关键词: 核糖体失活蛋白, Gelonin, 基因化学合成, 重组子, 表达

Abstract: 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 pUC 118 to construct the recombinants: pYW1, pYW2, pYW3 and pYW4 respectively by T₄DNA 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, pETgel which was ligated with pET-28a and gelonin gene was constructed and then expressed in host E.coli. The result showed that a 28000 band in expression products, equal to the apparent molecular weight of gelonin, significantly occurred on the 12% SDS-PAGE after incubation in soluble form.

Key words: Ribosome inactivating proteins, Gelonin, Chemical synthesis of gene, Recombinant, Expression