Pichia pastoris gene engineering bacteria for recombinant expression of human glutamic acid decarboxylase

A technology of glutamic acid decarboxylase and genetically engineered bacteria, applied in the field of genetic engineering, can solve the problems of formation of inclusion bodies, unstable expression of plasmids, and low enzyme activity

Inactive Publication Date: 2016-03-16
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] At present, although there have been a few reports that GAD of mammalian origin 65 Successfully expressed in microorganisms, but there a...

Method used

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  • Pichia pastoris gene engineering bacteria for recombinant expression of human glutamic acid decarboxylase
  • Pichia pastoris gene engineering bacteria for recombinant expression of human glutamic acid decarboxylase
  • Pichia pastoris gene engineering bacteria for recombinant expression of human glutamic acid decarboxylase

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Experimental program
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Embodiment 1

[0026] Embodiment 1: Construction of recombinant bacteria

[0027] (1) Recombinant plasmid pMD19-T-GAD 65 build

[0028] The GAD enzyme gene with nucleotides such as SEQIDNO:1 is connected to the pMD19-T plasmid, and transformed into Escherichia coli, and the correct transformant is verified to obtain the recombinant plasmid pMD19-T-GAD 65 . Extract pMD19-T-GAD 65 and pPICZα plasmids, and then double-enzyme pMD19-T-GAD with XhoI and NotI respectively 65 (Synthetic GAD 65 The gene was cloned into pMD19-T vector) and pPICZα plasmid for 4h, and analyzed by 0.7% agarose gel electrophoresis. Use the rubber tapping recovery kit to recover the target fragment, connect at 16°C for 4 hours, use the chemical transformation method to transform Escherichia coli DH5α, spread the transformed bacterial solution on a low-salt LB plate with a final concentration of 25 μg / mL zeocin, and store at 37°C After culturing for 24 hours, select the colonies grown on several plates, inoculate them...

Embodiment 2

[0045] Embodiment 2: Recombinant bacterial fermentation produces glutamic acid decarboxylase GAD 65

[0046] fusion protein hGAD 65 Induced expression and product identification

[0047] (1) Induced expression

[0048] Extraction and sequencing of the correct recombinant plasmid pPICZα-hGAD 65 Transform into Pichia pastoris X-33 to obtain gene engineering expression bacteria.

[0049] (a) Inoculate a single colony of the selected recombinant Pichia pastoris in a 50mL growth medium BMGY / 500mL Erlenmeyer flask, and culture at 30°C and 200r / min for 20h;

[0050] (b) Leave the bacterial solution in the above-mentioned BMGY growth medium at room temperature for 1 h, discard the supernatant, resuspend the bacterial cells with 30 mL of BMMY medium, add 100% methanol to a final concentration of 1% (v / v), Cultivate at 30°C, 200r / min, add 100% methanol every 24h to a final concentration of 1% (v / v) for induction;

[0051] (c) After methanol induction for 48h, centrifuge at 12000r / ...

Embodiment 3

[0087] Embodiment 3: the glutamic acid decarboxylase GAD of recombinant bacterial fermentation 65 activity detection

[0088] Identification of fusion protein hGAD by WesternBlot 65 .

[0089] (1) Use the serum of diabetic patients positive for GAD antibody as the primary antibody:

[0090]①Tricine-SDS-PAGE: Take 200 μl sink sample of shake flask horizontal protein sample for Tricine-SDS-PAGE;

[0091] ②Transfer: Take a nitrocellulose filter membrane (NC) with the same size as the gel and soak six pieces of 3mm filter paper in the electrotransfer buffer for 10-15min. After electrophoresis, remove the gel, cut off the stacking gel for marking, rinse with electrotransfer buffer and equilibrate for 20 minutes. Install in the following order: anode - sponge - 3 pieces of filter paper - NC membrane - gel - 3 pieces of filter paper - sponge - cathode. Every time a layer is placed, the air bubbles between the layers should be removed, so as not to affect the transfer effect. Cl...

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Abstract

The invention discloses pichia pastoris gene engineering bacteria for recombinant expression of human glutamic acid decarboxylase and belongs to the technical field of gene engineering. A human glutamic acid decarboxylase gene GAD65 and a vector pPICZalpha are connected to establish an expression vector pPICZalpha-hGAD65 which is placed in pichia pastoris X-33 in an electricity facing mode, and a recombinant strain for excessively expressing human glutamic acid decarboxylase (GAD65) is obtained. Through shake flask fermentation, Tricine-SDS-PAGE and Western Blot identification on the recombinant strain, it is proved that human glutamic acid decarboxylase (GAD65) achieves inducible expression, fusion protein is obtained after ferementaiton supernate is separated and purified, and the concentration of protein hGAD65 reaches 2.36 g/L through measurement on the protein concentration.

Description

technical field [0001] The invention relates to a Pichia pastoris genetic engineering bacterium for recombinantly expressing human glutamic acid decarboxylase, which belongs to the field of genetic engineering. Background technique [0002] Glutamic acid decarboxylase (glutamic acid decarboxylase, GAD, EC4.1.1.15) exists widely in organisms, which catalyzes glutamic acid to generate γ-aminobutyric acid (γ-aminobutyric acid, GABA), and GABA is an important inhibitory neurotransmitters. In the study of autoimmune diseases and diabetes, especially type I diabetes, the levels of GAD, GABA and glutamic acid decarboxylase antibody (GAD-Ab) are used as important parameters for pathological analysis, disease diagnosis and immunotherapy , has always attracted the attention of researchers. [0003] GAD exists in two isomeric forms: GAD with a molecular weight of 65,000 65 and GAD with a molecular weight of 67,000 67 , both of which are encoded by non-allelic genes on different chr...

Claims

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Application Information

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IPC IPC(8): C12N15/81C12N1/19C12N9/88A61K38/51A61P3/10C12R1/84
CPCC12N9/88A61K38/00C12Y401/01015
Inventor 刘立明罗秋玲刘佳
Owner JIANGNAN UNIV
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