化学与酶促相结合合成并克隆核糖体失活蛋白Gelonin基因

Synthesis and Clone of Ribosome Inactivating Protein-Gelonin Gene by the Combination of Chemical and Enzymatic Method

利用已知 Gelonin的氨基酸序列 ,逆向推算出整个基因的碱基序列 ,根据 E.coli的密码子偏爱性和后续基因克隆的需要 ,通过沉默突变设计相应的酶切位点和碱基序列 ,将整个基因分为四段 ,每个片段约1 75～ 2 2 0 bp,每一个片段中的互补链从 5′末端用化学合成 1 0 0～ 1 2 0的碱基单链 ,其中两个链的 3′末端有 2 0个互补碱基 .利用 T4 DNA聚合酶酶促添补成双链 DNA,用分子克隆技术 ,分别构建重组子 ,然后再构建成含整个 Gelonin基因的表达载体 p ET-gel进行表达 ,经诱导后 ,获得了一个 2 80 0 0的重组蛋白 。

An ambiguous DNA sequence for gelonin was inversely deduced from the amino acid residues of gelonin, a ribosome inactivating protein. Based on the favorable code of protein synthesis in E.coli and the requirement of gene cloning in the subsequent work, a modified base sequence, but no any change for amino acid residues, of gelonin was designed by inserting several restriction endonuclease sites and silent mutation. In order to make the chemical synthesis of the gene convenient, the total DNA sequence of gelonin was divided into four fragments about the length of 175 bp to 220 bp and two complementary strands in each fragment with a length of 100 to 120 nucleotides were chemically synthesized from the beginning of 5′\|terminus with a 20 bp overlapping in both 3′\|teminus. In such case, the two single polynucleotides for each fragment could anneal to form a double\|strand DNA fragment. Then each \{ds DNA\} fragment was separately cloned into the vector pUC118 to construct the recombinants: pYW1, pYW2, pYW3 and pYW4 respectively by T\-4DNA polymerase in the presence of dNTP and pyrophosphatase. With the relevant endonuclease sites of each fragment, a recombinant, pUC\|gel was formed by cloning technology. Finally, an expression vector, pET\|gel which was ligated with pET\|28a and gelonin gene was constructed and then expressed in host E.coli. The result showed that a 28 000 band in expression products, equal to the apparent molecular weight of gelonin, significantly occurred on the 12% SDS\|PAGE after incubation in soluble form.