A method for synthesizing low-molecular-weight heparin precursors by waar gene-deficient bacteria
A low-molecular-weight heparin, gene defect technology, applied in the field of reducing the molecular weight of polysaccharides, to achieve the effect of reducing the average molecular weight of polysaccharides
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Embodiment 1
[0025] Example 1: E.coli K5 waaR gene knockout
[0026]E.coli K5 genome was extracted according to the instructions (E.Z.N.A.TM Bacterial DNA Kit). According to the E.coli strain F632waaR gene (GenBank: AF019375.1) submitted by NCBI and the nearby base sequence of the gene, primers WY1 and WY2 were designed from the upstream 65bp and downstream 58bp of the gene to amplify E.coli K5waaR gene, and additional base sequences are provided at both ends of the gene for sequencing. Using the E. coli K5 genome as a template, under the action of primers WY1 and WY2, the E. coli K5 waaR gene (shown in SEQ ID NO.1) was amplified by PCR. Knockout primers P1 and P2 were designed to amplify the kanamycin resistance gene (underlined sequences are homology arms, and lowercase letters are primers for amplifying the kan gene on pKD4), and the amplified fragments are flanked by There is a 56bp homology arm of the waaR gene.
[0027] Extract plasmid pKD46 and plasmid pKD4 according to the instr...
Embodiment 2
[0038] Example 2: Electron microscope verification of cell morphology of mutant strains
[0039] The cell morphology of the mutant strain E.coli KT was observed by negative staining. The mutant strain E.coli KT prepared in Example 1 and the departure strain E.coli K5 were inoculated in LB agarose medium, cultivated at 37°C for 16-18h, and the departure strain and A small amount of mutant strain cells were stained with 1-2% phosphotungstic acid (PTA, pH 6.5-7.0) for 5-10 seconds, then observed under a transmission electron microscope, and photographed. The results are shown in figure 2 .
[0040] By comparing the cell surface morphology of the starting strain and the mutant strain, it was found that there was a layer of fluffy substance on the cell surface of the starting strain, which should be a capsular polysaccharide layer, while the surface of the mutant strain was relatively smooth without capsule. This is related to the deletion of the waaR gene in the mutant strain, ...
Embodiment 3
[0041] Embodiment 3: the fermentation situation of mutant strain
[0042] Use glucose synthesis medium (i.e. fermentation medium) to carry out the fermentation culture of the mutant strain E.coli KT, inoculate the mutant strain E.coli KT screened in Example 2 into the glucose synthesis medium, culture temperature 37°C, culture time 24 hours , rotating speed 200rpm, after the end of the fermentation, separate the fermentation liquid from the fermentation broth at 10000rpm for 30 minutes, discard the bacterial cells, and obtain the fermentation supernatant. The fermentation supernatant was decolorized with an aqueous hydrogen peroxide solution with a volume concentration of 1.5%. After staying overnight at 4°C, the precipitate was removed by centrifugation, and then the fermentation liquid was salted out with 65% saturated ammonium sulfate with a mass concentration of 65%, and stored overnight in an explosion-proof refrigerator. The next day, 10000rpm, centrifuged for 30min, the...
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