阅读说明：本技术 一种新冠病毒s1+s2抗独特型卵黄抗体疫苗的制备方法 (Preparation method of novel coronavirus S1+ S2 anti-idiotype yolk antibody vaccine ) 是由 黄威权 田超 赵锋 李丹 黄晓文 欧阳森 于 2020-05-25 设计创作，主要内容包括：本发明公开了一种新冠病毒S1+S2抗独特型卵黄抗体疫苗的制备方法,采用新冠病毒的S1+S2蛋白制备抗S1+S2抗体；用PBS对抗S1+S2抗体进行稀释后,与弗氏不完全佐剂等体积混合,获得第一混合溶液；将第一混合溶液肌肉注射产蛋鸡,对产蛋鸡进行第一免疫；判断产蛋鸡是否满足第二预定条件；若满足第二预定条件,产蛋鸡确定为高免鸡；按照第三预定条件采集高免鸡蛋；对高免鸡蛋进行消毒、卵黄分离、卵黄组织破碎后,获得卵黄液；去除卵黄液中的杂质蛋白,获得抗独特型卵黄抗体粗制品；对抗独特型卵黄抗体粗制品进行纯化,获得抗独特型卵黄抗体精制品。达到了使新冠病毒疫苗分子量大,进入人体不易被分解,体内留存时间长,稳定性强、效力强的技术效果。(The invention discloses a preparation method of a new coronavirus S1+ S2 anti-idiotype yolk antibody vaccine, which adopts S1+ S2 protein of the new coronavirus to prepare an anti-S1 + S2 antibody; diluting the anti-S1 + S2 antibody with PBS, and mixing with Freund incomplete adjuvant in an equal volume to obtain a first mixed solution; intramuscular injecting the first mixed solution into the laying hens, and carrying out first immunization on the laying hens; judging whether the laying hens meet a second preset condition; if the second preset condition is met, determining the laying hens as hyperimmune chickens; collecting high-immunity eggs according to a third preset condition; sterilizing high-immunity eggs, separating yolk, and crushing yolk tissues to obtain yolk liquid; removing impurity protein in yolk liquid to obtain crude product of anti-idiotype yolk antibody; purifying the anti-idiotype yolk antibody crude product to obtain an anti-idiotype yolk antibody refined product. Achieves the technical effects of large molecular weight, difficult decomposition after entering human body, long retention time in the body, strong stability and strong efficacy of the new coronavirus vaccine.)

1. A method for preparing a novel coronavirus S1+ S2 anti-idiotype yolk antibody vaccine, the method for preparing the novel coronavirus S1+ S2 anti-idiotype yolk antibody vaccine comprises the following steps:

preparing an anti-S1 + S2 antibody by adopting S1+ S2 protein of the novel coronavirus;

diluting the anti-S1 + S2 antibody by PBS, and mixing with Freund' S incomplete adjuvant in an equal volume to obtain a first mixed solution with a first concentration;

intramuscular injecting the first mixed solution into a laying hen, and carrying out first immunization on the laying hen for a first preset number of times according to a first preset condition;

judging whether the laying hens meet a second preset condition or not; if the second preset condition is met, determining the laying hen to be a hyperimmune chicken;

collecting high-immunity eggs according to a third preset condition, wherein the high-immunity eggs are eggs produced by the high-immunity chickens;

sterilizing the high-immunity eggs, separating yolk, and crushing yolk tissues to obtain yolk liquid;

removing impurity protein in the yolk liquid by using a water dilution and acidification method to obtain an anti-idiotype yolk antibody crude product;

purifying the crude anti-idiotype yolk antibody product by an alcohol precipitation method to obtain a refined anti-idiotype yolk antibody product.

2. The method of claim 1, wherein the first concentration is 80 μ g of the anti-S1 + S2 antibody per ml of the first solution.

3. The preparation method of claim 1, wherein said first predetermined conditions are that each of said laying hens injects 0.5mL of said first mixed solution per said first immunization, and said first immunization is performed every 7-10 days;

the first predetermined number of times is 3.

4. The preparation method according to claim 1, wherein the determining whether the laying hen satisfies a second predetermined condition is:

collecting eggs laid by the laying hens on the day of the third immunization, and judging whether the yolk of the eggs contains S1+ S2 anti-idiotype yolk antibodies;

if the S1+ S2 anti-idiotype yolk antibody is contained, judging whether the titer of the S1+ S2 anti-idiotype yolk antibody is more than or equal to 10^-4；

If the titer of the S1+ S2 anti-idiotype yolk antibody is more than or equal to 10^-4And if so, the laying hens meet the second preset condition.

5. The preparation method according to claim 1, wherein the collection of hyperimmune eggs according to the third predetermined condition is:

collecting the eggs produced by the hyperimmune chicken on the third day after the first immunization for 20 days continuously.

6. The preparation method of claim 1, wherein the impurity proteins in the egg yolk liquid are removed by water dilution and acidification, and the method comprises the following specific steps:

adding sterile distilled water with a first volume into the yolk liquid with a second volume, and uniformly stirring to obtain a yolk solution; wherein the first volume is equal to 15-30 times the second volume;

adjusting the pH value of the yolk solution to 5.2-6.0 by using hydrochloric acid and sodium hydroxide solution;

standing the yolk solution for 14-18 hours at 4-8 ℃, and taking the upper clear liquid to obtain a first clear liquid;

centrifuging the bottom turbid solution, and taking the upper clear solution to obtain a second clear solution;

mixing the first clear liquid and the second clear liquid to obtain a third clear liquid;

the third clear solution was filtered through a 0.4 μm microfiltration membrane.

7. The method according to claim 1, wherein the anti-S1 + S2 antibody is a rabbit anti-S1 + S2 antibody, and the rabbit anti-S1 + S2 antibody is prepared by:

uniformly mixing the S1+ S2 protein solution and Freund' S incomplete adjuvant in equal volume to obtain a second mixed solution;

subcutaneously injecting M rabbits with the second mixed solution, and performing second immunization on the rabbits for a second predetermined number of times according to a fourth predetermined condition;

judging whether the rabbit meets a fifth preset condition or not;

if the fifth preset condition is met, determining the rabbit as a high-immunity rabbit;

5-7 days after the third immunization, bleeding the hyperimmune rabbit to obtain first plasma;

centrifuging the first plasma, and taking serum to obtain the rabbit anti-S1 + S2 antibody;

wherein M is more than or equal to 2 and is a positive integer.

8. The method according to claim 7, wherein the determining whether the rabbit satisfies a fifth predetermined condition is:

on the day of carrying out the second immunization for the third time, taking blood of the rabbit to obtain second plasma;

centrifuging the second plasma to obtain a first serum;

measuring the titer of said first serum by an ELISA assay;

when the titer of the first serum is more than 10^-4Said rabbit fulfils said fifth predetermined condition.

9. The method of claim 1, wherein the anti-S1 + S2 antibody is a murine anti-S1 + S2 antibody, and the murine anti-S1 + S2 antibody is prepared by a method comprising:

uniformly mixing the S1+ S2 protein solution and Freund' S incomplete adjuvant in equal volume to obtain a third mixed solution;

carrying out intraperitoneal injection on N mice by using the third mixed solution, and carrying out third immunization on the mice for a third preset time according to a sixth preset condition;

judging whether the mouse meets a seventh preset condition;

if the seventh predetermined condition is met, the mouse is determined to be a hyperimmune mouse;

bleeding the hyperimmune mouse 5-7 days after the third immunization to obtain third plasma;

centrifuging the third plasma, and taking serum to obtain the mouse anti-S1 + S2 antibody;

wherein N is more than or equal to 2 and is a positive integer.

10. The method according to claim 9, wherein the determining whether the mouse satisfies a seventh predetermined condition is specifically:

on the day of the third immunization, blood of the mouse is taken to obtain fourth plasma;

centrifuging the fourth plasma to obtain a second serum;

measuring the titer of said second serum using an ELISA assay;

when the second serum has a potency of greater than 10^-4When the mouse satisfies the seventh predetermined condition.

Technical Field

The invention relates to the technical field of epidemic prevention, in particular to a preparation method of a novel coronavirus S1+ S2 anti-idiotype yolk antibody vaccine.

Background

The new coronavirus is currently prevalent in almost all countries around the world, the number of infected people is over millions, and the number of infected people is continuously increased so far, so that the new coronavirus has great destructive effect on human life safety and social economy. The main methods for dealing with the epidemic at present are early diagnosis, early isolation and early treatment. These measures have had a positive effect on preventing the spread of new coronaviruses, but also have a great negative impact on various industries, and countries in the world have paid a great deal of cost for this.

To stop the spread of this disease and eventually eliminate it, it is best to develop an effective vaccine as soon as possible. So far, no new coronavirus vaccine is available all over the world, and only two vaccines are clinically tested, namely adenovirus vaccine which is hosted by Chenwei academy in China and mRNA vaccine which is developed by American scholars. There are dozens of new coronavirus vaccines currently under investigation, which are grouped together into the following categories: DNA vaccine, mRNA vaccine, and gene recombinant vaccine.

The adenovirus vaccine is actually a gene recombinant vaccine, and is characterized in that pathogenic genes of new coronavirus are cloned, recombined and transformed, then the new coronavirus is transfected into adenovirus, the adenovirus is inoculated to immunize a human body, the adenovirus expresses a new coronavirus pathogenic antigen with low toxicity while amplifying and propagating in the human body, and the later stimulates the human body to generate antibodies, so that the human body can generate immune protection capability for infection of the new coronavirus.

The mRNA vaccine is prepared by modifying mRNA related to the new coronavirus disease and then immunizing a human body, wherein the mRNA is replicated in the human body and expresses a disease antigen with low toxicity, and the later stimulates the human body to generate an antibody so that the human body has immune protection capability on the infection of the new coronavirus.

The DNA vaccine is prepared through cloning and modifying virus and pathogenic DNA, in vitro amplification, intramuscular injection to immunize human body, amplification of virus DNA in muscle cell to express mRNA, and expression of pathogenic antigen with mRNA as template to stimulate human body to produce antibody and to make human body to produce immune protection to new coronavirus infection.

However, the present inventors have found that the above prior art has at least the following technical problems:

Disclosure of Invention

The invention provides a preparation method of a new coronavirus S1+ S2 anti-idiotype yolk antibody vaccine, solves the technical problems that the new coronavirus vaccine in the prior art has small molecular weight, is difficult to decompose after entering a body, has stability difference, has a long in-vivo retention time period and weak efficacy, and achieves the technical effects that the new coronavirus vaccine has large molecular weight, is difficult to decompose after entering the body, has long in-vivo retention time, and has strong stability and strong efficacy.

In order to solve the above problems, in a first aspect, the embodiment of the present invention provides a method for preparing a novel coronavirus S1+ S2 anti-idiotype yolk antibody vaccine, which comprises: preparing an anti-S1 + S2 antibody by adopting S1+ S2 protein of the novel coronavirus; diluting the anti-S1 + S2 antibody by PBS, and mixing with Freund' S incomplete adjuvant in an equal volume to obtain a first mixed solution with a first concentration; intramuscular injecting the first mixed solution into a laying hen, and carrying out first immunization on the laying hen for a first preset number of times according to a first preset condition; judging whether the laying hens meet a second preset condition or not; if the second preset condition is met, determining the laying hen to be a hyperimmune chicken; collecting high-immunity eggs according to a third preset condition, wherein the high-immunity eggs are eggs produced by the high-immunity chickens; sterilizing the high-immunity eggs, separating yolk, and crushing yolk tissues to obtain yolk liquid; removing impurity protein in the yolk liquid by using a water dilution and acidification method to obtain an anti-idiotype yolk antibody crude product; purifying the crude anti-idiotype yolk antibody product by an alcohol precipitation method to obtain a refined anti-idiotype yolk antibody product.

Further, the first concentration is 80 μ g of the anti-S1 + S2 antibody per ml of the first solution.

Further, the first preset condition is that each laying hen injects 0.5mL of the first mixed solution every time of the first immunization, and the first immunization is carried out every 7-10 days; the first predetermined number of times is 3.

Further, the determining whether the laying hen meets a second predetermined condition specifically includes: collecting eggs laid by the laying hens on the day of the third immunization, and judging whether the yolk of the eggs contains S1+ S2 anti-idiotype yolk antibodies; if the S1+ S2 anti-idiotype yolk antibody is contained, judging whether the titer of the S1+ S2 anti-idiotype yolk antibody is more than or equal to 10^-4(ii) a If the titer of the S1+ S2 anti-idiotype yolk antibody is more than or equal to 10^-4And if so, the laying hens meet the second preset condition.

Further, the acquiring of the high-immunity eggs according to a third preset condition specifically comprises: collecting the eggs produced by the hyperimmune chicken on the third day after the first immunization for 20 days continuously.

Further, the method for removing impurity proteins in the egg yolk liquid by using water dilution and acidification methods specifically comprises the following steps: adding sterile distilled water with a first volume into the yolk liquid with a second volume, and uniformly stirring to obtain a yolk solution; wherein the first volume is equal to 15-30 times the second volume; adjusting the pH value of the yolk solution to 5.2-6.0 by using hydrochloric acid and sodium hydroxide solution; standing the yolk solution for 14-18 hours at 4-8 ℃, and taking the upper clear liquid to obtain a first clear liquid; centrifuging the bottom turbid solution, and taking the upper clear solution to obtain a second clear solution; mixing the first clear liquid and the second clear liquid to obtain a third clear liquid; the third clear solution was filtered through a 0.4 μm microfiltration membrane.

Further, the anti-S1 + S2 antibody is a rabbit anti-S1 + S2 antibody, and the preparation method of the rabbit anti-S1 + S2 antibody specifically comprises the following steps: uniformly mixing the S1+ S2 protein solution and Freund' S incomplete adjuvant in equal volume to obtain a second mixed solution; subcutaneously injecting M rabbits with the second mixed solution, and performing second immunization on the rabbits for a second predetermined number of times according to a fourth predetermined condition; judging whether the rabbit meets a fifth preset condition or not; if the fifth preset condition is met, determining the rabbit as a high-immunity rabbit; 5-7 days after the third immunization, bleeding the hyperimmune rabbit to obtain first plasma; centrifuging the first plasma, and taking serum to obtain the rabbit anti-S1 + S2 antibody; wherein M is more than or equal to 2 and is a positive integer.

Further, the judging whether the rabbit meets a fifth predetermined condition specifically includes: on the day of carrying out the second immunization for the third time, taking blood of the rabbit to obtain second plasma; centrifuging the second plasma to obtain a first serum; measuring the titer of said first serum by an ELISA assay; when the titer of the first serum is more than 10^-4Said rabbit fulfils said fifth predetermined condition.

Further, the anti-S1 + S2 antibody is a murine anti-S1 + S2 antibody, and the preparation method of the murine anti-S1 + S2 antibody comprises: uniformly mixing the S1+ S2 protein solution and Freund' S incomplete adjuvant in equal volume to obtain a third mixed solution; carrying out intraperitoneal injection on N mice by using the third mixed solution, and carrying out third immunization on the mice for a third preset time according to a sixth preset condition; judging whether the mouse meets a seventh preset condition; if the seventh predetermined condition is met, the mouse is determined to be a hyperimmune mouse; bleeding the hyperimmune mouse 5-7 days after the third immunization to obtain third plasma; centrifuging the third plasma, and taking serum to obtain the mouse anti-S1 + S2 antibody; wherein N is more than or equal to 2 and is a positive integer.

Further, the determining whether the mouse satisfies a seventh predetermined condition is specifically: on the day of the third immunization, blood of the mouse is taken to obtain fourth plasma; centrifuging the fourth plasma to obtain a second serum; measuring the titer of said second serum using an ELISA assay; when the second serum has a potency of greater than 10^-4When the mouse satisfies the seventh predetermined condition.

One or more technical solutions in the embodiments of the present invention at least have one or more of the following technical effects:

the embodiment of the invention provides a preparation method of a novel coronavirus S1+ S2 anti-idiotype yolk antibody vaccine, which comprises the following steps: preparing an anti-S1 + S2 antibody by adopting S1+ S2 protein of the novel coronavirus; diluting the anti-S1 + S2 antibody by PBS, and mixing with Freund' S incomplete adjuvant in an equal volume to obtain a first mixed solution with a first concentration; intramuscular injecting the first mixed solution into a laying hen, and carrying out first immunization on the laying hen for a first preset number of times according to a first preset condition; judging whether the laying hens meet a second preset condition or not; if the second preset condition is met, determining the laying hen to be a hyperimmune chicken; collecting high-immunity eggs according to a third preset condition, wherein the high-immunity eggs are eggs produced by the high-immunity chickens; sterilizing the high-immunity eggs, separating yolk, and crushing yolk tissues to obtain yolk liquid; removing impurity protein in the yolk liquid by using a water dilution and acidification method to obtain an anti-idiotype yolk antibody crude product; purifying the crude anti-idiotype yolk antibody product by an alcohol precipitation method to obtain a refined anti-idiotype yolk antibody product. The preparation method solves the technical problems of small molecular weight, easy decomposition when entering a human body, poor stability, short in vivo retention time and weak efficacy of the new coronavirus vaccine in the prior art, and achieves the technical effects of large molecular weight, difficult decomposition when entering the human body, long in vivo retention time, strong stability and strong efficacy of the new coronavirus vaccine.

The foregoing description is only an overview of the technical solutions of the present invention, and the embodiments of the present invention are described below in order to make the technical means of the present invention more clearly understood and to make the above and other objects, features, and advantages of the present invention more clearly understandable.

Drawings

FIG. 1 is a flow chart of the preparation method of a new coronavirus S1+ S2 anti-idiotype yolk antibody vaccine in the embodiment of the invention;

FIG. 2 is a standard curve of chicken IgY of a new coronavirus S1+ S2 anti-idiotype yolk antibody vaccine in the embodiment of the invention.

Detailed Description

The embodiment of the invention provides a preparation method of a new coronavirus S1+ S2 anti-idiotype yolk antibody vaccine, solves the technical problems that the new coronavirus vaccine in the prior art has small molecular weight, is easy to decompose after entering a human body, has poor stability, short in vivo retention time and weak efficacy, and achieves the technical effects that the new coronavirus vaccine has large molecular weight, is difficult to decompose after entering the human body, has long in vivo retention time, strong stability and strong efficacy.

The technical scheme in the embodiment of the invention has the following overall structure:

a method for preparing a novel coronavirus S1+ S2 anti-idiotype yolk antibody vaccine, the method for preparing the novel coronavirus S1+ S2 anti-idiotype yolk antibody vaccine comprises the following steps: preparing an anti-S1 + S2 antibody by adopting S1+ S2 protein of the novel coronavirus; diluting the anti-S1 + S2 antibody by PBS, and mixing with Freund' S incomplete adjuvant in an equal volume to obtain a first mixed solution with a first concentration; intramuscular injecting the first mixed solution into a laying hen, and carrying out first immunization on the laying hen for a first preset number of times according to a first preset condition; judging whether the laying hens meet a second preset condition or not; if the second preset condition is met, determining the laying hen to be a hyperimmune chicken; collecting high-immunity eggs according to a third preset condition, wherein the high-immunity eggs are eggs produced by the high-immunity chickens; sterilizing the high-immunity eggs, separating yolk, and crushing yolk tissues to obtain yolk liquid; removing impurity protein in the yolk liquid by using a water dilution and acidification method to obtain an anti-idiotype yolk antibody crude product; purifying the crude anti-idiotype yolk antibody product by an alcohol precipitation method to obtain a refined anti-idiotype yolk antibody product. The preparation method solves the technical problems of small molecular weight, difficult decomposition in vivo, stable phase difference, time period for in vivo retention and weak efficacy of the new coronavirus vaccine in the prior art, and achieves the technical effects of large molecular weight, difficult decomposition in vivo, long in vivo retention time, strong stability and strong efficacy of the new coronavirus vaccine.

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.