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Porcine diarrhea associated virus tandem epitope protein induced egg yolk antibody and application thereof

A technology of egg yolk antibody and pig diarrhea, applied in the field of egg yolk antibody, to achieve the effect of high antibody titer

Pending Publication Date: 2022-02-11
重庆申金生物工程研究院有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In the prior art, there is no relevant information about the antibody induced by the combination of three pathogenic biological protein epitopes, such as porcine transmissible gastroenteritis virus, porcine epidemic diarrhea virus, and porcine rotavirus, as an effective drug. to report

Method used

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  • Porcine diarrhea associated virus tandem epitope protein induced egg yolk antibody and application thereof
  • Porcine diarrhea associated virus tandem epitope protein induced egg yolk antibody and application thereof
  • Porcine diarrhea associated virus tandem epitope protein induced egg yolk antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Preparation of Egg Yolk Antibodies Induced by Three Inactivated Viruses of TGEV, PEDV and PoRV

[0044] 1.1 Cultivate TGEV, PEDV, and PoRV and inactivate three viruses to prepare antigens

[0045] Three viruses, including TGEV, PEDV, and PoRV, were cultured in VERO cells at 37°C and CO 2 The concentration is 5%, and the calf serum concentration is 1-5%. When the number of cells reaches 1×10 9 / mL, inactivate the virus, dilute to 1×10 with normal saline after inactivation 8 / mL to obtain inactivated TGEV, PEDV, PoRV and other three viral antigens, and mix them in a ratio of 1:1:1.

[0046] 1.2 Immune laying hens and collect eggs

[0047] The initial immunization steps are as follows: prepare antigens from the inactivated TGEV, PEDV, and PoRV viruses, add the same amount of Freund’s complete adjuvant, and immunize hens after fully emulsifying. The optimal solution is to use rod-shaped nano-aluminum hydroxide as an adjuvant. Agent, emulsified according to th...

Embodiment 2

[0065] Example 2 Preparation of Egg Yolk Antibody Induced by Three Kinds of Viral Epitope Proteins of TGEV, PEDV and PoRV

[0066] 2.1 Express the S protein gene of TGEV virus, the S protein gene of PEDV virus, and the VP7 protein gene of PoRV virus in eukaryotic vectors.

[0067] Add the recombinant expression plasmids pET22b-S-S-VP7, pET32a-Cap and pGEX6P-1-S-S-VP7-Cap with correct sequencing and digestion to 100 μL prepared DH5α, TOP10, DH5α(contain vector pTARA), TOP10( contain vector pTARA), BL21, BL21(plus) and Rosetta(DE3) competent cells, mix gently, place on ice for 30min; then heat shock at 42°C for 90s, quickly ice bath for 5min, add 600ul antibiotic-free LB liquid culture base, placed on a shaker at 37°C, 120rpm / min, and incubated for 45min. Centrifuge at 10,000 rpm / min for 2 min, discard 600 μL of the supernatant, resuspend the cells with 100 μL of LB liquid medium, spread LB solid medium containing ampicillin (final concentration: 50 μg / ml), and culture overnigh...

Embodiment 3

[0089] The titer detection of embodiment 3 egg yolk antibody

[0090] Indirect enzyme-linked immunosorbent assay (ELISA) was used to detect the titer, and the antigens of TGEV, PEDV, and PoRV were used to form a mixture in a ratio of 1:1:1: the concentration of the mixture was 10mg / ml, and the coating ELISA plate, 100 μL per well, coated overnight at 4°C, washed 5 times with PBST, then added blocking solution containing 1% BSA, blocked at 37°C for 4h. After washing 5 times, add the egg yolk antibody induced by the "multi-linked multivalent whole antigen" (Example 1) of three viruses such as TGEV, PEDV, and PoRV at multiple dilutions, and add TGEV, PEDV, and PoRV with multiple dilutions to the other group Etc. recombinant protein (S-S-VP7) "multi-link multivalent epitope antigen" (Example 2). A blank control and a negative control were set at the same time, and the negative control group used PBS solution; the blank control group used egg yolk liquid, and incubated at 37°C for...

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Abstract

The invention belongs to the field of biological pharmacy, and relates to a preparation method and application of a porcine diarrhea associated virus tandem epitope protein induced antibody. The egg yolk antibody is obtained by immunizing laying hens with a'multi-union multivalent epitope antigen 'formed by combining main protein epitopes of inactivated TGEV, PEDV and PoRV, collecting eggs of the immunized laying hens, and separating and purifying from egg yolk. The titer of the egg yolk antibody is superior to that of an antibody induced by a single virus antigen, and the egg yolk antibody can be used for preventing and treating diarrhea caused by infection of TGEV, PEDV and PoRV viruses of pigs. Eggs with antibodies are shelled and then prepared into a pH-sensitive coating material preparation, the pH-sensitive coating material preparation is used for resisting pig gastric acid and pepsin damage and is further released in intestinal tracts to neutralize enteroviruses, the pH-sensitive coating material preparation can be used as a pig feed additive, and the antibodies can be purified and then prepared into an oral medicine of the pH-sensitive coating material, so that pig gastric acid damage can be resisted, and thepreparation is released in the intestinal tract to neutralize enteroviruses.

Description

technical field [0001] The invention belongs to the technical field of biopharmaceuticals, in particular to egg yolk antibodies induced by porcine diarrhea-associated virus tandem epitope proteins and applications thereof. Background technique [0002] Porcine transmissible gastroenteritis virus (TGEV) causes infectious diseases of the digestive tract of pigs. The onset is frequent in seasons with low temperatures, and it is transmitted through food, water, feces, respiratory tract and secretions. The main symptoms are dehydration, vomiting, and diarrhea. The morbidity and mortality of TGEV are very high, and the symptoms of different ages are different. The dehydration symptoms of piglets are more serious, and long-term dehydration will lead to death. Adult pigs have abdominal distension, lack of appetite, watery diarrhea, etc., and indigestion. The phenomenon that there are feed residues in the feces. [0003] Porcine epidemic diarrhea virus (PEDV) infects pigs of all ag...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10C07K16/02C07K19/00C07K1/36C07K1/34C07K1/30C12N15/85A61K39/42A61P31/14A23K50/30A23K20/147A23K20/163A23K20/24A23K10/20
CPCC07K16/10C07K16/02C07K14/005C12N15/85A61P31/14A23K50/30A23K20/147A23K20/163A23K20/24A23K10/20C07K2317/11C07K2317/76C07K2317/94C07K2319/00C12N2770/20022C12N2720/12322C12N2800/107A61K2039/505Y02P60/87
Inventor 吕凤林陈彩宇罗彦凤
Owner 重庆申金生物工程研究院有限公司
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