Process of synthesizing 6-methylpurine-2'-deoxyncleoside with gene engineering bacterium

A technology of genetically engineered bacteria and methyl purine, applied in the field of biopharmaceuticals, can solve the problems of difficult separation, a large number of isomers, chemical catalyst personnel and environmental poisoning, and achieve the effects of cost saving, short cycle and low cost

Inactive Publication Date: 2010-11-17
FUDAN UNIV
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Problems solved by technology

[0006] Chemical method: In 1996, Janet E et al. reported a method of synthesizing MePdR from MeP by chemical method, and the yield rate was 13% after two-step reaction. The disadvantage of this method is that a large number of isomers are produced during the synthesis process, which is difficult to separate , and chemical catalysts are highly toxic to personnel and the environment, so it is still not possible to form large-scale production to meet scientific research and clinical needs

Method used

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  • Process of synthesizing 6-methylpurine-2'-deoxyncleoside with gene engineering bacterium
  • Process of synthesizing 6-methylpurine-2'-deoxyncleoside with gene engineering bacterium
  • Process of synthesizing 6-methylpurine-2'-deoxyncleoside with gene engineering bacterium

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Experimental program
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Embodiment Construction

[0034] 1. Construction of genetically engineered bacteria.

[0035] Primers were designed according to the PNP and TP gene sequences of E.coli provided in GeneBank as follows:

[0036] PNP

[0037] 5' primer: 5'cat gccatgg ctaccccacacattaa-3' (Ncol)

[0038] 3' primer: 5'-at gtcgac ttactctttatcgcccagcag-3 (Sal I)

[0039] TP

[0040] 5' Primer: 5'-tttt gtcgac catgtttctcgcacaa-3'(SaI)

[0041] 3' Primer: 5'-aaaa ctgcag ttattcgctgatacgg-3' (PstI)

[0042] PCR was performed using the chromosomal DNA of E.coli DH5α as a template: 95°C for 5 min, 30× (95°C for 40 s, 62°C for 40 s, 72°C for 1 min), 72°C for 10 min. The PCR product and the pBV220 vector were digested with restriction enzymes and ligated with T4DNA ligase to transform E.coli DH5α competent cells. The pBV220 vector general primer PCR and restriction enzyme digestion were used to identify the transformants to obtain the genetically engineered bacteria pBV220-PNP and pBV220-TP. DNA sequencing found that th...

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Abstract

The present invention belongs to the field of biomedicine preparing technology, and is especially the process of constructing gene engineering bacterium by means of DNA recombining technology and synthesizing 6-methylpurine-2'-deoxyriboside (MePdR) with the gene engineering bacterium. The process includes the following steps: 1. constructing purine nucleoside phosphorylase and pyrimidine nucleoside phosphorylase expressing vector, and transforming to colibacillus to obtain gene engineering bacterium with high efficiency expression of purine nucleoside phosphorylase and pyrimidine nucleoside phosphorylase; 2. catalyzing the reaction between pyrimidine deoxyriboside and 6-methylpurine with the purine nucleoside phosphorylase and pyrimidine nucleoside phosphorylase to synthesize MePdR; and 3. separating and purifying MePdR. The catalytic synthesis process of MePdR has the features of simplicity, high efficiency, ets, and is suitable for industrial production.

Description

technical field [0001] The invention belongs to the technical field of biopharmaceuticals, and specifically relates to a genetically engineered bacterium constructed by DNA recombination technology, and the genetically engineered bacterium is used to synthesize 6-methylpurine-2'-deoxyriboside (6-Methylpurine-2'-Deoxyriboside, MePdR) method. Background technique [0002] MePdR is the prodrug of Escherichia coli PNP / 6-methylpurine-2′-deoxynucleoside (ePNP / MePdR) anti-tumor therapy system. 2.4.2.1, purinenucleoside phosphorylase, PNP) decomposes into 6-methylpurine (6-Methylpurine, MeP), MeP can inhibit RNA and protein synthesis, leading to cell death; mammalian PNPase cannot decompose MePdR. E.coli PNP gene is transferred to tumor cells to express E.coli PNP. When the drug precursor MePdR exists, E.coli PNP will decompose it into toxic MeP to kill tumor cells efficiently. [0003] Compared with other suicide gene / prodrug anti-tumor therapeutic systems, the ePNP / MePdR anti-tu...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P19/38C12N15/63C12N9/10
Inventor 梁胜华李文周高彤李晓晖任大明
Owner FUDAN UNIV
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