Pigeon circovirus-like particle, its preparation method and application
A pigeon circovirus and particle technology, which is applied in the fields of immunology and genetic engineering, can solve problems such as no reports on pigeon circo vaccine research, and achieve the effect of low cost and high safety.
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Embodiment 1
[0041] Example 1 Construction of recombinant transfer vector
[0042] 1. Acquisition of pigeon ring structure gene
[0043] The nucleotide sequence of pigeon circovirus ORF C1 gene was obtained from GenBank (accession number DQ090945), the nucleotide sequence was synthesized by Shanghai Sangong, the length was 822bp, and the target sequence was connected to the PUC57 vector (purchased from Thermo FisherScientific Company) , named PUC57-ORF C1.
[0044] The original nucleotide sequence of the pigeon circovirus ORF C1 gene is shown in SEQ ID NO:1, and the amino acid sequence encoded by it is shown in SEQ ID NO:2.
[0045] 2. Digestion, ligation, transformation and identification
[0046] The pFastBac1 vector (purchased from Invitrogen) and the PUC57-ORF C1 plasmid were subjected to enzyme digestion treatment, and the enzyme digestion system was 50 μl, and the composition was as follows:
[0047] DNA 10 μl, restriction enzyme BamHI / restriction enzyme NotI 2 μl each, 10×M buffe...
Embodiment 2
[0051] Example 2 Construction of recombinant baculovirus
[0052] 1. Construction of recombinant baculovirus genome
[0053] Transform the pFastBac1-ORF C1 transfer vector into E.coli DH10Bac competent cells (containing the Autographa californica nuclear polyhedrosis baculovirus AcMNPV genome, Invitrogen Cat NO.10359-016), and the transformation conditions are as follows:
[0054] Mix 2μl of pFastBac1-ORF C1 with E.coli DH10Bac, put it in ice bath for 35min, heat stress at 42°C for 50s, then put it in ice bath for 3min, then add 1ml of non-resistant LB medium, incubate at 37°C, 200rpm After 4 to 5 hours of under-cultivation, the bacterial solution was carried out for 10 -1 、10 -2 、10 -3 Doubly serially dilute, take 100 μl from each dilution and smear it on the culture plate containing three-antibody-resistant LB bacteria (containing 50 μg / ml kanamycin, 7 μg / ml gentamicin, 10 μg / ml tetracycline , concentration 40μg / mlX-gal), after culturing at 37°C for 48 hours, the blue an...
Embodiment 3
[0057] Example 3 Rescue of recombinant baculovirus
[0058] The extracted recombinant baculovirus genomic DNA was transfected into insect cell Sf21, and the transfection process was as follows:
[0059] (1) Add 4 μg of recombinant DNA to 250 μl of serum- and double-antibody-free Grace medium (purchased from Invitrogen), mix well, and let stand at room temperature for 5 minutes;
[0060] (2) Add 6 μl liposome Lipfectin 2000 (purchased from Invitrogen) to 250 μl Grace medium without serum and double antibody, mix well, and let stand at room temperature for 5 minutes;
[0061] (3) Mix equal volumes of the above (1) and (2) solutions, and let stand at room temperature for 20 minutes;
[0062] (4) During the stationary period of the above (3), the Sf21 cell line of Spodoptera frugiperda (Spodoptera frugiperda) cultured in a 6-well plate (covering 70-80% of the bottom area of the well, purchased from Invitrogen) was treated with serum-free and Gently wash the double-antibody Gra...
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