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Purification method of immunodominant membrane protein of recombinant phytoplasma and application thereof

A purification method and membrane protein technology are applied in the field of purification of recombinant phytoplasma immunity-dominated membrane proteins, which can solve the problems of difficulty in expression, hydrophobicity of membrane proteins, and difficulty in separating and purifying membrane proteins by solubility.

Inactive Publication Date: 2014-12-24
深圳市兰科植物保护研究中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, due to the specificity of different phytoplasma membrane proteins, some membrane proteins can be completely expressed in the E. coli system, while others are difficult to express and easily form inclusion bodies. On the other hand, even if the membrane proteins are expressed in the E. Due to the hydrophobicity, solubility and other properties of membrane proteins, it is difficult for subsequent downstream separation and purification of membrane proteins

Method used

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  • Purification method of immunodominant membrane protein of recombinant phytoplasma and application thereof
  • Purification method of immunodominant membrane protein of recombinant phytoplasma and application thereof
  • Purification method of immunodominant membrane protein of recombinant phytoplasma and application thereof

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Embodiment 1

[0066] For purification methods of recombinant phytoplasma immunodominant membrane proteins, see figure 1 shown, including the following steps:

[0067] S11, obtaining a recombinant bacterium expressing a phytoplasma immune-dominant membrane protein, the recombinant bacterium contains a phytoplasma immune-dominant membrane protein gene with a 6×His Tag sequence, and induces expression;

[0068] S12, freeze and thaw 1 g of the recombinant bacteria in step S11 twice at -20°C, add 5ml of BugBuster Master Mix extraction reagent, resuspend at 25°C, shake and incubate for 30 minutes, and obtain the above Supernatant one and sediment one;

[0069] S13, weigh 1ml of 50% Ni-NTA-His.Bind resin suspension and mix with 4ml of 1×Ni-NTA binding buffer, the 1×Ni-NTA binding buffer does not contain imidazole, and then mix it with 4ml of Once the supernatant was mixed, the pH value was 7, shaken at 4°C for 1 hour, and then allowed to settle; then rinsed with 1×Ni-NTA washing buffer, and elut...

Embodiment 2

[0073] For purification methods of recombinant phytoplasma immunodominant membrane proteins, see figure 1 shown, including the following steps:

[0074] S21, obtaining a recombinant bacterium expressing a phytoplasma immune-dominant membrane protein, the recombinant bacterium contains a phytoplasma immune-dominant membrane protein gene with a 6×His Tag sequence, and induces expression;

[0075] S22, freeze and thaw 1 g of the recombinant bacteria in step S11 three times at -20°C, add 5ml of BugBuster Master Mix extraction reagent, resuspend at 25°C, shake and incubate for 30 minutes, and obtain the above Supernatant one and sediment one;

[0076] S23, weigh 1ml of 50% Ni-NTA-His.Bind resin suspension and mix with 4ml of 1×Ni-NTA binding buffer, the 1×Ni-NTA binding buffer does not contain imidazole, and then mix it with 4ml of the Once the supernatant was mixed, the pH value was 7, shaken at 4°C for 1 hour, and then allowed to settle; then rinsed with 1×Ni-NTA washing buffer...

Embodiment 3

[0080] For purification methods of recombinant phytoplasma immunodominant membrane proteins, see figure 1 shown, including the following steps:

[0081] S31, obtaining a recombinant bacterium expressing a phytoplasma immune-dominant membrane protein, the recombinant bacterium contains a phytoplasma immune-dominant membrane protein gene with a 6×His Tag sequence, and induces expression;

[0082] S32, freeze and thaw 1 g of the recombinant bacteria in step S11 twice at -20°C, add 5ml of BugBuster Master Mix extraction reagent, resuspend at 25°C, shake and incubate for 30 minutes, and obtain the above Supernatant one and sediment one;

[0083] S33, weigh 1ml of 50% Ni-NTA-His.Bind resin suspension and mix with 4ml of 1×Ni-NTA binding buffer, the 1×Ni-NTA binding buffer does not contain imidazole, and then mix with 4ml of Once the supernatant was mixed, the pH value was 7, shaken at 4°C for 1 hour, and then allowed to settle; then rinsed with 1×Ni-NTA washing buffer, and eluted ...

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Abstract

The invention provides a purification method of an immunodominant membrane protein of recombinant phytoplasma and an application thereof. According to the purification method of the immune leading membrane protein of recombinant phytoplasma provided by the invention, the soluble immunodominant membrane protein of recombinant phytoplasma expressed by recombinant bacteria is purified by using affinity chromatography, particularly an inclusion body is split, insoluble immunodominant membrane protein of recombinant phytoplasma in the inclusion body is purified by a dialysis method and affinity chromatography, and the purification conditions and process parameters are optimized, so that the purification effect of the immunodominant membrane protein of recombinant phytoplasma is greatly improved. The purification degree can reach 95% while the purification degree in the conventional method is 60%. The method is simple in process, easy to operate and high in yield, and the production cost is lowered.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for purifying a recombinant phytoplasma immunodominant membrane protein and an application thereof. Background technique [0002] Phytoplasma (phytoplasma) is a kind of prokaryotic microorganism with relatively low GC content, no cell wall, and only three-layer unit membrane coating, obligately parasitic on plants and insects. The main symptoms of plant disease include arbuscules, yellowing, flower change and leaf degradation, etc. Due to the strong infectivity of phytoplasma and the extensive damage to hosts, it has caused a large number of economic crops and trees to die, causing serious economic losses. Because phytoplasmas are prokaryotic microorganisms without cell walls, they cannot be cultured in vitro on artificial medium, which brings many difficulties to the research of classification and identification, transmission mechanism and pathogenic mechanism. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/02C07K1/22C07K1/14C12R1/19
Inventor 刘仲健李利强肖新菊张丽君罗焕亮梁楠楠
Owner 深圳市兰科植物保护研究中心
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