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Optimized DNA (Deoxyribonucleic Acid) molecule for coding ADI (Aiginine Deiminase) and engineering bacteria for expressing ADI

A DNA molecule, engineering bacteria technology, applied in the field of engineering bacteria, can solve the problems of restricting the development of large-scale industrialization and low expression, and achieve the effects of high specific activity and genetic stability

Active Publication Date: 2014-12-10
BEIJING KAWIN TECH SHARE HLDG
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the problem of low expression level, this seriously restricts the large-scale industrialization of ADI. Therefore, it is essential to find engineering bacteria that can efficiently express arginine deiminase to solve the problem of low expression level and realize industrial production. Problems that domain technicians have been eager to solve

Method used

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  • Optimized DNA (Deoxyribonucleic Acid) molecule for coding ADI (Aiginine Deiminase) and engineering bacteria for expressing ADI
  • Optimized DNA (Deoxyribonucleic Acid) molecule for coding ADI (Aiginine Deiminase) and engineering bacteria for expressing ADI
  • Optimized DNA (Deoxyribonucleic Acid) molecule for coding ADI (Aiginine Deiminase) and engineering bacteria for expressing ADI

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Embodiment 1 Experimental material

[0027] adi full sequence and adi Amplification primer

[0028] Entrusted Beijing Sanbo Yuanzhi Biotechnology Co., Ltd. to synthesize the optimized sequence of SEQ ID No: 1 adi sequence, and cloned into the pMD18-T simple vector to obtain adi Sequence vector pMD18-T simple- adi .

[0029] Entrust Sangon Bio (Shanghai) Co., Ltd. to synthesize adi PCR amplification primers.

[0030] Primer name sequence adi F ctgaaatcggtgaactg adi R gagacagcggcatagac

[0031] The nucleic acid sequence determination of this experiment was entrusted to Beijing Liuhe Huada Gene Technology Co., Ltd. to complete.

[0032] The expression vector pET30a was purchased from Novagen, and the cloning host strain DH5α and the expression host strain BL21 (DE3) were both products of Invitrogen.

[0033] Tool enzymes used in the experiment: xho I, Nde I, T4 DNA ligase is the product of Fermentas Company, and Trans...

Embodiment 2

[0035] The construction of embodiment 2 ADI expression vector

[0036] Digestion of vectors and fragments

[0037] Take pMD18-T Simple- adi Put 2 μg each of pET30a plasmid (100ng / μL) in different EP tubes, and add ddH to them 2 O 60 μL, Buffer O 10 μL, xho I 5μL and Nde I 2.5 μL. After the samples were mixed, they were centrifuged in a centrifuge for 5 seconds, and then placed in a constant temperature water bath at 37°C for 3 hours.

[0038] Ligation of vectors and fragments

[0039] Digested pMD18-T Simple- adi Add 20 μL Loading Buffer (6×) to the pET30a plasmid sample, load the sample on a wide-well agarose gel (0.9%), and electrophoresis at a constant flow of 80 mA for 40 min.

[0040] Take pMD18-T Simple- adi A fragment of about 1224bps was cut from the electrophoresis gel, and a fragment of about 5256bps was cut from the pET30a electrophoresis gel. After the gel was cut and weighed, it was recovered with an agarose gel electrophoresis DNA recovery kit, an...

Embodiment 3

[0055] Example 3 strain screening

[0056] ADI expression vector transformation host bacteria

[0057] Take 0.2 μL pET30a- adi plasmid, according to E. coli BL21 (DE3) Chemical Transformation Competent Use instructions for transformation.

[0058] Pick 6 colonies from the transformed LB (Kan 50 μg / mL) resistant plate, transfer them to a 10 mL LB (Kan 50 μg / mL) test tube, and incubate at 37°C for 3.5 hours. The strains were preserved with 30% glycerol.

[0059] Screening of ADI engineering bacteria

[0060] Transfer pET30a- adi / BL21 (DE3) transformants were transferred to LB (Kan 50 μg / mL) test tubes, cultured at 37°C for 2 hours, then induced by adding IPTG with a final concentration of 0.5 mM, and induced expression for 6 hours.

[0061] SDS-PAGE electrophoresis detection of each bacteria before and after induced expression is shown in Figure 4. According to the electrophoresis results in Figure 4, the pair pET30a- adi / Analysis data of protein expression in ea...

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Abstract

The invention relates to an optimized DNA (Deoxyribonucleic Acid) molecule for coding ADI (Aiginine Deiminase), an expression vector pET-30a-ADI containing the optimized DNA molecule and engineering bacteria pET-30a-adi / BL21(DE3) containing the expression vector, wherein the collection serial number for the engineering bacteria is CGMCC No. 5199. The engineering bacteria strain has the effect of efficiently expressing the ADI; the expression rate of the engineering bacteria reaches over 40 percent; and the specific activity can reach 110IU / mg after purification.

Description

technical field [0001] The invention relates to an optimized DNA molecule encoding ADI and engineering bacteria containing the optimized DNA molecule, belonging to the field of biotechnology. Background technique [0002] L-arginine is an important nutrient for mammalian cells, and it is also used by mycoplasma as a major non-glycolytic energy source. Arginine deiminase (Aiginine deiminase, ADI, EC 3.5.3.6) obtained from cell cultures infected with mycoplasma can degrade arginine into citrulline and ammonia, which is present in many types of microorganisms , ADI has been shown to inhibit the growth of tumor cells in vitro and has anti-angiogenic activity. [0003] Normal human cells can synthesize arginine through the cytoplasmic enzymes argininosuccinate synthase (ASS) and argininosuccinate lyase (ASL) through citrulline. The metabolic pathway of arginine has been well described. During dietary intake, arginine is absorbed by the liver from the hepatic portal vein and rap...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/55C12N15/70C12N1/21C12N9/80C12R1/19
Inventor 侯建华杨璐
Owner BEIJING KAWIN TECH SHARE HLDG
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