生长因子-Ⅰ 化学合成 植物表达载体 重组农杆菌 药用植物 |
| DOI： |
| 分类号: |
| 基金项目:博士后科学基金资助项目(8784) |
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| The construction of recombinational agrobacterium of huIGF-Ⅰ |
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| Abstract: |
| Four oligonucleotides were synthesized according to human insulin-like growth factor Ⅰ DNA sequence by means of the solid-phase phosphotriester method.Two pairs of oligos 1 and 2,3 and 4 had respectively been annealed by their 15 base-complementarity,and then polymerized into two intact double-stranded gene,which was then inserted into PGEM T-Easy vector site of EcoRⅠ/PstⅠ,DNA sequencing show that the sequence of synthesized gene of human IGF-Ⅰ was the same as that of the designed one.After sequencing,the partial codons of plant were choosed for correcting 6 codons behind ATG,and Kozak was added,all for reconstructing IGF-Ⅰ, which would be inserted into BamHⅠ/SacⅠ site of pBI121,then pBI121 was digested with HindⅢ /EcoRⅠ and the fragment of 35S-Kozak-IGF-Ⅰ-Nos(ter).was inserted into Hind Ⅲ /EcoRⅠ site of pCMBIA2300,a new plant expression vector was formed,transferred into EHA105,finally,a kind of recombinational agrobacterum strain was reconstructed,these results had been identified in this study. |
| Key words: human insulin-like growth factor-Ⅰ(huIGF-Ⅰ) chemical synthesis plant expression vector recombinational agrobacterium identification |
