阅读说明：本技术 一种鸡胚培养法来源的流感病毒纯化方法、四价流感病毒亚单位疫苗及其应用 (Chicken embryo culture method-derived influenza virus purification method, tetravalent influenza virus subunit vaccine and application thereof ) 是由 安有才 张玉辉 王帅 贾春玉 陈晓芬 于 2021-09-29 设计创作，主要内容包括：本发明公开了一种鸡胚培养法来源的流感病毒纯化方法、四价流感病毒亚单位疫苗及其应用,方法对原始工艺进一步改良,首先扩增病毒后增加一步去卵清蛋白处理改良步骤,大大降低成品中卵清蛋白残留量；然后改良分离纯化四种亚型流感病毒血凝素HA的步骤,进一步降低裂解剂与灭活剂甲醛残留量,增强疫苗的安全性；最后将四种亚型流感病毒疫苗原液按一定比例混合,从而制备成四价流感病毒亚单位疫苗成品。从而获得一种裂解剂与灭活剂残留量更少、抗原纯度更高好且免疫原性良好的四价流感病毒亚单位疫苗。(The invention discloses an influenza virus purification method from chick embryo culture method, a tetravalent influenza virus subunit vaccine and application thereof, wherein the method further improves the original process, firstly, a step of removing ovalbumin is added after virus amplification, and the residual amount of ovalbumin in a finished product is greatly reduced; then the step of separating and purifying four subtype influenza virus hemagglutinin HA is improved, the residual quantity of formaldehyde of a cracking agent and an inactivating agent is further reduced, and the safety of the vaccine is enhanced; and finally, mixing the four subtype influenza virus vaccine stock solutions according to a certain proportion to prepare a tetravalent influenza virus subunit vaccine finished product. Thereby obtaining the quadrivalent influenza virus subunit vaccine with less residual amounts of the cracking agent and the inactivating agent, higher antigen purity and good immunogenicity.)

1. A method for purifying influenza virus from chick embryo culture method comprises amplification, clarification, concentration and cracking, and is characterized in that: after clarification, removing ovalbumin, concentrating, cracking and purifying;

the egg white protein removing method specifically comprises the following steps: placing the clarified monovalent influenza virus mixed solution in a warm water bath at 30 ℃, slowly adding a 0.1M NaOH solution to pH9.5 under the condition of continuous stirring, filtering by a filter screen to remove a large amount of white floccules formed by aggregation of ovalbumin, collecting filtrate, and slowly neutralizing by a 0.3M HCl solution to pH 7.2 to obtain the monovalent influenza virus mixed solution for removing ovalbumin;

the purification treatment comprises the following specific steps: eluting and then inactivating, collecting a sample, and removing 95% of a lytic agent through a lytic agent removing medium, firstly, centrifuging at 1500 Xg and room temperature for 1min to remove a storage buffer solution in the lytic agent removing medium, then adding a proper amount of a balancing solution, centrifuging at 1500 Xg and room temperature for 1min to balance the lytic agent removing medium, then incubating at room temperature for 2min to ensure that the lytic agent is fully combined with the lytic agent removing medium, centrifuging at 1500 Xg and room temperature for 2min, collecting a flow-through protein sample, and obtaining a lytic agent monovalent influenza virus antigen removing liquid;

the purification treatment comprises the following specific steps: and (3) eluting and then inactivating, and dialyzing the collected sample to remove residual cracking agent and formaldehyde in the solution.

2. The method of claim 1, wherein the amplification is specifically: preparing 2.0-5.0 log EID50/mL of various influenza virus working seed batches, inoculating the seeds into a clean-grade chick embryo allantoic cavity at the dose of 0.2 mL/embryo, and standing and culturing at the constant temperature of 33-35 ℃ for 48-72 h; carefully screening live chick embryos by an egg candler, standing and cooling the chick embryos for 10-20 h at 4-10 ℃, and then harvesting chick embryo allantoic fluid, namely influenza virus allantoic fluid.

3. The method of claim 1, wherein the clarification specifically comprises: the allantoic fluid of influenza virus is harvested, centrifuged at 5000rpm for 10min, and the supernatant is collected and combined into monovalent influenza virus combined fluid.

4. The method of claim 1, wherein the concentration is specifically: and (3) carrying out ultrafiltration concentration on the ovalbumin-removed monovalent influenza virus combined solution by using a 100kD ultrafiltration membrane, wherein the concentration multiple of the influenza virus solution is 30-60 times, and thus obtaining the monovalent influenza virus concentrated solution.

5. The method of claim 1, wherein the splitting is specifically: preparing a sucrose solution of a Triton N-101 cracking agent with the final concentration of less than or equal to 1.5%, adding a monovalent influenza virus concentrated solution, centrifuging at 25000rpm for 3.5-4.5 h at 15-25 ℃, and collecting a target protein peak with the sucrose concentration of 10-18% according to the OD280 value of an ultraviolet absorption curve.

6. The method for purifying an influenza virus derived from chick embryo culture according to claim 1, wherein: and (3) purifying the influenza virus antigen solution again after the purification treatment, specifically, repeatedly washing and filtering the purified influenza virus antigen solution, sterilizing and filtering, sampling and detecting the content and purity of hemagglutinin, and finally preparing the influenza virus monovalent antigen solution.

7. A tetravalent influenza virus subunit vaccine, characterized by: the kit consists of four subtype influenza virus hemagglutinin proteins prepared by the method of any one of claims 1 to 6 and a Tris buffer solution system, wherein the antigen content of each subtype influenza virus hemagglutinin protein is 30 to 39 mu g/mL.

8. A tetravalent influenza virus subunit vaccine of claim 7, wherein: the weight part ratio of four subtype influenza virus hemagglutinin proteins is 1:1:1: 1.

9. A tetravalent influenza virus subunit vaccine of claim 7, wherein: four influenza virus subtype hemagglutinin proteins are respectively H1N1, H3N2, B/Victoria and B/Yamagata; the amino acid sequences of the four subtype influenza virus HA proteins are respectively shown in SEQID NO. 1-4.

10. Use of a tetravalent influenza virus subunit vaccine of any of claims 7-9 in the preparation of a medicament for simultaneous prevention of four subtypes of influenza virus.

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to an influenza virus purification method derived from a chick embryo culture method, a tetravalent influenza virus subunit vaccine and application thereof.

Background

Influenza is a short for influenza, is a disease of respiratory tract and other organ injury caused by influenza virus infection, and is epidemic in winter and spring to different degrees every year. Influenza viruses are mainly divided into four subtypes, namely A (A), B (B), C (C) and D (D), wherein A (A) and B (B) are easy to cause wide-range outbreak infection. Hospitalization and death after influenza virus infection and hair dyeing mainly occur in infants, the elderly, patients with chronic basic diseases, pregnant women and other people with low immunity. For the protection against influenza in the above-mentioned population, yearly vaccination with influenza vaccine is the most effective method for preventing influenza.

The inactivated influenza virus vaccine comprises an influenza virus split vaccine and an influenza virus subunit vaccine. The preparation process of quadrivalent influenza virus split vaccine includes culturing virus in chicken embryo to prepare virus liquid, collecting allantoic liquid, clarifying, concentrating, purifying, deactivating and splitting to prepare quadrivalent influenza virus split vaccine semi-finished product, and packing to obtain quadrivalent influenza virus split vaccine finished product. The tetravalent influenza virus subunit vaccine is prepared by further purifying antigen protein hemagglutinin on the basis of tetravalent influenza virus split vaccine.

At present, the influenza vaccine market in China has the following problems to be solved urgently: 1) the influenza vaccine market supply is severely deficient; the prediction difficulty of the market demand of the influenza vaccine is high, and the specificity of influenza for one year and one season, the production period of the vaccine is nearly 5 months, and the quality guarantee period is only 1 year, so that the accurate judgment of influenza vaccine demand of influenza vaccine enterprises is increased. 2) Cracking agent, ovalbumin, inactivating agent and the like in the influenza virus cracking vaccine cause that the influenza virus vaccine has low purity; the purity of the current influenza virus split vaccine is about 75-80%; 3) the cracking agent, ovalbumin and the inactivating agent existing in the influenza virus cracking vaccine seriously affect the safety of the vaccine; due to purity and safety reasons, current vaccines cannot be suitable for people with low immunity, such as children and the elderly.

In order to solve the problems, the development of an influenza virus subunit vaccine with less residual amount of a lytic agent, high antigen purity and good immunogenicity is urgently needed.

Disclosure of Invention

In order to solve the technical problems, the invention aims to provide an influenza virus purification method derived from a chick embryo culture method, a tetravalent influenza virus subunit vaccine and application thereof.

The purpose of the invention is realized as follows:

the method for purifying the influenza virus from the chicken embryo culture method comprises amplification, clarification, concentration and cracking, and is characterized in that: after clarification, removing ovalbumin, concentrating, cracking and purifying; the purity of the purified influenza virus antigen liquid is more than 85 percent;

the egg white protein removing method specifically comprises the following steps: placing the clarified monovalent influenza virus mixed solution in a warm water bath at 30 ℃, slowly adding a 0.1M NaOH solution to pH9.5 under the condition of continuous stirring, filtering by a filter screen to remove a large amount of white floccules formed by aggregation of ovalbumin, collecting filtrate, and slowly neutralizing by a 0.3M HCl solution to pH 7.2 to obtain the monovalent influenza virus mixed solution for removing ovalbumin;

because the chicken embryo is adopted to amplify four subtype influenza viruses in the preparation process of the inactivated influenza virus vaccine, a small amount of ovalbumin is mixed in a finished product finally, and the ovalbumin influences the purity of the vaccine and the storage of the vaccine, the ovalbumin is removed by adopting the method.

The purification treatment is mainly used for removing a cracking agent, an inactivating agent and virus structural proteins; the three sources are mainly: because the complete structure of the influenza virus is destroyed by the cracking agent in the preparation process of the influenza virus split vaccine, a part of the cracking agent is remained in the finished product; meanwhile, because the inactivated virus is adopted in the preparation process of the influenza virus inactivated vaccine, a part of the inactivated agent is remained in the finished product finally; in addition, virus structural proteins are not removed in the preparation process of the inactivated influenza virus vaccine, and finally, a part of influenza virus structural proteins are remained in a finished product. The purification treatment can therefore be carried out in two ways:

the method comprises the following steps: eluting and then inactivating, collecting a sample, removing 95% of a lytic agent through a lytic agent removing medium, firstly, centrifuging at 1500 Xg and room temperature for 1min to remove a storage buffer solution in the lytic agent removing medium, then adding a proper amount of a balancing solution, centrifuging at 1500 Xg and room temperature for 1min to balance the lytic agent removing medium, then incubating at room temperature for 2min to ensure that the lytic agent is fully combined with the lytic agent removing medium, centrifuging at 1500 Xg and room temperature for 2min, collecting a flow-through protein sample, and obtaining a lytic agent monovalent influenza virus antigen removing liquid;

the second is as follows: and (3) eluting and then inactivating, and dialyzing the collected sample to remove residual cracking agent and formaldehyde in the solution.

Preferably, the amplification is specifically: preparing 2.0-5.0 log EID50/mL of various influenza virus working seed batches, inoculating the seeds into a clean-grade chick embryo allantoic cavity at the dose of 0.2 mL/embryo, and standing and culturing at the constant temperature of 33-35 ℃ for 48-72 h; carefully screening live chick embryos by an egg candler, standing and cooling the chick embryos for 10-20 h at 4-10 ℃, and then harvesting chick embryo allantoic fluid, namely influenza virus allantoic fluid.

Preferably, the clarification is in particular: the allantoic fluid of influenza virus is harvested, centrifuged at 5000rpm for 10min, and the supernatant is collected and combined into monovalent influenza virus combined fluid.

Preferably, the above-mentioned concentration is specifically: and (3) carrying out ultrafiltration concentration on the ovalbumin-removed monovalent influenza virus combined solution by using a 100kD ultrafiltration membrane, wherein the concentration multiple of the influenza virus solution is 30-60 times, and thus obtaining the monovalent influenza virus concentrated solution.

Preferably, the above lysis is in particular: preparing a sucrose solution of a Triton N-101 cracking agent with the final concentration of less than or equal to 1.5%, adding a monovalent influenza virus concentrated solution, centrifuging at 25000rpm for 3.5-4.5 h at 15-25 ℃, and collecting a target protein peak with the sucrose concentration of 10-18% according to the OD280 value of an ultraviolet absorption curve.

Preferably, the purification treatment is followed by purification, specifically, the purified influenza virus antigen solution is repeatedly washed and filtered, then is subjected to sterilization and filtration, and the hemagglutinin content and purity are sampled and detected, so that the influenza virus monovalent antigen solution is finally prepared, wherein the purity of the influenza virus monovalent antigen solution can reach more than or equal to 85%.

The key point of the tetravalent influenza virus subunit vaccine is as follows:

the kit consists of four subtype influenza virus hemagglutinin proteins prepared by the method of any one of claims 1 to 6 and a Tris buffer solution system, wherein the antigen content of each subtype influenza virus hemagglutinin protein is 30 to 39 mu g/mL.

Preferably, the ratio of the four influenza virus subtype hemagglutinin proteins in parts by weight is 1:1:1: 1.

Preferably, four of the influenza virus subtype hemagglutinin proteins are H1N1, H3N2, B/Victoria and B/Yamagata, respectively; the amino acid sequences of the four subtype influenza virus HA proteins are respectively shown in SEQID NO. 1-4.

And thirdly, the application of the tetravalent influenza virus subunit vaccine in preparing a medicament for simultaneously preventing four subtype influenza viruses.

Has the advantages that:

compared with the existing influenza virus vaccines in the current market, the influenza virus subunit vaccine prepared by the invention has the advantages that the residual quantity of the cracking agent and the inactivating agent is less, the residual quantity of free formaldehyde is as low as 4-5ug/ml, the residual quantity of the cracking agent is as low as 84-101ug/ml, the antigen purity is higher, the hemagglutinin purity in the semi-finished product of the vaccine is more than or equal to 85%, the residual quantity of ovalbumin is as low as 21-35ng/ml, the immunogenicity is better, and the high safety enables the influenza virus subunit vaccine to help people of all ages, especially children and old people, and effectively prevent seasonal influenza virus infection every year by increasing the dosage or adding an adjuvant.

Drawings

FIG. 1 is SDS-PAGE gel of hemagglutinin products of experimental and control compositions.

FIG. 2 is a comparison chart of the immunogenicity test results of three batches of finished products of the experimental group and the control group.

Detailed Description

The invention is further illustrated by the following examples and figures.

Example 1 a tetravalent influenza subunit vaccine preparation method:

the tetravalent influenza subunit vaccine contains H1N1, H3N2, B/Victoria and B/Yamagata antigen proteins, and the content of each valence antigen is 30-39 mu g/mL.

Amplification of influenza viruses of respective subtypes

Preparing 2.0-5.0 lgEID50/mL of various influenza virus working seed batches of virus seeds, inoculating the virus seeds into a clean-grade chick embryo allantoic cavity at the dose of 0.2 mL/embryo, and standing and culturing at the constant temperature of 33-35 ℃ for 48-72 h.

2. Harvesting of influenza virus liquid of each subtype

Carefully screening live chick embryos by an egg candler, standing and cooling the chick embryos for 10-20 h at 4-10 ℃, then harvesting chick embryo allantoic fluid and sampling and detecting.

3. Centrifugal clarification and combination of influenza virus liquid

And (3) centrifuging allantoic harvest liquid of each subtype influenza virus at 5000rpm for 10min, collecting supernatant, and combining the supernatant into monovalent influenza virus combined liquid.

4. Removal of ovalbumin

Placing the monovalent influenza virus merged solution in a warm water bath at 30 ℃, slowly adding 0.1M NaOH solution to pH9.5 under the condition of continuous stirring, generating a large amount of white floccules formed by aggregation of ovalbumin in the solution, filtering by a filter screen to remove the floccules, collecting filtrate, and slowly neutralizing by 0.3M HCl solution to pH 7.2 to obtain the monovalent influenza virus merged solution for removing ovalbumin.

5. Concentration, influenza virus splitting and antigen purification

1) Concentration of influenza virus

And (3) carrying out ultrafiltration concentration on the ovalbumin-removed monovalent influenza virus combined solution by using a 100kD ultrafiltration membrane, wherein the concentration multiple of the influenza virus solution is 30-60 times, and thus obtaining the monovalent influenza virus concentrated solution.

2) Influenza virus splitting

Preparing a sucrose solution of a Triton N-101 cracking agent with the final concentration of less than or equal to 1.5%, adding a monovalent influenza virus concentrated solution, centrifuging at 25000rpm for 3.5-4.5 h at 15-25 ℃, and collecting a target protein peak with the sucrose concentration of 10-18% according to the OD280 value of an ultraviolet absorption curve.

3) Influenza virus antigen purification

And (3) eluting by using Sephadex-G25 as a medium and 0.01 mol/L phosphate buffer solution in gel chromatography, collecting target protein according to an ultraviolet absorption curve OD280 value, further diluting, sterilizing, filtering and collecting a sample, adding formaldehyde with the final concentration of 200 mu G/mL into the sample, and standing and inactivating the sample for 36-48h at 4-10 ℃. The collected sample was then loaded onto a dialysis membrane (Spectra/Pro 7, molecular weight cut-off > 50 kDa) and dialyzed to remove residual lysis agent and formaldehyde from the solution.

4) Repurification of influenza virus antigens

And finally, repeatedly washing and filtering each subtype influenza virus inactivated solution to reduce the residual amount of free formaldehyde to 4-5ug/ml, the residual amount of a cracking agent to 84-101ug/ml and the residual amount of ovalbumin to 21-35ng/ml, then performing sterilization and filtration, sampling and detecting the content of bacterial endotoxin, the microbial limit and the content and purity of hemagglutinin (the purity is required to be more than or equal to 85%), and finally preparing each subtype influenza virus monovalent stock solution.

6. Preparation of semi-finished product

According to the hemagglutinin content of the influenza virus monovalent stock solution of each subtype, the influenza virus of each subtype is prepared into a semi-finished product of the tetravalent influenza subunit vaccine according to the antigen content proportion of the hemagglutinin of each subtype in a ratio of 1:1:1:1, wherein the preparation amount of the hemagglutinin is within the range of 30-39 mu g/mL.

Example 2 a method of preparing a tetravalent influenza subunit vaccine:

the tetravalent influenza subunit vaccine contains H1N1, H3N2, B/Victoria and B/Yamagata antigen proteins, and the content of each valence antigen is 30-39 mu g/mL.

Amplification of influenza viruses of respective subtypes

Preparing 2.0-5.0 log EID50/mL of various influenza virus working seed batches, inoculating the seeds into a clean chick embryo allantoic cavity at the dose of 0.2 mL/embryo, and standing and culturing at the constant temperature of 33-35 ℃ for 48-72 h.

2. Harvesting of influenza virus liquid of each subtype

Carefully screening live chick embryos by an egg candler, standing and cooling the chick embryos for 10-20 h at 4-10 ℃, then harvesting chick embryo allantoic fluid and sampling and detecting.

3. Centrifugal clarification and combination of influenza virus liquid

And (3) centrifuging allantoic harvest liquid of each subtype influenza virus at 5000rpm for 10min, collecting supernatant, and combining the supernatant into monovalent influenza virus combined liquid.

4. Removal of ovalbumin

Placing the monovalent influenza virus merged solution in a warm water bath at 30 ℃, slowly adding 0.1M NaOH solution to pH9.5 under the condition of continuous stirring, generating a large amount of white floccules formed by aggregation of ovalbumin in the solution, filtering by a filter screen to remove the floccules, collecting filtrate, and slowly neutralizing by 0.3M HCl solution to pH 7.2 to obtain the monovalent influenza virus merged solution for removing ovalbumin.

5. Concentration, influenza virus splitting and antigen purification

1) Concentration of influenza virus

And (3) carrying out ultrafiltration concentration on the ovalbumin-removed monovalent influenza virus combined solution by using a 100kD ultrafiltration membrane, wherein the concentration multiple of the influenza virus solution is 30-60 times, and thus obtaining the monovalent influenza virus concentrated solution.

2) Influenza virus splitting

Preparing a sucrose solution of a Triton N-101 cracking agent with the final concentration of less than or equal to 1.5%, adding a monovalent influenza virus concentrated solution, centrifuging at 25000rpm for 3.5-4.5 h at 15-25 ℃, and collecting a target protein peak with the sucrose concentration of 10-18% according to the OD280 value of an ultraviolet absorption curve.

3) Influenza virus antigen purification

Firstly, performing gel chromatography by using Sephadex-G25 as a medium, eluting with 0.01 mol/L phosphate buffer solution, collecting target protein according to an ultraviolet absorption curve OD280 value, further diluting, sterilizing, filtering and collecting a sample, adding formaldehyde with the final concentration of 200 mu G/mL, and standing and inactivating for 36-48h at 4-10 ℃.

Secondly, the collected sample is treated once by a lytic agent removing medium, 95% of the residual Triton N-101 lytic agent in the solution can be removed, and the specific operation steps are as follows:

1500 Xg, room temperature, centrifugation for 1min, thereby removing the lysis buffer in the clearing medium.

Adding appropriate amount of balancing solution, 1500 Xg, centrifuging at room temperature for 1min to balance the lysis agent scavenging medium.

Adding a collected sample containing a cracking agent, and incubating at room temperature for 2min to ensure that the cracking agent is fully combined with a cracking agent removing medium.

1500 Xg, room temperature, centrifugation for 2min, collection of flow through protein samples. Thus obtaining the univalent influenza virus antigen solution eliminated by the cracking agent. The lysis medium used was Thermo Scientific, Pierce Detergent Removal Resin, cat: 87780.

4) Repurification of influenza virus antigens

And finally, repeatedly washing and filtering the monovalent influenza virus antigen removing liquid of each subtype cracking agent, then performing sterilization and filtration, sampling and detecting the content and purity of hemagglutinin (the purity is required to be more than or equal to 89%), and finally preparing the monovalent influenza virus antigen removing liquid of each subtype.

6. Preparation of semi-finished product

According to the hemagglutinin content of the influenza virus monovalent stock solution of each subtype, semi-finished product preparation is carried out on each type of influenza virus according to the hemagglutinin antigen content ratio of 1:1:1:1 (the hemagglutinin preparation amount can be in the range of 30-39 mu g/mL), and then the tetravalent influenza subunit vaccine semi-finished product is prepared.

Example 3 the tetravalent vaccine prepared in example 2 was used as experimental group. Compared with the experimental group, the preparation method of the control group vaccine lacks the following steps: 1) removing and collecting ovalbumin in allantoic fluid by an alkaline solution precipitation method; 2) the residual cracking agent is reduced by 95% by removing the cracking agent medium; 3) the dialysis method reduces the residual quantity of the cracking agent and the inactivating agent.

1. Finished product hemagglutinin content detection

A one-way immunodiffusion method: standard antigen and finished product samples with different concentrations are respectively added on a 1% agarose gel plate containing standard antibody of the specified subtype influenza virus, 6 mu L of each well is added, and the standing culture is carried out for more than 18 h. After being soaked in PBS for 1h, the mixture is dried, dyed and decolored. The diameters of the precipitation rings formed after the standard antigens with different concentrations and the finished product samples are diffused are accurately measured, the diameters of the precipitation rings formed by the standard antigens with different concentrations are subjected to linear regression to obtain a linear regression equation, and the linear regression equation is substituted into the diameters of the precipitation rings of the finished product samples to obtain the corresponding subtype influenza virus hemagglutinin contents of the three batches of finished product samples of the experimental group and the control group, wherein the results are shown in table 1.

TABLE 1 statistical table of hemagglutinins content (μ g/mL) of four subtypes of three batches of finished products in experimental group and control group

2. Residual quantity detection of ovalbumin

Double antibody sandwich ELISA: and respectively adding an ovalbumin standard substance and a sample to be tested into the anti-ovalbumin multi-antibody coated microporous enzyme label plate, adding an HRP coupled secondary antibody, standing and incubating for 1h at 37 ℃. And (3) adding a TMB substrate after washing the plate for three times, standing for dark color development for 10min, and adding 1M H2SO4 stop solution. The detection wavelength was set at 450nm and the reference wavelength at 620 nm. And (3) substituting the OD value of the sample into the linear regression equation according to the OD value of the standard sample to calculate the concentration of the ovalbumin of the three batches of finished products of the test experimental group and the control group. The results are shown in Table 2.

TABLE 2 statistical table of residual amounts (ng/mL) of ovalbumin in three batches of finished products of experimental group and control group

3. Detection of residual amount of cracking agent in finished product

A colorimetric method: firstly, a standard curve is made to obtain a linear regression equation. Then calculating the residual amount of the cracking agent of the sample to be tested. Adding 2mL of dichloromethane solution into a Triton N-101 standard substance solution tube (the concentration is 40/100/200/300/400 mug/mL respectively) and a sample tube respectively, mixing uniformly, adding 3mL of thiocyanocoamide solution, mixing uniformly, standing for 1.5 h at room temperature, and performing vortex oscillation once at an interval of 15 min. Standing for 30min, detecting the absorbance at OD620nm by using a lower layer sample, substituting the OD value of the sample into a linear regression equation according to the OD value of the standard sample to calculate the residual amounts of the cracking agents of the three batches of finished products of the test experimental group and the control group, wherein the results are shown in Table 3.

TABLE 3 statistics of cracking agent residue (μ g/mL) in three batches of experimental and control groups

4. Finished hemagglutinin HA antigen purity detection

Respectively taking 16 mu L of finished protein samples of the experimental group and the control group, adding 4 mu L of 5 × loading buffer solution, and boiling for 5min in boiling water; a further 1. mu.L of glycosidase F was added and incubated at 37 ℃ for 18 hours. Putting the prepared gel into an electrophoresis tank, adding 500ml of 1 × electrophoresis buffer solution, and adding 20 μ L of sample or 10 μ L of Marker into each hole; and (3) under the voltage of 100V for 40min, taking out the gel after the front edge of the bromophenol blue dye runs out of the glass plate, and marking. The protein gel is put into a proper amount of protein gel staining solution. Placing the mixture in a horizontal shaking table, slowly shaking the mixture, and dyeing the mixture for 5-10 min at room temperature. The stained protein gel was then placed in purified water and slowly shaken on a horizontal shaker and decolorized for 10min at room temperature. During the period, the purified water is replaced at least 1-2 times until the blue background in the protein gel is basically removed and the protein band is clearly visible (figure 1). And (3) photographing by using a full-function imager, and finally calculating the gray value of the strip in the picture to measure and calculate the HA purity of the influenza virus hemagglutinin (Table 4).

TABLE 4 detection of hemagglutinin end product purity for the experimental and control compositions

5. Immunogenicity testing

The research method is to take blood to detect the titer of the specific anti-influenza virus antibody generated by the induction after the experimental vaccine is inoculated to the Balb/c mice by muscle inoculation. Balb/c mice are immunized respectively by an experimental group, a control vaccine group (samples without being subjected to ovalbumin removal, past lytic agent medium and repeated washing and filtering treatment) and a PBS control group, blood is collected in groups before immunization (d 0), at 28 days after immunization (d 28) and at 42 days after immunization (d 42), mouse serum is prepared by separation, the titer of the antibody of the mouse serum after the immunization of the experimental group, the control vaccine group and the PBS control group is respectively determined by adopting a hemagglutination inhibition test method, and the result is shown in figure 2.

In summary, the following steps: compared with the existing influenza virus vaccines in the current market, the tetravalent influenza virus subunit vaccine has the advantages that the residual quantity of the cracking agent is less, the residual quantity of the cracking agent is as low as 8-15ug/ml, the antigen purity is higher, the hemagglutinin purity in a semi-finished vaccine product is more than or equal to 85%, the residual quantity of ovalbumin is as low as 21-35ng/ml, and the immunogenicity is better.

Finally, it should be noted that the above-mentioned description is only a preferred embodiment of the present invention, and those skilled in the art can make various similar representations without departing from the spirit and scope of the present invention.

Sequence listing

<110> Hui Biotechnology (Shanghai) Co., Ltd

<120> influenza virus purification method derived from chick embryo culture method, tetravalent influenza virus subunit vaccine and application thereof

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420 425 430

Lys Ile Asp Leu Trp Ser Tyr Asn Ala Glu Leu Leu Val Ala Leu Glu

435 440 445

Asn Gln His Thr Ile Asp Leu Thr Asp Ser Glu Met Asn Lys Leu Phe

450 455 460

Glu Lys Thr Lys Lys Gln Leu Arg Glu Asn Ala Glu Asp Met Gly Asn

465 470 475 480

Gly Cys Phe Lys Ile Tyr His Lys Cys Asp Asn Ala Cys Ile Gly Ser

485 490 495

Ile Arg Asn Glu Thr Tyr Asp His Asn Val Tyr Arg Asp Glu Ala Leu

500 505 510

Asn Asn Arg Phe Gln Ile Lys Gly Val Glu Leu Lys Ser Gly Tyr Lys

515 520 525

Asp Trp Ile Leu Trp Ile Ser Phe Ala Met Ser Cys Phe Leu Leu Cys

530 535 540

Ile Ala Leu Leu Gly Phe Ile Met Trp Ala Cys Gln Lys Gly Asn Ile

545 550 555 560

Arg Cys Asn Ile Cys Ile

565

<210> 2

<211> 566

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Met Lys Ala Ile Leu Val Val Met Leu Tyr Thr Phe Thr Thr Ala Asn

1 5 10 15

Ala Asp Thr Leu Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Asp Thr

20 25 30

Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His Ser Val Asn

35 40 45

Leu Leu Glu Asp Lys His Asn Gly Lys Leu Cys Lys Leu Arg Gly Val

50 55 60

Ala Pro Leu His Leu Gly Lys Cys Asn Ile Ala Gly Trp Ile Leu Gly

65 70 75 80

Asn Pro Glu Cys Glu Ser Leu Ser Thr Ala Arg Ser Trp Ser Tyr Ile

85 90 95

Val Glu Thr Ser Asn Ser Asp Asn Gly Thr Cys Tyr Pro Gly Asp Phe

100 105 110

Ile Asn Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser Val Ser Ser Phe

115 120 125

Glu Arg Phe Glu Ile Phe Pro Lys Thr Ser Ser Trp Pro Asn His Asp

130 135 140

Ser Asp Asn Gly Val Thr Ala Ala Cys Pro His Ala Gly Ala Lys Ser

145 150 155 160

Phe Tyr Lys Asn Leu Ile Trp Leu Val Lys Lys Gly Lys Ser Tyr Pro

165 170 175

Lys Ile Asn Gln Thr Tyr Ile Asn Asp Lys Gly Lys Glu Val Leu Val

180 185 190

Leu Trp Gly Ile His His Pro Pro Thr Ile Ala Asp Gln Gln Ser Leu

195 200 205

Tyr Gln Asn Ala Asp Ala Tyr Val Phe Val Gly Thr Ser Arg Tyr Ser

210 215 220

Lys Lys Phe Lys Pro Glu Ile Ala Thr Arg Pro Lys Val Arg Asp Gln

225 230 235 240

Glu Gly Arg Met Asn Tyr Tyr Trp Thr Leu Val Glu Pro Gly Asp Lys

245 250 255

Ile Thr Phe Glu Ala Thr Gly Asn Leu Val Ala Pro Arg Tyr Ala Phe

260 265 270

Thr Met Glu Arg Asp Ala Gly Ser Gly Ile Ile Ile Ser Asp Thr Pro

275 280 285

Val His Asp Cys Asn Thr Thr Cys Gln Thr Pro Glu Gly Ala Ile Asn

290 295 300

Thr Ser Leu Pro Phe Gln Asn Val His Pro Ile Thr Ile Gly Lys Cys

305 310 315 320

Pro Lys Tyr Val Lys Ser Thr Lys Leu Arg Leu Ala Thr Gly Leu Arg

325 330 335

Asn Val Pro Ser Ile Gln Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly

340 345 350

Phe Ile Glu Gly Gly Trp Thr Gly Met Val Asp Gly Trp Tyr Gly Tyr

355 360 365

His His Gln Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Leu Lys Ser

370 375 380

Thr Gln Asn Ala Ile Asp Lys Ile Thr Asn Lys Val Asn Ser Val Ile

385 390 395 400

Glu Lys Met Asn Thr Gln Phe Thr Ala Val Gly Lys Glu Phe Asn His

405 410 415

Leu Glu Lys Arg Ile Glu Asn Leu Asn Lys Lys Val Asp Asp Gly Phe

420 425 430

Leu Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn

435 440 445

Glu Arg Thr Leu Asp Tyr His Asp Ser Asn Val Lys Asn Leu Tyr Glu

450 455 460

Lys Val Arg Asn Gln Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly

465 470 475 480

Cys Phe Glu Phe Tyr His Lys Cys Asp Asn Thr Cys Met Glu Ser Val

485 490 495

Lys Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ala Lys Leu

500 505 510

Asn Arg Glu Lys Ile Asp Gly Val Lys Leu Asp Ser Thr Arg Ile Tyr

515 520 525

Gln Ile Leu Ala Ile Tyr Ser Thr Val Ala Ser Ser Leu Val Leu Val

530 535 540

Val Ser Leu Gly Ala Ile Ser Phe Trp Met Cys Ser Asn Gly Ser Leu

545 550 555 560

Gln Cys Arg Ile Cys Ile

565

<210> 3

<211> 576

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Met Val Val Thr Ser Asn Ala Asp Arg Ile Cys Thr Gly Ile Thr Ser

1 5 10 15

Ser Asn Ser Pro His Val Val Lys Thr Ala Thr Gln Gly Glu Val Asn

20 25 30

Val Thr Gly Val Ile Pro Leu Thr Thr Thr Pro Thr Lys Ser Tyr Phe

35 40 45

Ala Asn Leu Lys Gly Thr Arg Thr Arg Gly Lys Leu Cys Pro Asp Cys

50 55 60

Leu Asn Cys Thr Asp Leu Asp Val Ala Leu Gly Arg Pro Met Cys Val

65 70 75 80

Gly Thr Thr Pro Ser Ala Lys Ala Ser Ile Leu His Glu Val Arg Pro

85 90 95

Val Thr Ser Gly Cys Phe Pro Ile Met His Asp Arg Thr Lys Ile Arg

100 105 110

Gln Leu Pro Asn Leu Leu Arg Gly Tyr Glu Lys Ile Arg Leu Ser Thr

115 120 125

Gln Asn Val Ile Asp Ala Glu Lys Ala Pro Gly Gly Pro Tyr Arg Leu

130 135 140

Gly Thr Ser Gly Ser Cys Pro Asn Ala Thr Ser Lys Ile Gly Phe Phe

145 150 155 160

Ala Thr Met Ala Trp Ala Val Pro Lys Asp Asn Tyr Lys Asn Ala Thr

165 170 175

Asn Pro Leu Thr Val Glu Val Pro Tyr Ile Cys Thr Glu Gly Glu Asp

180 185 190

Gln Ile Thr Val Trp Gly Phe His Ser Asp Asn Lys Thr Gln Met Lys

195 200 205

Ser Leu Tyr Gly Asp Ser Asn Pro Gln Lys Phe Thr Ser Ser Ala Asn

210 215 220

Gly Val Thr Thr His Tyr Val Ser Gln Ile Gly Asp Phe Pro Asp Gln

225 230 235 240

Thr Glu Asp Gly Gly Leu Pro Gln Ser Gly Arg Ile Val Val Asp Tyr

245 250 255

Met Met Gln Lys Pro Gly Lys Thr Gly Thr Ile Val Tyr Gln Arg Gly

260 265 270

Val Leu Leu Pro Gln Lys Val Trp Cys Ala Ser Gly Arg Ser Lys Val

275 280 285

Ile Lys Gly Ser Leu Pro Leu Ile Gly Glu Ala Asp Cys Leu His Glu

290 295 300

Glu Tyr Gly Gly Leu Asn Lys Ser Lys Pro Tyr Tyr Thr Gly Lys His

305 310 315 320

Ala Lys Ala Ile Gly Asn Cys Pro Ile Trp Val Lys Thr Pro Leu Lys

325 330 335

Leu Ala Asn Gly Thr Lys Tyr Arg Pro Pro Ala Lys Leu Leu Lys Glu

340 345 350

Arg Gly Phe Phe Gly Ala Ile Ala Gly Phe Leu Glu Gly Gly Trp Glu

355 360 365

Gly Met Ile Ala Gly Trp His Gly Tyr Thr Ser His Gly Ala His Gly

370 375 380

Val Ala Val Ala Ala Asp Leu Lys Ser Thr Gln Glu Ala Ile Asn Lys

385 390 395 400

Ile Thr Lys Asn Leu Asn Ser Leu Ser Glu Leu Glu Val Lys Asn Leu

405 410 415

Gln Arg Leu Ser Gly Ala Met Asp Glu Leu His Asn Glu Ile Leu Glu

420 425 430

Leu Asp Glu Lys Val Asp Asp Leu Arg Ala Asp Thr Ile Ser Ser Gln

435 440 445

Ile Glu Leu Ala Val Leu Leu Ser Asn Glu Gly Ile Ile Asn Ser Glu

450 455 460

Asp Glu His Leu Leu Ala Leu Glu Arg Lys Leu Lys Lys Met Leu Gly

465 470 475 480

Pro Ser Ala Val Asp Ile Gly Asn Gly Cys Phe Glu Thr Lys His Lys

485 490 495

Cys Asn Gln Thr Cys Leu Asp Arg Ile Ala Ala Gly Thr Phe Asn Ala

500 505 510

Gly Glu Phe Ser Leu Pro Thr Phe Asp Ser Leu Asn Ile Thr Ala Ala

515 520 525

Ser Leu Asn Asp Asp Gly Leu Asp Asn His Thr Ile Leu Leu Tyr Tyr

530 535 540

Ser Thr Ala Ala Ser Ser Leu Ala Val Thr Leu Met Leu Ala Ile Phe

545 550 555 560

Ile Val Tyr Met Val Ser Arg Asp Asn Val Ser Cys Ser Ile Cys Leu

565 570 575

<210> 4

<211> 582

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Met Lys Ala Ile Ile Val Leu Leu Met Val Val Thr Ser Asn Ala Asp

1 5 10 15

Arg Ile Cys Thr Gly Ile Thr Ser Ser Asn Ser Pro His Val Val Lys

20 25 30

Thr Ala Thr Gln Gly Glu Val Asn Val Thr Gly Val Ile Pro Leu Thr

35 40 45

Thr Thr Pro Thr Lys Ser His Phe Ala Asn Leu Lys Gly Thr Glu Thr

50 55 60

Arg Gly Lys Leu Cys Pro Lys Cys Leu Asn Cys Thr Asp Leu Asp Val

65 70 75 80

Ala Leu Gly Arg Pro Lys Cys Thr Gly Lys Ile Pro Ser Ala Arg Val

85 90 95

Ser Ile Leu His Glu Val Arg Pro Val Thr Ser Gly Cys Phe Pro Ile

100 105 110

Met His Asp Arg Thr Lys Ile Arg Gln Leu Pro Asn Leu Leu Arg Gly

115 120 125

Tyr Glu His Val Arg Leu Ser Thr His Asn Val Ile Asn Ala Glu Asp

130 135 140

Ala Pro Gly Arg Pro Tyr Glu Ile Gly Thr Ser Gly Ser Cys Pro Asn

145 150 155 160

Ile Thr Asn Gly Asn Gly Phe Phe Ala Thr Met Ala Trp Ala Val Pro

165 170 175

Lys Asn Lys Thr Ala Thr Asn Pro Leu Thr Ile Glu Val Pro Tyr Ile

180 185 190

Cys Thr Glu Gly Glu Asp Gln Ile Thr Val Trp Gly Phe His Ser Asp

195 200 205

Asn Glu Thr Gln Met Ala Lys Leu Tyr Gly Asp Ser Lys Pro Gln Lys

210 215 220

Phe Thr Ser Ser Ala Asn Gly Val Thr Thr His Tyr Val Ser Gln Ile

225 230 235 240

Gly Gly Phe Pro Asn Gln Thr Glu Asp Gly Gly Leu Pro Gln Ser Gly

245 250 255

Arg Ile Val Val Asp Tyr Met Val Gln Lys Ser Gly Lys Thr Gly Thr

260 265 270

Ile Thr Tyr Gln Arg Gly Ile Leu Leu Pro Gln Lys Val Trp Cys Ala

275 280 285

Ser Gly Arg Ser Lys Val Ile Lys Gly Ser Leu Pro Leu Ile Gly Glu

290 295 300

Ala Asp Cys Leu His Glu Lys Tyr Gly Gly Leu Asn Lys Ser Lys Pro

305 310 315 320

Tyr Tyr Thr Gly Glu His Ala Lys Ala Ile Gly Asn Cys Pro Ile Trp

325 330 335

Val Lys Thr Pro Leu Lys Leu Ala Asn Gly Thr Lys Tyr Arg Pro Pro

340 345 350

Ala Lys Leu Leu Lys Glu Arg Gly Phe Phe Gly Ala Ile Ala Gly Phe

355 360 365

Leu Glu Gly Gly Trp Glu Gly Met Ile Ala Gly Trp His Gly Tyr Thr

370 375 380

Ser His Gly Ala His Gly Val Ala Val Ala Ala Asp Leu Lys Ser Thr

385 390 395 400

Gln Glu Ala Ile Asn Lys Ile Thr Lys Asn Leu Asn Ser Leu Ser Glu

405 410 415

Leu Glu Val Lys Asn Leu Gln Arg Leu Ser Gly Ala Met Asp Glu Leu

420 425 430

His Asn Glu Ile Leu Glu Leu Asp Glu Lys Val Asp Asp Leu Arg Ala

435 440 445

Asp Thr Ile Ser Ser Gln Ile Glu Leu Ala Val Leu Leu Ser Asn Glu

450 455 460

Gly Ile Ile Asn Ser Glu Asp Glu His Leu Leu Ala Leu Glu Arg Lys

465 470 475 480

Leu Lys Lys Met Leu Gly Pro Ser Ala Val Glu Ile Gly Asn Gly Cys

485 490 495

Phe Glu Thr Lys His Lys Cys Asn Gln Thr Cys Leu Asp Arg Ile Ala

500 505 510

Ala Gly Thr Phe Asp Ala Gly Glu Phe Ser Leu Pro Thr Phe Asp Ser

515 520 525

Leu Asn Ile Thr Ala Ala Ser Leu Asn Asp Asp Gly Leu Asp Asn His

530 535 540

Thr Ile Leu Leu Tyr Tyr Ser Thr Ala Ala Ser Ser Leu Ala Val Thr

545 550 555 560

Leu Met Ile Ala Ile Phe Val Val Tyr Met Val Ser Arg Asp Asn Val

565 570 575

Ser Cys Ser Ile Cys Leu

580