A kind of duck virus hepatitis egg yolk antibody with anti-inflammatory effect and its preparation method and application
A yolk antibody and positive technology, which is applied in the field of poultry disease immune control, can solve the problems of rapid death, failure to eliminate inflammation, failure of duck viral hepatitis, etc., and achieve the effect of low cost, convenient operation and high-efficiency expression
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Embodiment 1
[0100] Example 1 Construction of recombinant plasmid pPIC9-VP1-IL6
[0101] Include the following steps:
[0102] (1) Determine the enzyme cleavage site
[0103] According to the sequencing results of the VP1 gene sequence (SEQ ID NO: 1) of the standard strain of duck hepatitis virus type I from NCBI and the duck IL-6 gene sequence (SEQ ID NO: 3) obtained by PCR amplification, and the pPIC9 plasmid map The information was compared, and the double enzyme cleavage sites Xho I and Not I were determined.
[0104] (2) Construction of recombinant plasmid pPIC9-VP1-IL6
[0105] ① PCR amplification of duck hepatitis virus type I VP1 gene:
[0106] The following primers were designed according to the type I duck hepatitis virus VP1 published by GenBank:
[0107] Primer 1:5'-GGTGATTCTAACCAGTTAGG-3', as shown in SEQ ID NO: 6 of the sequence listing;
[0108] Primer 2:5'-TTCAATTTCCAGATTGAGTT-3', as shown in SEQ ID NO:7 of the Sequence Listing.
[0109] Extract the RNA of the standar...
Embodiment 2
[0165] Example 2 Expression and purification of pPIC9-VP1-IL6 plasmid Pichia pastoris
[0166] (1) Expression and identification of VP1-IL6 fusion protein in Pichia pastoris
[0167] ①The constructed recombinant plasmid was linearized with Sal I endonuclease and recovered by gel. At the same time, the empty vector was linearized with the same enzyme and recovered by gel. After dephosphorylation, phenol / chloroform was extracted and recovered, and dissolved in TE buffer. The recombinant plasmid was transformed into Pichia pastoris GS115 competent cells by conventional LiCl method, coated on RDB plate, and positive transformants were selected.
[0168] The enzyme digestion reaction system is as follows: 1.0 μg of pPIC9-VP1-IL6 recombinant plasmid, 1.0 μL of Sal I endonuclease, 2.5 μL of 10×buffer, and sterilized ultrapure water to make up to 20 μL. 2h in a water bath at 37°C;
[0169] ②Identification of positive transformants: Extract the genome of the transformants, and use th...
Embodiment 3
[0185] Example 3 Preparation of anti-VP1-IL6 egg yolk antibody
[0186] (1) Preparation of vaccine: Induce the expression of pPIC9-VP1-IL6 recombinant Pichia pastoris in large quantities, and harvest the medium supernatant after culturing for 96 hours; measure the VP1-IL6 protein concentration with a spectrophotometer, and store at -20°C; use sterile water Adjust the final protein concentration of VP1-IL6 to 100 μg / mL, add 5.0 μg / mL LPS to the solution as a polyclonal antibody activator, use the above prepared protein solution as the water phase, and an equal volume of white oil adjuvant. Mix, and then fully emulsify with a homogenizer for 20 minutes to prepare a vaccine;
[0187] (2) Use the prepared vaccine to immunize the laying hens about 1 month before the start of production in 4 times, with an interval of 2 weeks for each immunization; The secondary immunization dose is 1.0-2.0mL / only;
[0188] (3) The immunized eggs were collected two weeks after the fourth immuniz...
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