Monoclonal antibody capable of recognizing natural-structure sika prion and preparation method
A monoclonal antibody and sika deer technology, applied in botany equipment and methods, microorganism-based methods, biochemical equipment and methods, etc., can solve problems such as food safety threats and ineffective inactivation of prions, and achieve strong specificity , the effect of accurate experimental results
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Embodiment 1
[0036] Example 1 Recombinant prion protein PrP res preparation of
[0037] (1) Preparation of recombinant prion protein PrP res Gene synthetic primers are:
[0038] FW: GGG AAT TCC ATA TGA AGA AGC GAC CAA AAC CTG
[0039] RV: CGGATC CGA ACT TGC CCC TCG TTG GTA;
[0040](2) Extract the total genomic DNA of sika deer as a template, and carry out PCR amplification. The amplification conditions are: pre-denaturation at 95°C for 2 minutes, denaturation at 93°C for 50 seconds, annealing at 56°C for 45 seconds, extension at 72°C for 1 minute, 30 cycles, Finally, extend at 72°C for 10 minutes to obtain the target gene, clone it into the PMD-18T-Simple vector, and identify it by enzyme digestion. The nucleotide sequence of the target gene is shown in SEQ ID 1. PrP res Insert the gene fragment into the expression vector pET-TRX and PET-HIS to construct the recombinant expression plasmid, pET-TRX-PrP res , pET-HIS-PrP res ;
[0041] (3) The recombinant expression plasmid pET-Trs...
Embodiment 2
[0042] Example 2 Recombinant prion protein PrP res Bulk preparation and cut gel recovery
[0043] The expression condition is that the positive clone culture is inoculated in LB medium (50mg / L antibiotic) at 1%. When cultured with vigorous shaking at 37°C until OD600=0.6, IPTG with a final concentration of 1 mmol / L was added for induction, and cultured with vigorous shaking at 220 r / min for 4 hours. The bacteria were collected by centrifugation, re-suspended in PBS and lysed by ultrasonic waves, and the supernatant and precipitate were collected by centrifugation, and the whole bacteria, supernatant and precipitate were detected by SDS-PAGE, and PrP was found res -Trx and PrP res -His were abundantly expressed in the form of inclusion bodies, respectively.
[0044] For the gel-cutting purification method, take 500 μL of the inclusion body extract solution and load it directly without inserting a sample comb, and perform SDS-PAGE according to the conventional method. The ge...
Embodiment 3
[0045] Example 3 Animal Immunization
[0046] renatured recombinant prion protein PrP res -Trx immunization of 8-week-old Balb / c female mice, 100 μg / mouse, subcutaneous injection, the first immunization with Freund's complete adjuvant (1:1) to emulsify the antigen, after an interval of 3 weeks, use Freund's incomplete adjuvant (1:1) emulsified antigen, for the second and third immunizations, and for the final booster immunization, the immunogen was directly injected intraperitoneally.
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