Study Of Cloning And Expression Of Vitreoscilla Hemoglobin Gene (vgb) In Cephalosporium Acremonium

Cephalosporins are one of the anti-infective drugs used in clinical. Their market share among anti-infective drugs has been steadily increasing year by year.Cephalosporin C as a raw material, was produced with Cephalosporium acremonium. Its ferment requires lots of oxygen, the contradiction between oxygen demands and provides is quite obvious.Vitreoscilla hemoglobin (VHb) is a kind of prokaryote hemoglobin. The research showed that the expression of VHb was modulated by oxygen if Vitreoscilla hemoglobin gene (vgb) was placed under the control of its natural promoters, and the amount of expressed VHb was great increased in poor oxygen conditions.According to DNA sequence reported from GenBank designed a pair of primers with KpnI and SacI restriction site, and vgb was amplified from plasmid pMK4-LV. Vgb gene and vector pUCATPH were digested by KpnI and SacI. The two parts were ligated by T4 DNA ligase and constructed the recombinant plasmid pUCATPH-LV. Transformed pUCATPH-LV into E.coli DH5α. The result showed that the sequence inserted didn't mutant. Induced the transformants in low-oxygen ferment and inspected it by the protein electrophoresis. The result showed that the Vitreoscilla hemoglobin protein was successfully expressed in E.coli DH5α.Cephalosporium acremonium mycelium was cultured in cellophane flat culture for 4～5days. Protoplasm was produced by using the penetrative stabilizer (0.6mol/L KCl+25mmol/L CaCl2·2H2O) and compound enzyme for 3.5 hours in 30℃. The compound enzyme was composed of 1% cellulase and 1% snailase. The number of protoplasm was up to 4.62×107 per milliliter. The regeneration rate was 10.8%. The vgb gene was transformed into Cephalosporium acremonium by spherplast electroporation, and some hygromycin B resistant fungus were filtrated on the medium. The vgb was successfully recombined into Cephalosporium acremonium by PCR inspection, and the transformed successfully expressed the Vitreoscilla hemoglobin protein in low-oxygen ferment, CO different spectrum showed a special peak in 420nm, which proved VHb was active, vgb had already expressed in Cephalosporium acremonium.Another recombined plasmid pCAMBIA1301-LV was constructed for the way of the Agrobacterium tumefaciens-mediated transformation to achieve the expression of vgb in Cephalosporium acremonium. According to polyclonal sites of plasmid pCAMBIA1301 and vgb gene reported in GenBank, designed a pair of primers with BamHI and SacI restriction site, amplified vgb gene from pMk4-LV. Vgb gene and plasmid pCAMBIA1301 were digested by BamHI and Sad. The two parts were ligated by T4 DNA ligase and constructed the recombinant plasmid pCAMBIA1301-LV. Transformed pCAMBIA1301-LV into E.coli DH5a.The sequencing results showed that the inserted parts were the same as GenBank reports. pCAMBIA1301-LV transformed into A. tumefaciens, making ready for A.tumefaciens-mediated transformation.The research provided a new direction of stains modification about Cephalosporin C, and had crucial theoretical and practical direction function on genetically modification of other antibiotic-producing stains.