人胰岛素样生长因子-Ⅰ基因农杆菌工程菌株的构建

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－Ｉ）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ｈｕＩＧＦ－ＩＤＮＡ片段，用 ＥｃｏＲ　Ｉ／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 Ｈｉｎｄ ＩＩ／ＥｃｏＲＩ切下“３５Ｓ－Ｋｏｚａｋ－ＩＧＦ－Ｉ－ＮＯＳ（ｔｅｒ．）”插入 ｐＣＡＭＢＩＡ２３００之Ｈｉｎｄ ＩＩ／ＥｃｏＲ　Ｉ位点，构建成ｈｕＩＧＦ－Ｉ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株为培育ｈｕＩＧＦ－Ｉ豆药用植物奠定了基础。

The construction of recombinational agrobacterium of huIGF-

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 EcoR I /Pst I,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 BamH I /Sac I site of pBI121,then pBI121 was digested with Hind III /EcoR I and the fragment of 35S-Kozak-IGF-I -Nos(ter). was inserted into Hind III /EcoR I 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.