关键词: 透明颤菌血红蛋白, 植物偏爱密码子, 人工合成, 大肠杆菌, 诱导表达

Abstract: According to the amino acid sequence of Vitreoscilla hemoglobin (VHb), a novel gene encoding the VHb protein was designed with plant preferential codon. First 22 oligodeoxynucleotide fragments were synthesized based on the design, meanwhile, certain restriction enzyme sites for further cloning were considered. Three small DNA fragments, which were formed by annealing certain oligonucleotide fragments, were sub-cloned respectively. Then the vgbM gene, which is 450 bp in length, was cloned into pUC19 by ligating the three small DNA fragments. The recombinant plasmid was named pGSVHB. The pBV221SVHB was further constructed by inserting the vgbM gene into the plasmid pBV221 after a Nco I site adding at 5'-terminal through PCR. Subsequently, the vector was introduced into E. coli, BL21 strain. The 16 kD VHb protein was detected after hot-shock inducing expression of the engineered E. coli. The biological activity of the expressed VHb was demonstrated by CO difference spectrum analysis. This work has laid a foundation for further studies to transfer vgbM gene into plants.

Key words: Vitreoscilla hemoglobin, Plant preferential codon, Synthesis, E. coli, Inducing expression