Establishment of hybridoma cell strain for secreting duck NDV (newcastle disease virus)-resisting isolate monoclonal antibody
A hybridoma cell line and monoclonal antibody technology, applied in the fields of molecular immunology and virology, can solve tedious and time-consuming problems
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Embodiment 1
[0031] Cloning and sequencing of NDV-HN gene
[0032] Referring to the HN gene sequence of NDV duck-derived isolate SD03, design and synthesize primers for HN gene expression. The forward primer is 5′-CCGGAATTCATACCCTCATTTGACATGAG-3′, as shown in SEQ ID NO.2, which contains the EcoRI restriction site, and the reverse primer is 5'- CCCAAGCTTTTAAACTCTATTCATCCTTGAGG-3' as shown in SEQ ID NO.3, containing Hind Ⅲ Restriction sites. Use the above primers to PCR amplify nucleotides 523-1716 in the open reading frame of the HN gene of SD03, purify and recover the PCR product with a DNA purification kit, insert it into the pET28(a) vector, and obtain the pET28(a)-HN recombinant plasmid. Transform the recombinant plasmid into CaCl 2 Competent cells BL21 prepared by the enzyme digestion method identified positive clones, and the positive clones were sent to a sequencing company for sequencing. The gene sequence of the NDV-HN gene is shown in SEQ ID NO.1.
[0033] The PCR amplificat...
Embodiment 2
[0035] Expression and purification of His-HN protein
[0036]1) Inoculate the BL21 bacteria containing the recombinant plasmid in LB solid medium containing Kanamycin (Kan), culture at 37°C, pick a single colony and inoculate it in 500ml LB liquid medium containing Kan (final concentration: 50μg / ml) medium, shake (200rpm) at 37°C, culture until OD600=0.6, add IPTG to a final concentration of 0.6mmol / L, continue to culture at 37°C for 6h, and induce the expression of HN protein in large quantities.
[0037] 2) Centrifuge 12,000g of the above bacterial solution at 4°C for 20min, discard the supernatant, and resuspend in 15ml of PBS. Ultrasonic disruption of bacteria.
[0038] 3) Centrifuge at 12,000g for 20min at 4°C to collect the precipitate.
[0039] 4) Fully wash the precipitate obtained in the previous step with Washimg buffer, centrifuge at 12,000g at 4°C for 20min, discard the supernatant, and remove bacterial debris as much as possible.
[0040] 5) Sufficiently susp...
Embodiment 3
[0044] animal immunity
[0045] The above-mentioned purified His-HN fusion protein was used to immunize 6-week-old Balb / C female mice, each time the immunization dose was 50ug, and the immunization method was intraperitoneal injection, and immunized four times in total. The interval between each immunization was 2 weeks. The protein was emulsified with Freund's complete adjuvant at a volume ratio of 1:1 for the first immunization, and the protein was emulsified with Freund's incomplete adjuvant for the second to fourth immunization at a volume ratio of 1:1. One week after the three times of immunization, blood was collected from the orbit of the mice to measure its titer, and the serum antibody titer was monitored. Two weeks after the fourth immunization, the mouse with the highest titer was selected and immunized once by intraperitoneal injection of protein without adjuvant. After 3 days, the spleen lymphocytes of the mice were fused with SP2 / 0 cells.
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