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A kind of purification method of igy

A purification method and high-purity technology, applied in the purification field of affinity purification of high-purity IgY, can solve problems such as unfavorable IgY, complex process, backward purification process, etc., and achieve the effect of simple steps, high purity, and simple purification steps

Active Publication Date: 2020-10-13
XINJIANG ACADEMY OF AGRI & RECLAMATION SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Egg yolk antibody has many advantages, but because its purification process is relatively backward, there are many steps in the purification method, and the process is complicated, which is not conducive to the rapid and large amount of high-purity IgY, which has also become a limiting factor in the practical application of IgY

Method used

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  • A kind of purification method of igy
  • A kind of purification method of igy
  • A kind of purification method of igy

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1: This embodiment discloses a method for purifying IgY, and its specific steps are as follows:

[0035] ①. Use IgY as an antigen to immunize Bactrian camels. After the immunization, obtain anti-IgY nanobodies through phage display and bio-affinity panning, and purify the nanobodies to a concentration of 0.5 mg / mL or more;

[0036] ② Take 4g of 4FF gel filler, wash it with DMSO solution with a volume ratio of 20%, 50%, and 70% for 3 minutes, that is, dimethyl sulfoxide solution, then wash the filler with distilled water, and then use a vacuum filtration device to remove Drain the water for 6 minutes;

[0037] ③. Add 3 mL of DMSO with a volume ratio of 70% and 2 mL of NaBH with a concentration of 2 mg / mL to the filler treated in step ②. 4 , 5mL of NaOH with a concentration of 0.8mol / L and 1.5mL of epichlorohydrin, then reacted in a constant temperature shaker for 2h, the reaction temperature was 35°C, and the rotation speed was 150rpm, and washed with distill...

Embodiment 2

[0044] Embodiment 2: This embodiment discloses a method for purifying IgY, and its specific steps are as follows:

[0045] ①. Use IgY as antigen to immunize Bactrian camels. After immunization, obtain anti-IgY nanobodies through phage display and bio-affinity panning, and purify the nanobodies to a concentration of 0.5 mg / mL or more;

[0046] ② Take 2g of 4FF gel filler, wash it with 20%, 50%, and 70% DMSO solution for 10 minutes in turn, that is, dimethyl sulfoxide solution, then wash the filler with distilled water, and then use a vacuum filtration device to remove Drain the water for 8 minutes;

[0047] ③. Add 2 mL of DMSO with a volume ratio of 70% and 4 mL of NaBH with a concentration of 2 mg / mL to the filler treated in step ②. 4 , 3mL of NaOH with a concentration of 0.8mol / L and 1.0mL of epichlorohydrin, and then reacted in a constant temperature shaker for 2h, the reaction temperature was 40°C, and the rotation speed was 170rpm. After the reaction, it was washed with d...

Embodiment 3

[0054] Embodiment 3: This embodiment discloses a method for purifying IgY, and its specific steps are as follows:

[0055] ①. Use IgY as an antigen to immunize Bactrian camels. After the immunization, obtain anti-IgY nanobodies through phage display and bio-affinity panning, and purify the nanobodies to a concentration of 0.5 mg / mL or more;

[0056] ② Take 3g of 4FF gel filler, wash it with DMSO solution with a volume ratio of 20%, 50%, and 70% for 7 minutes, that is, dimethyl sulfoxide solution, then wash the filler with distilled water, and then use a vacuum filtration device to remove Drain the water for 5 minutes;

[0057] ③. Add 2 mL of DMSO with a volume ratio of 70% and 3 mL of NaBH with a concentration of 2 mg / mL to the filler treated in step ②. 4 , 5mL of NaOH with a concentration of 0.8mol / L and 1.8mL of epichlorohydrin, and then reacted in a constant temperature shaker for 3h, the reaction temperature was 40°C, and the rotation speed was 160rpm. After the reaction ...

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Abstract

An IgY purification method comprises following steps: obtaining an anti-IgY nano antibody by subjecting an IgY immunized two-humped camel to phage display and biocompatibility screening; fetching 2 to4 grams of 4FF gel filler, washing 4FF gel filler by DMSO solutions with volume ratios of 20%, 50%, and 70% in sequence, then washing 4FF gel filler by water, drying; then adding DMSO, NaBH4, NaOH, and epoxy chloropropane in sequence to obtain a mixed filler; coupling the nano antibody and a Na2CO3 solution with the mixed filler; after coupling, adding glycine, using a NaCl solution to wash a column to obtain an affinity column that is coupled with the nano antibody, wherein the volume ratio of the NaCl solution to the column is 10-20:1; adding an IgY sample, which needs to be purified, intothe nano antibody affinity column to carry out coupling, after coupling, washing the column by PBS; finally washing IgY from the column by using glycinate, and then neutralizing separated IgY by glycinate (pH=8) so as to avoid the situation that IgY is inactivated under an acidic condition. The provided method can obtain high purity IgY.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to a purification method for affinity purification of high-purity IgY by using a functional nanometer fragment. Background technique [0002] IgY is ubiquitous in birds, reptiles, and amphibians and is functionally equivalent to mammalian IgG, but many of its biological properties remain undiscovered. Recently, many researchers have analyzed the gene composition and biological function of IgY, and confirmed that it is the direct origin of mammalian IgG and IgE. [0003] IgY consists of two heavy chains (H) and a light chain (L), and the heavy chain (H) consists of a variable region (V) and four constant regions (C), without a hinge structure. The complete molecular weight is 180KDa (7.8S), the heavy chain is 67-70KDa, and the light chain is 25KDa, but there is also a small copy, 20KDa (5.7S), found in wild ducks and some turtles. Birds (chickens) and mammals (humans, mice) b...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/00C07K1/22C07K1/14
CPCC07K16/00
Inventor 张小莺钟发钢杨艳丽吴桐忠张星星韩猛立黄新何延华
Owner XINJIANG ACADEMY OF AGRI & RECLAMATION SCI
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