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.
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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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