天花粉胰蛋白酶抑制剂基因的合成与克隆

天花粉胰蛋白酶抑制剂是从中药天花粉中分离出来的一种新的胰蛋白酶抑制剂,其结构最近被测定为含有三对二硫键的27肽,它也是目前发现的最小的多肽类蛋白酶抑制剂。本文报导了天花粉胰蛋白酶抑制剂基因的合成及克隆。设计的合成基因采用了大肠杆菌偏爱的密码子,全长为108个碱基对,它包括了起始密码子ATG,终止密码子TAG和两端的HindⅢ和BamH Ⅰ的识别顺序。整个基因的合成分为两步,首先用化学方法合成四个DNA片段(F1,38mer;F2,30mer;F3,30mer和F4A,46mer),再经连接酶连接成为两个3′端彼此互补的DNA片段(F1+F2,68mer和F3+F4,76mer),最后从两个互为引物的3′端用Klenow酶聚合补齐得到双链基因。同时,用在基因3′端有两个碱基改变的F4B代替F4A,使基因的终止密码子(TAG)变为甲硫氨酸密码子(ATG),并使基因的阅读框架与质粒pUC19中的LaeZ基因相一致,从而实现该基因与LaeZ基因的融合表达。合成基因经DNA序列分析证明其结构正确。

Chemical Synthesis and Cloning of the Trichosanthes Trypsin Inhibitor Gene

The trichosenthes trypsin inhibitor (TTI) is newly isolated from Trichosan-thes and composed of 27 amino acid residues with three pairs of disulfide bonds. It is also the smallest polypeptide proteinase inhibitor discovered so far. The synthesis and cloning of TTI gene are described. The 108 base pair nucleotide sequence of synthetic gene was designed by choosing codons favored for expression in E.coli.It contains the initiation codon ATC and the stop codon TAG, and also recognition sequence of Hind Ⅲ and BamH J at each end, respectively. The gene was synthesized in two stages. First,four polynucleotide fragments (F1,38mer; F2,30mer; F3,30mer and F4A, 46mer) were synthesized by chemical method. Then they were ligated to form two larger fragments (F1+F2, 68mer and F3 + F4A, 76mer) which can be complemented on 3'end and converted into a complete double-stranded molecule by mutually primed extention with Klenow fragment. Meanwhile the F4A was replaced by F4B which have two bases different from F4As,o that the stop codon (TAG) changed into Met codon (ATG)to make the gene identical in reading frame with LacZ when fused to 5' region of LacZ for cloning in plasmid pUC19. A fused protein composed of TTI and β-gal-actosidase was expressed. The DNA sequence was proved by sequenceing analysis.