Reconstruction And Expression Of Fusion Recombinant Vector Of Vitreoscilla Hemoglobingene In Escherichia Coli

The biotin is one of the growth factors of the main supersession with essential course in the organism , which has irreplaceable function on the biological physiology. It is used extensivly in animal husbandry, molecular biology, biological ferment and medical. The biotin can't be got from the nature directly , only from chemosynthesis. Because it is complicated that the process of the production technology of chemosynthesis and it is difficult to sublimate, the costs of biotin tremain high.With the development of fermented industry and the industrialization application of and biosynthesis of riboflavin (VB2 ), the ways of synthesizing biotin shift from chemosynthesis to biosynthesis gradually. But dissolving oxygen is the bottleneck of industry fermates ,which restricts the growing and purification of the biotin seriously. So seeing that VHb can raise the content of oxygen in the fermented environment , this research aims at structuring the merging express vector with vgb.Then it get VHb of the high purity by metal ion (Ni2 +), and offer more data for structuring the fermented project bacterium in order to solve the problem that industry ferments and produces the biotin to dissolve oxygen further and improve output and purity of the biotin .In the study, at first ,it is easilier for the operation of genetic engineering to put EcoRI and Xbal to both ends of the primer in addition,respectively. Then vgb through PCR technology is cloned to express vector PET28a with protein merging label His6. The express vector PET28a-vgb expressed in colon bacillus BL21 (DE3 ). Optimizing the condition of expressing, the result showed: Recombinate protein can obtain the soluble expression in 30℃, the inducement of the end density of 2.5mmol IPTG, and expression amount of VHb account for 27.7% of the whole albumen.The fragment can be saw obviously by SDS-PAGE electrophoresis. For recombinate VHb by purification of Ni~2+, there is strong peak of absorbing under visible light wavelength 400nm of district, which displays the same nature of hemoglobin. To VHb of the purification by molecular Screen, two peaks appear altogether on the record sheet. The first peak obvious and high than second peak, and the first peak correspondto gather the dimer of hemoglobin, it was gathering the monomer of VHb that the second peak answered.