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A technology of adenosine monophosphate and uridine phosphorylase, which is applied in the field of bioengineering and can solve the problems of low substrate amount and large amount of bacterial cells.
Inactive Publication Date: 2015-02-25
GUANGDONG XIANQIANG PHARMA
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The above methods all have the disadvantage of using too much bacteria and low substrate amount.
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Embodiment 1
[0039] Construction of E. coli strains co-expressing purine nucleoside phosphorylase and uridine phosphorylase.
[0040] (1) Design primers according to the sequence published in genebank
[0041] Design of primers for Escherichia coli purine nucleoside phosphorylase gene
[0042] Primer (A): 5′-CCCATGGCTACCCCACACATTAATGCAG-3′
[0043] Primer (B): 5′-CCAAGCTTGGTTACTCTTTATCGCCCAGCAGAAC-3′
[0044] Primer Design of Escherichia coli Uridine Phosphorylase Gene
[0045] Primer (C): 5′-CGCCATATGTCCAAGTCTGATGTTTTTC-3′
[0046] Primer (D): 5′-CCGCTCGAGTTACAGCAGACGACGCGCCG-3′
[0047] (2) PCR amplification gene
[0048] PCR reaction template is Escherichia coli DH5α genomic DNA
[0049] PCR reaction system (30μl):
[0050]
[0051] PCR reaction conditions:
[0052]
[0053] The PCR products were purified using Tiangen PCR Product Recovery Kit.
[0054] (3) Construction of expression plasmid
[0055] Use NcoI and HindIII to double-enzyme digest the plasmid pETDuet-1 and pu...
Embodiment 2
[0059] Shake Flask Fermentation of UP-I
[0060] Culture conditions: Pick the monoclonal strain UP-I and inoculate it into 50 ml LB liquid medium containing ampicillin (100 μg / ml), 37° C., 220 rpm. When the OD600 reached 0.6, IPTG (final concentration: 0.5 mM) was added for induction, and culture was continued for 4 hours after induction, and 0.3 g of bacterial cells were collected by centrifugation, and stored at -20°C.
Embodiment 3
[0062] Carry out biotransformation reaction with the thalline that embodiment 2 obtains
[0063] 30mM potassium phosphate buffer pH 7.4 contains 100mM vidarabine and 100mM adenine in 100ml, add 0.3g of the bacteria obtained by fermentation in Example 1, shake and react at 50°C for 6 hours, and obtain 2.48 grams of vidarabine after crystallization and purification . The molar yield was 92.8%.
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Abstract
The invention discloses a preparation method of vidarabine monophosphate. The method first prepares genetically engineered bacterium with co-expression of purine nucleoside phosphorylase and uridine phosphorylase, and then uses the genetically engineered bacterium to prepare vidarabine monophosphate. The method is characterized in that coexpression of purine nucleoside phosphorylase and uridine phosphorylase is realized in genetically engineered bacterium by using a molecular cloning method, so as to obtain vidarabine biotransformation bacteria with high enzyme specific activity; the thalli obtained by the invention is used to produce vidarabine; and the reaction substrate concentration can be increased to more than 100 mM, the reaction conversion rate is more than 90%, the dosage of reaction thalli is less than 0.3% (thalli wet weight / reaction volume ml), and reaction time is shortened to 6 h.
Description
technical field [0001] The invention relates to a method for preparing a bioengineering bacterium by a biological method, and then using the bioengineering bacterium to prepare vidarabine monophosphate, belonging to the technical field of bioengineering. Background technique [0002] Adenosine Arabinoside (araA) is a natural antiviral compound with broad-spectrum antiviral activity. Clinically used adenosine arabinoside monophosphate is mainly used to treat chronic hepatitis B and other viral infections such as Herpes zoster, herpes simplex, genital herpes, etc. In addition, it also has certain curative effects on hand, foot and mouth disease and children's chickenpox. [0003] In the prior art, the synthesis method of vidarabine includes chemical synthesis and biotransformation synthesis. The chemical method has complicated process, harsh reaction conditions, high cost of raw materials, and the use of heavy metal reagents, which pollutes the environment. The basic princip...
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