阅读说明：本技术 一种高产量甲型h3n2流感病毒抗体的制备方法 (Preparation method of high-yield H3N2 influenza A virus antibody ) 是由 卢晓露 李海明 郑卓君 于 2019-09-27 设计创作，主要内容包括：本发明提供了一种高产量甲型H3N2流感病毒抗体的制备方法。包括以下工艺方法：在蛋鸡20周龄及以后,在每日普通饲料中添加3-7%海藻粉和4-7%蝗虫粉；制备甲型H3N2流感病毒免疫抗原、免疫产蛋母鸡,获得免疫鸡蛋、从免疫鸡蛋蛋黄液水稀释法提取甲型H3N2流感病毒抗体粗品；并对粗品进行纯化、冷冻喷雾干燥处理后,制得甲型H3N2流感病毒抗体,并进行纯度、活性检测。该工艺方法通过在鸡饲料中增加蝗虫粉和海藻粉,提高了甲型H3N2流感病毒抗体产量,并且使用蝗虫粉具有环境保护和良好的经济效益,本方法成本低、产量高、具有高特异性,可实现产业化。(The invention provides a preparation method of a high-yield H3N2 influenza A virus antibody. The method comprises the following steps: adding 3-7% of seaweed powder and 4-7% of locust powder into common feed every day after the laying hens are 20 weeks old; preparing influenza A H3N2 virus immune antigen, immunizing laying hens to obtain immune eggs, and extracting influenza A H3N2 virus antibody crude products from the immune eggs by a liquid-water dilution method; and purifying, freezing, spraying and drying the crude product to prepare the influenza A H3N2 virus antibody, and detecting the purity and the activity. The method increases locust powder and seaweed powder in chicken feed, improves H3N2 influenza A virus antibody yield, and has environmental protection and good economic benefit.)

1. A preparation method of high-yield influenza A H3N2 virus antibody is characterized by comprising the following steps:

feeding the laying hens: adding 3-7% of seaweed powder and 4-7% of locust powder into common feed every day after the laying hens are 20 weeks old;

immunizing the laying hens: preparing influenza A H3N2 virus, determining TCID50 value, diluting, mixing at equal proportion, emulsifying to obtain influenza A H3N2 virus immune antigen, and immunizing laying hen with influenza A H3N2 virus antigen;

extraction and purification of yolk antibody IgY: sterilizing the shell of an egg, breaking, removing egg white and an egg yolk membrane, reserving an egg yolk part, adding 1:6-12 purified water, adjusting the pH to 5.2-6.0, standing overnight at 4 ℃, centrifuging, taking a supernatant, adding PEG66000 or kieselguhr accounting for 4-8% of the mass of the supernatant, fully stirring, centrifuging at 10 ℃, pouring off the supernatant, and taking a precipitate part which is a crude product of the chicken egg yolk antibody specific to the influenza A virus H3N 2;

equilibrating DEAE-TOYOPEAL 650M ion exchange resin with 0.075mol/L PBS, and solubilizing the crude product with 0.075mol/L PBS to a protein concentration of about 20mg/ml, about 200ml per load;

eluting the hybrid protein which is not combined with the filler by using an equilibrium buffer solution after the sample is loaded, and then eluting by using 0.15mol/l PBS buffer solution with the pH value of 7.0;

dialyzing, desalting, freeze drying to obtain pure product of yolk antibody, and storing at 4 deg.C;

the recovery rate of the antibody is 70.3-71.4%, and the purity of the obtained antibody finished product is 94.8-95.5%.

2. The method of claim 1, wherein the layer chicken is one of Beijing white chicken, Hailan white chicken, Isha brown layer chicken, Hailan brown layer chicken, Romanbrown layer chicken, ya kang layer chicken, Hailan pink layer chicken, and Jingbai 939 pink layer chicken.

3. The method for producing a high-yield influenza a H3N2 virus antibody according to claim 1, wherein the locust is one or more of a rice locust, an eastern asian migratory locust, a red-reared locusts, a taiwan rice locust, and a desert locust.

4. The method of claim 1, wherein the algae is one or more of Sargassum horneri, Palmaria palmata, Chlorella, Red algae, and Brown algae.

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a preparation method of a high-yield H3N2 influenza A virus antibody.

Background

Influenza a viruses can be classified into 16 subtypes (H1-H16) according to the Hemagglutinin (HA) antigen on their surface, and 9 subtypes (N1-N9) according to the Neuraminidase (NA) antigen. Influenza viruses propagate and propagate in an infected population by producing a new influenza virus (antigen shift) through cumulative changes in the antigenicity of surface proteins (antigen drift) or through gene rearrangement of different influenza viruses to escape immune recognition of infected hosts. Influenza a H3N2 is a respiratory disease caused by the influenza a H3N2 virus. H3N2 flu outbreaks in hong kong in 1968 lost lives to millions of people. Early 2013, with H3N2 seasonal influenza outbreaks in multiple states in the eastern united states, the government in boston declared a public health emergency on day 1 and 9. At present, chemical drugs and vaccines are mainly adopted for prevention and control measures aiming at the interpersonal H3N2 influenza A pandemic, but side effects caused by excessive use of chemical drugs and the disadvantage that vaccine development is usually lagged behind epidemic outbreak and the vaccine can only play a role in preventing susceptible people are obvious.

The chicken egg yolk antibody is obtained by effectively transferring IgY in serum of a hen in the egg development process. The egg yolk antibody is safe, convenient, simple and easily obtained, has high yield and does not have cross serological reaction with IgG; the egg yolk antibody has the advantages of no combination with rheumatoid factors or Fc receptors, no generation of false negative or false positive results in the immunoassay process, heat resistance, acid resistance, no inactivation at pasteurization temperature and stable activity at the pH value of less than 3, and plays an important role in the clinical and basic scientific application of the egg yolk antibody, so the application of the egg yolk antibody in the field of medical prevention is more and more concerned by human beings, and the development of the H3N2 influenza A virus antibody brings a new breakthrough in the prevention of the H3N2 influenza A.

The ingestion of chicken protein includes animal protein and vegetable protein, such as various insects and plant leaves, stems, root blocks and seeds, and the active seeking competitive ingestion maintains the balance of various amino acids constituting protein and provides guarantee for the formation of immunoglobulin, which is one of the reasons why wild or stocking chicken has stronger immunity than cage-raised chicken. The locust is the main pest of crops, the locust powder contains 74.88% of protein and 0.809% of phosphorus, and is a high-quality animal protein raw material, and the locust powder is added to supplement animal protein of the chicken feed, so that the animal protein and plant protein of the chicken feed are reasonably proportioned, the chicken ingests balanced amino acid, and the yield of immunoglobulin can be improved. Meanwhile, the method has environmental protection and economic benefits.

The seaweed is rich in seaweed polysaccharide, mannitol, vitamins, and various major and trace elements such as potassium, iron, calcium, phosphorus, iodine, selenium, cobalt, etc. The addition of 3-7% of seaweed powder into the layer feed can improve the laying rate by more than 10% and the iodine content in eggs by more than 15%. The xu Bao Hua is obtained by adding nano selenium into the daily ration of the broiler chicken, so that the serum IgG level is obviously improved, and the IgG level can reach 26mg/kg at most. Guyuli et al report that high iodine obviously enhances the immune response of broilers, and appears to increase epithelial-like lymphocytes among small intestine epithelia, and the number of lymphocytes in small intestine solid layer and skin block, thyroid tissue, ovary tissue and pancreas tissue is obviously increased.

Disclosure of Invention

The invention aims to provide a preparation method of high-yield H3N2 influenza A virus antibody

In order to realize the purpose, the following technical scheme is adopted: when the laying hens grow for 20 weeks, adding 4-7% of locust powder and 3-7% of seaweed powder into the common chicken feed; preparing influenza A H3N2 virus, determining TCID50 value, diluting, mixing in equal proportion, and emulsifying to obtain influenza A H3N2 virus immunizing antigen; injecting the antigen into egg laying hens to obtain immune eggs; sterilizing egg shell, breaking, removing egg white and egg yolk membrane, collecting yolk, adding 1:6-12 purified water, adjusting pH to 5.2-6.0, standing overnight at 4 deg.C, centrifuging, collecting supernatant, adding PEG66000 or diatomaceous earth 4-8% of the supernatant, stirring, centrifuging at 10 deg.C, and removing supernatant to obtain precipitate as crude H3N2 influenza A virus antibody. DEAE-TOYOPEAL 650M ion exchange resin was equilibrated with 0.075mol/LPBS and the crude product was solubilized with 0.075mol/L PBS to a protein concentration of about 20mg/ml and about 200ml per load. After loading, the heteroproteins not bound to the filler were eluted with equilibration buffer and then with 0.15mol/l PBS buffer pH 7.0. Dialyzing, desalting, freeze drying to obtain pure product of yolk antibody, storing at 4 deg.C, and detecting purity and activity. The recovery rate of the antibody is 70.3-71.4%, and the purity of the obtained antibody finished product is 94.8-95.5%.

Compared with the prior art, the invention has the beneficial effects that: by adding locust powder and seaweed powder into the chicken feed, the yield of the H3N2 influenza A virus antibody is improved, and the use of the locust powder has environmental protection and good economic benefit.

Detailed Description

The present invention is further described below by way of specific examples, but the present invention is not limited thereto.