整合型vgb基因载体的构建和研究

运用染色体-质粒同源基因重组的方法将外源Vitreoscilla血红蛋白基因(vgb)整合在大肠杆菌JM105染色体的苏氨酸操纵子位置上，构建单拷贝的vgb表达载体VGl。VGl保持了vgb基因的生理功能，可在低氧条件下表达Citreoscilla血红蛋白使细胞适应贫氧环境;同时它克服了以质粒作为表达载体时vgb基因剂量和表达量过高带来的生理负担，因此可作为一种适于高密度培养的基因工程宿主菌。实验结果还显示vgb基因的表达使VGl与普通大肠杆菌相比在低氧条件下仍能维持较高的呼吸强度，说明vgb基因产物参与了细胞处于低氧水平时的代谢过程。

Construction and Study of an Integrated Expression Vector of Vitreoscilla Hemoglobin Gen e (vgb) by Duble Homologous Recombination Between Plasmid and Chromosome

A method of double homologous recombination between plasmid and chromosome is introduced to construct an integrated expression vector of Vitreoscilla hemoglobin gene(vgb) ,which has advantages of positive selection and determinable integrated site. The vgb gene is inserted in the threonine operon of the chromosome DNA of E. colt JM105 and expressed under its native promoter. This vgb integrated cell, VG1, maintains the physiological functions of vgb, moreover it decreases expression amount of cellular hemoglobin by lowering the gene dose, which is overproduced using multicopy plasmid as an expression vector. Thus, the new developed strain VG1 can be used as a host cell suitable for high cell density fermentation. Comparison of the respiratory kinetics of VG1 and JM105 suggests that the hemoglobin participates in some steps of the cellular metabolism.