Method for extracting and purifying plague bacillus profibrinolysin activating factor Pla

A technology of plasminogen and activator, applied in biochemical equipment and methods, hydrolytic enzymes, microorganisms, etc., can solve problems such as unproven, target protein flow-through, and protein quality impact.

Inactive Publication Date: 2013-04-03
云南省地方病防治所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, when using urea extraction to extract Pla protein, it is difficult to remove urea by dialysis, and its existence will affect the subsequent extraction and purification; the proportion of the target protein in the crude extract obtained by this method is not high enough, This makes the less expressed Pla protein difficult to obtain a sufficient amount of harvest after purification
[0006] There are following deficiencies in the purification of Pla protein by preparative electrophoresis: 1) the method obtains data through pre-experiment, and adopts the method of distance measurement to locate the gel cutting position, which has errors; 2) in the process of preparative electrophoresis and eluting protein, surfactant , reducing agent, heat treatment, electric field and other conditions have a great impact on the protein quality; 3) The background of the position of the target protein obtained by this method is relatively dark, indicating that there are some other proteins; The gel is then eluted to obtain the target protein, the recovery rate is low, it is difficult to meet the needs, and the operation is cumbersome
[0007] The separation and purification of Pla protein by high performance liquid chromatography has the following disadvantages: using ion exchange purification, most of the target protein flows through, and only a small part remains; if combined with gel filtration for two-step chromatography, the purification of Pla can be achieved, However, the yield is extremely low, concentration is required to detect protein content, and the operation is cumbersome
Simultaneously, although three bands can account for 80% of protein total amount, only illustrate that it is Pla from relative molecular mass and enzymatic activity, have not been further confirmed (the inventor of this case once carried out mass spectrometric analysis confirmation to the protein purified by this method in research, These 3 bands are not all Pla protein)

Method used

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  • Method for extracting and purifying plague bacillus profibrinolysin activating factor Pla
  • Method for extracting and purifying plague bacillus profibrinolysin activating factor Pla
  • Method for extracting and purifying plague bacillus profibrinolysin activating factor Pla

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Embodiment 1

[0095] Extraction, purification and identification of embodiment 1, Pla protein

[0096] 1 Preparation of Yersinia pestis EV76 bacterial powder

[0097]The plague EV76 bacteria subcultured on LB plates were inoculated densely in the eggplant-shaped flasks prepared with nutrient agar, incubated at 28°C for 48 hours, washed with PBS (0.01M, pH7.2) and centrifuged at 7000r / min for 20min Wash twice, add 4 times the volume of acetone pre-cooled at -40°C to the precipitate, mix well, place at -40°C overnight, centrifuge at 4000r / min for 10min, discard the supernatant, and treat the bacteria in the same way with pre-cooled acetone 3 times, discard the acetone, and make bacterial powder in a 37°C incubator overnight.

[0098] 2 Preliminary extraction of Pla protein from Yersinia pestis

[0099] 1) Weigh 1.5g of EV76 bacterial powder, add 60ml of PBS (0.01M, pH7.2) and 3mg of lysozyme, at 4°C, stir magnetically (120 rpm) for 3-4 hours, and ultrasonically break the bacterial suspensio...

Embodiment 2

[0168] Embodiment 2, the application example of Pla protein in hybridoma strain establishment and screening

[0169] The recombinant Pla protein (rPla) that is cloned and expressed is used to immunize animals, the hybridoma technology is used to prepare cell lines, and the hybridoma cell strains that can secrete monoclonal antibodies against Yersinia pestis Pla antigen are screened. In the screening process of tumor strains, the present invention is applied Example 1 extracted and purified natural Pla protein (nPla) as an antigen, and screened 3 hybridoma cell lines capable of recognizing the natural antigen, laying the foundation for establishing plague diagnostic technology and carrying out related research.

[0170] (1) Screening of hybridoma strains by indirect ELISA:

[0171] Using rPla, nPla, and rGST as coating antigens, detect the OD value of the fusion cell supernatant. As determined by the checkerboard, the coating concentrations of rPla, nPla, and rGST were 10 μg / m...

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Abstract

The invention relates to a method for extracting and purifying a plague bacillus profibrinolysin activating factor Pla, which comprises the following steps: treating plague bacillus with lysozyme, and carrying out ultrasonication and ammonium sulfate salting-out to obtain a crude Pla protein extract; and purifying by carrying out chromatography on the crude Pla protein extract with hydroxyapatitechromatographic columns, including: adding Ca+ into a loading buffer, carrying out gradient elution with a phosphate buffer to remove non-target proteins, eluting the target protein with 1N NaOH, collecting the sample, dialyzing with a Tris-Cl buffer to remove NaOH so as to obtain the purified Pla protein, and verifying by mass spectrometric analysis. The invention also provides a Pla protein product which is extracted and purified according to the method. The method provided by the invention is easy to operate; and the obtained protein has the advantages of high abundance and high purity.

Description

technical field [0001] The invention relates to a method for extracting and purifying Pla, the plasminogen activator of Yersinia pestis, specifically, treating Yersinia pestis with lysozyme to prepare crudely extracted Pla protein, and then using a hydroxyapatite chromatography column for chromatographic purification The method for preparing and purifying the Pla protein, the invention also relates to the Pla protein product extracted and purified by the method and its application in the establishment and screening of hybridoma strains. Background technique [0002] Yersinia pestis plasminogen activator (Plasminogen activator, Pla) is a cell membrane surface protease encoded by the unique pPCP1 plasmid of Yersinia pestis. Studies have shown that the Pla protein of Yersinia pestis is the key factor for the spread of Yersinia pestis from subcutaneous infection to the circulatory system. An important virulence factor (Sodeinde OA, Subrahmanyam YV, Stark K, et al.A surface prote...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/52C12N5/20
Inventor 宋志忠杜春红石丽媛王鹏杨光璨钟佑宏董珊珊
Owner 云南省地方病防治所
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