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CDNA (Complementary Deoxyribose Nucleic Acid) nucleotide of monascus ruber GAD (Glutamic Acid Decarboxylase) gene and synthetic method thereof and corresponding protein

A technology of Monascus ruberus and nucleotides, applied in the biological field, can solve the problems of no non-specific amplification and low amplification efficiency, and achieve the effect of broad application prospects and multi-purpose application prospects

Active Publication Date: 2015-07-08
艾吉泰康(嘉兴)生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

First, amplify at a higher temperature. Although the amplification efficiency is low at this time, there is basically no non-specific amplification.

Method used

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  • CDNA (Complementary Deoxyribose Nucleic Acid) nucleotide of monascus ruber GAD (Glutamic Acid Decarboxylase) gene and synthetic method thereof and corresponding protein
  • CDNA (Complementary Deoxyribose Nucleic Acid) nucleotide of monascus ruber GAD (Glutamic Acid Decarboxylase) gene and synthetic method thereof and corresponding protein
  • CDNA (Complementary Deoxyribose Nucleic Acid) nucleotide of monascus ruber GAD (Glutamic Acid Decarboxylase) gene and synthetic method thereof and corresponding protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Monascus ruber total RNA extraction and cDNA synthesis

[0024] 1.1 Media, strains and kits

[0025] Potato dextrose agar medium (PDA): 200g potato; 20g glucose; 20g agar; 1000mL distilled water; pH6.0. For the cultivation of Monascus ruber.

[0026] Monascus ruber (Monascus ruber) is a high-yield γ-aminobutyric acid Monascus ruber MR-5 strain screened by this microorganism (application number: 200810061419.2 application date: 2008-05-09), and its preservation number is: CCTCC NO: M208043 , and the place of preservation is: China Center for Type Culture Collection.

[0027] The fungal RNA extraction kit is E.Z.N.A. of Omega Company. TM Fungal RNA Kit.

[0028] 1.2 Method

[0029] 1.2.1 Total RNA extraction

[0030] ①Take 2-3 flat plates of Monascus red that have been cultured for 15 days, pick mycelia and grind them into powder in a mortar filled with liquid nitrogen.

[0031] ②Take 100mg mycelium powder into a 1.5mL centrifuge tube, add 500μL Buffer R...

Embodiment 2

[0050] Embodiment 2 drop-down PCR and product recovery, purification

[0051] 2.1 Method

[0052] 2.1.1 Landing PCR

[0053] The designed upstream and downstream degenerate primers were used for touchdown PCR amplification.

[0054] The sequences of the upstream and downstream degenerate primers are as follows:

[0055] The upstream primer is: 5'-ATGGTY(C / T)CAY(C / T)CTY(C / T)GCY(C / T)M(A / C)V(G / A / C)R(A / G )GTB(G / T / C)M(A / C)AS(G / C)M(A / C)S(G / C)CGC-3'; the downstream primer is 5'-Y(C / T) TAR(A / G)CAAACN(A / T / C / G)CCR(A / G)TGV(G / A / C)GTCTTR(A / G)CC-3'.

[0056] PCR reaction system (25 μL): 2.5 μL of 10×LA Taq buffer (Mg2+plus), 4 μL of dNTP Mixture (2.5 mM each), 1 μL of upstream and downstream primers, 1 μL of cDNA template, 0.2 μL of TaKaRa LA Taq enzyme, plus sterile double-distilled Make up to 25 μL with water.

[0057] Drop-down PCR program: Annealing temperature dropped from 65°C to 55°C, 1°C drop per cycle, a total of 15 cycles; followed by routine procedures: 94°C pre-denaturatio...

Embodiment 3

[0088] Example 3PCR product cloning and sequencing

[0089] 3.1 Method

[0090] 3.1.1 Ligation of PCR products

[0091] Ligate the recovered and purified target fragment to the vector, and operate according to the instructions of TaKaRa's pMD-18T Vector kit. The ligation system is as follows: pMD-18T Vector 0.5 μL, PCR purified product 5 μL, Solution Ⅰ 4.5 μL. The operation was carried out on ice, centrifuged and mixed, and connected in a water bath at 16°C for more than 3 hours.

[0092] 3.1.2 Conversion of Ligation Products

[0093] ① Add the ligation product to the competent cells pre-placed on ice, mix gently, and place on ice for 30 minutes.

[0094] ②After heat-shocking in a water bath at 42°C for 90s, immediately move it to an ice bath and place it for 3 minutes.

[0095] ③ Add 800 μL LB liquid medium, and incubate for 40 minutes in a shaker at 37°C and 220 rpm / min.

[0096] ④ Centrifuge the bacterial solution at room temperature at 3,000rpm / min for 10min, and disc...

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Abstract

The invention relates to the field of biotechnology, and in particular relates to a cDNA (Complementary Deoxyribose Nucleic Acid) nucleotide of monascus ruber GAD (Glutamic Acid Decarboxylase) gene and a synthetic method thereof, and a corresponding protein. The base sequence of the nucleotide at the coding region of monascus ruber glutamic acid decarboxylase gene is shown as SEQ ID NO: 1. According to the cDNA nucleotide of monascus ruber GAD gene and the synthetic method thereof and the corresponding protein, the complete nucleotide sequence at the cDNA coding region of monascus ruber GAD (Glutamic Acid Decarboxylase) gene is cloned, the nucleotide can be applied to the immune prediction and early diagnosis of Type 1 Diabetes Mellitus, and GABA (Gamma-Aminobutyric Acid) can be prepared by using the biocatalysis of the nucleotide, thus having multiple applications and wide application prospect.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a complete nucleotide sequence of the coding region of Monascus ruber (Monascus ruber) glutamic acid decarboxylase (Glutamate decarboxylase; GAD) gene, its synthesis method and the corresponding protein. Background technique [0002] Glutamate Decarboxylase (Glutamate Decarboxylase, GAD; EC4.1.1.15) catalyzes the α-position decarboxylation of L-glutamic acid to generate the important inhibitory neurotransmitter γ-aminobutyric acid (γ-Aminobutyric Acid, GABA) , GAD is the key rate-limiting enzyme that catalyzes the decarboxylation of glutamate to GABA. Existing studies have shown that: GABA is a physiologically active ingredient that has various physiological functions such as anti-anxiety, anti-convulsion, lowering blood pressure, increasing neurotrophy, improving brain function, promoting long-term memory, promoting secretion of growth hormone, activating kidney function, and liver ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/60C12N9/88C12N15/11C12N15/10C12R1/645
Inventor 蒋冬花齐育平孙蕾陈璨谢祥聪
Owner 艾吉泰康(嘉兴)生物科技有限公司
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