阅读说明：本技术 口服六价重配轮状病毒活疫苗 (Oral hexavalent reassortant rotavirus live vaccine ) 是由 杨晓明 徐葛林 李庆亮 何泗涛 白萱 姜志军 程满荣 张久威 马涛 董犇 胡蓉 于 2014-12-08 设计创作，主要内容包括：本发明涉及一种口服六价重配轮状病毒活疫苗,含有六种主要流行株血清型G1、G2、G3、G4、G8及G9,涵盖了A群轮状病毒99.6％的G血清型病毒,预计对轮状病毒腹泻有很好的预防保护效果。本发明的目的旨在提供一种口服接种的、由六价轮状病毒血清型组成的活疫苗,用以预防因轮状病毒导致的婴幼儿腹泻,所述的六价重配口服轮状病毒疫苗成分为,(1)G1、G2、G3、G4、G8及G9血清型活疫苗,各血清型原液滴度为1～5×10～(6)FFU/ml；(2)保护剂：枸橼酸0.5～2g/L、枸橼酸钠50～150g/L、蔗糖250～450g/L、氯化锌5～10mM、氯化钙10～20mM。(The invention relates to an oral hexavalent reassortant rotavirus live vaccine, which contains six main epidemic strains of serotype G1, G2, G3, G4, G8 and G9, covers the G serotype viruses of group A rotavirus of 99.6 percent, and is expected to have good prevention and protection effects on rotavirus diarrhea. Hair brushThe hexavalent reassortant oral rotavirus vaccine comprises the components of (1) serotype G1, G2, G3, G4, G8 and G9 live vaccines, wherein the titer of each serotype stock solution is 1-5 multiplied by 10 6 FFU/ml; (2) a protective agent: 0.5-2 g/L citric acid, 50-150 g/L sodium citrate, 250-450 g/L sucrose, 5-10 mM zinc chloride and 10-20 mM calcium chloride.)

1. An oral hexavalent reassorted rotavirus live vaccine is characterized in that,

(1) the vaccine contains six main epidemic strains of serotypes G1, G2, G3, G4, G8 and G9, and the titer of stock solution of each serotype is 1-5 multiplied by 106 FFU/ml;

(2) a protective agent: 0.5-2 g/L of citric acid, 50-150 g/L of sodium citrate, 250-450 g/L of cane sugar, 5-10 mM of zinc chloride and 10-20 mM of calcium chloride, wherein the volume ratio of the protective agent to the mixed solution of the hexavalent vaccine is 2: 1;

the live virus of G1 serotype NIH is numbered as follows: MVS-BRV-1;

the live virus of G2 serotype NIH is numbered as follows: MVS-BRV-2;

the live virus of G3 serotype NIH is numbered as follows: MVS-BRV-3;

the live virus of G4 serotype NIH is numbered as follows: MVS-BRV-4;

the live virus of G8 serotype NIH is numbered as follows: 1290 XUK;

the live virus of G9 serotype NIH is numbered as follows: MVS-BRV-10.

2. The process for the preparation of an oral hexavalent reassortant rotavirus live vaccine of claim 1, comprising the steps of:

(1) preparing each serotype monovalent virus stock solution, preparing each serotype virus stock solution according to the target titer, sequentially adding each stock solution into the same container sterilized in advance, and fully mixing and sterilizing to obtain a hexavalent mixed solution;

(2) preparing a protective agent mother solution and sterilizing;

(3) and (3) mixing the mother solution of the protective agent and the hexavalent mixed solution prepared in the step (1), and subpackaging according to the specification of 2 ml/bottle to obtain a finished product.

3. The method of claim 2, wherein the sterilization is performed by 0.2 μm sterilizing filter filtration sterilization.

4. The method according to claim 2, wherein the step of preparing each serotype monovalent virus stock solution is:

(1) cell passage: the culture medium of each serotype virus is a Vero cell, Vero cell working seeds are firstly recovered, after being cultured for 3-5 days at 37 ℃, subculture is carried out according to a certain proportion, culture containers are respectively a T25, a T75, a T175 cell culture bottle, a 10-layer cell factory and a 40-layer cell factory in sequence, and after being cultured for 3-5 generations in the 40-layer cell factory, G1, G2, G3, G4, G8 and G9 serotype rotaviruses can be respectively inoculated;

(2) and (3) virus culture: and (4) performing virus inoculation operation after the cell confluency in the cell factory reaches more than 90%. Before virus inoculation, a cell factory is selected for cell counting, and according to a counting result, the number of virus infection is calculated according to 0.005-0.2, so that the required virus amount is obtained; activating the virus by using trypsin, wherein the activation condition is that the temperature is 37 ℃, the concentration of the trypsin is 10-20 mu g/ml, and the activation is 15-60 min; inoculating the activated viruses into a cell factory, and culturing each inoculated serotype virus at 35-37 ℃ for 2-5 days;

(3) and (3) harvesting viruses: collecting the liquid in each cell factory in a container after the cytopathy is completed, and carrying out downstream treatment;

(4) downstream operations: firstly, carrying out cell disruption treatment, disrupting cells by adopting a freeze-thaw or high-pressure homogenization mode, wherein the freeze-thaw temperature is-30-37 ℃, the high-pressure homogenization pressure range is 100-1000bar, after the high-pressure homogenization treatment is finished, carrying out cell debris clarification filtration operation, adopting two-stage filtration, wherein the first stage is a 1.0-0.5 mu m deep filter, the second stage is a 0.2 mu m sterilization filter, after the clarification filtration is finished, carrying out ultrafiltration concentration on virus liquid, adopting a 300K pore diameter ultrafiltration membrane, and carrying out 10-time concentration on the treated virus harvest liquid to obtain the monovalent serotype vaccine stock solution.

5. Use of an oral hexavalent reassortant rotavirus live vaccine according to claim 1 for the preparation of an oral rotavirus vaccine.

Technical Field

The invention belongs to the field of vaccine preparations, and particularly relates to an oral hexavalent reassortant rotavirus live vaccine.

Background

The human rotavirus belongs to the genus rotavirus of reoviridae, is a non-enveloped RNA virus, has a particle diameter of about 75nm, and consists of three layers of icosahedral protein capsids. The genome is double-stranded RNA comprising 11 segments, and encodes 6 structural proteins (VP1, VP2, VP3, VP4, VP6 and VP7) and 5 non-structural proteins (NSP1, NSP2, NSP3, NSP4 and NSP5), the outer-layer proteins are VP4 and VP7, the inner-shell protein is VP6, and the nuclear protein is VP1, VP2 and VP 3. Gene reassortment can occur between viruses of the same group. Based on the antigenic difference of the viral inner shell protein VP6, HRV was divided into 7 groups (A-G), and only A, B, C groups resulted in diarrhea in humans. The group A is the most main reason of the infantile diarrhea, and the rotavirus vaccine of the item is also designed according to the group A; group B becomes human diarrhea rotavirus, which has been epidemic in China; group C infections are widespread throughout the world and are mostly sporadic. HRV coat protein VP7 (glycoprotein or G protein) determines the G serotype, coat protein VP4 (protease sensitive protein or P protein) determines the P serotype, common human group a rotavirus G serotypes are G1, G2, G3, G4, G8, G9, and P serotypes are P4, P8. Epidemic disease monitoring in Europe shows that the serotypes of strains are respectively G1 (63%), G2 (8%), G3 (5%), G4 (15%) and unshypted (8%), the Indian epidemic strain is G9, the Brazil is G5, and G8 gradually becomes the African dominant epidemic strain in recent years. HRV epidemiological monitoring data of 11 regions nationwide in 2008 in China show that the serotypes of strains of the HRV strain are G1 (32.4%), G2 (10%), G3 (44.8%), G4 (0.5%), G5 (0.4%) and G9 (11.9%) in sequence, a dominant epidemic strain is gradually changed from a G1 serotype in 1999 to a G3 serotype in 2007, and the G9 also becomes the dominant epidemic strain in 2013.

Rotavirus infection is a common disease spread worldwide, often occurring in infants and young children of 6-24 months of age, and also infecting adults, mainly causing Rotavirus Enteritis (Rotavirus enterititis), resulting in dehydrated diarrhea. The peak of onset is in autumn, so it is also called "diarrhea in autumn". Rotavirus is closely contacted with infection mainly through a fecal oral route, watery diarrhea and vomiting appear after 2 to 4 days of latency, dehydration symptoms are caused, fever and abdominal pain are accompanied, viruses are discharged from feces 3 to 9 days after the onset of diseases, and the virus discharge reaches a peak at 3 days.

Almost all children are infected with rotavirus 3-5 years after birth. Global rotavirus causes about 1.1 million people under 5 years old to have gastroenteritis disease requiring home care, 2.5 million people outpatient treatment, 2 million people hospitalization and 60 million people death each year. Although diarrhea is common in developed countries today, the mortality rate is low, and over 85% of deaths occur in low-income countries, both countries and non-countries. 94.5% of infants within 2 years of age in China are infected with rotavirus, the detection rate of rotavirus in excrement of infants with 0-5 months of age is 34%, the detection rate of infants with 6-11 months of age is 53%, the detection rate of infants with 12-23 months of age is 57.7%, the detection rate of infants with 24-35 months of age is 39%, and the detection rate of infants with 36-59 months of age is 15%. The gender ratio of male to female in children with rotavirus diarrhea was 1.77: 1. Rotavirus is the first pathogenic ranking list of severe diarrhea of infants under 2 years old because of its extensive and frequent causes of severe diarrhea. The season distribution is characterized in that the disease peak is 10 months per year to 1 month next year in China. Recent studies have found that rotavirus can also cause other systemic infections outside the intestinal tract.

Improvements in public health conditions do not reduce the incidence of rotavirus diarrhea. At present, no specific prevention and treatment method for rotavirus infection exists, which mainly controls the infection source, cuts off the transmission path and closely disinfects the articles which are possibly polluted. In the aspect of clinical treatment, the support therapy such as timely transfusion, acid correction and water electrolyte balance correction is mainly adopted to reduce the death rate of the infants. Worldwide, through the increasing depth of research on rotavirus, the development of rotavirus vaccines is widely considered as the most effective way for preventing severe rotavirus infection in human beings.

Currently, rotavirus vaccines on the market at home and abroad mainly comprise unit price sheep strain attenuated live vaccine rottwey of Lanzhou biological product Limited liability company, pentavalent human-bovine recombined attenuated live vaccine Rotateq of Merck company and unit price human strain attenuated live vaccine Rotarix of GSK company.

The characteristics of the diversity of rotavirus epidemic strains determine that a multivalent vaccine is the most effective method for preventing rotavirus diarrhea, however, the main rotavirus vaccines in the market at present, rottwey of Lanzhou biological product research institute and Rotarix of GSK company are monovalent serotype rotavirus vaccines, the serotype coverage range is small, Rotateq of Merck company does not contain G8 and G9 serotypes although being the pentavalent rotavirus vaccine, the coverage range of the main serotype epidemic strains is small, and the clinical test result of the Rotateq in African maraviron (G8 serotype is the dominant epidemic strain) shows that the protection rate of the heavy rotavirus diarrhea is only 40%.

Thus, there is a need for a more comprehensive multivalent rotavirus vaccine.

Disclosure of Invention

The oral hexavalent reassortant rotavirus live vaccine contains six main epidemic strains of serotype G1, G2, G3, G4, G8 and G9, covers the G serotype viruses of group A rotavirus of 99.6 percent, and is expected to have good prevention and protection effects on rotavirus diarrhea.

The invention aims to provide an oral vaccination live vaccine composed of hexavalent rotavirus serotype, which is used for preventing infantile diarrhea caused by rotavirus and belongs to the field of vaccine production and preparation. The invention provides a preparation method of each serotype stock solution, research and development of an oral rotavirus protective agent and a preparation method of a rotavirus vaccine.

According to rotavirus infection and an immunological mechanism, the rotavirus vaccine disclosed by the invention adopts an oral administration route for immunization, is consistent with a virus infection route, and is more rapid and convenient in oral administration and beneficial to operation compared with injection administration.

The hexavalent reassortment oral rotavirus vaccine comprises the following components,

(1) g1, G2, G3, G4, G8 and G9 serotype live vaccines, and the titer of each serotype stock solution is 1-5 multiplied by 10⁶FFU/ml；

(2) A protective agent: 0.5-2 g/L of citric acid, 50-150 g/L of sodium citrate, 250-450 g/L of cane sugar, 5-10 mM of zinc chloride and 10-20 mM of calcium chloride;

the live virus of G1 serotype NIH is numbered as follows: MVS-BRV-1;

the live virus of G2 serotype NIH is numbered as follows: MVS-BRV-2;

the live virus of G3 serotype NIH is numbered as follows: MVS-BRV-3;

the live virus of G4 serotype NIH is numbered as follows: MVS-BRV-4;

the live virus of G8 serotype NIH is numbered as follows: 1290 XUK;

the live virus of G9 serotype NIH is numbered as follows: MVS-BRV-10.

The invention also relates to a preparation method of the hexavalent reassortment oral rotavirus vaccine, which comprises the following steps:

(1) preparing each serotype monovalent virus stock solution, preparing each serotype virus stock solution according to the target titer, sequentially adding each stock solution into the same container sterilized in advance, and fully mixing and sterilizing to obtain a hexavalent mixed solution;

(2) preparing a protective agent mother solution and sterilizing;

(3) and (3) mixing the mother solution of the protective agent and the hexavalent mixed solution prepared in the step (1), and subpackaging according to the specification of 2 ml/bottle to obtain a finished product.

The sterilization is performed by filtering and sterilizing through a 0.2 mu m sterilization filter.

The preparation method of the univalent virus stock solution of each serotype comprises the following steps:

(1) cell passage: the culture medium of each serotype virus is a Vero cell, Vero cell working seeds are firstly recovered, after being cultured for 3-5 days at 37 ℃, subculture is carried out according to a certain proportion, culture containers are respectively a T25, a T75, a T175 cell culture bottle, a 10-layer cell factory and a 40-layer cell factory in sequence, and after being cultured for 3-5 generations in the 40-layer cell factory, G1, G2, G3, G4, G8 and G9 serotype rotaviruses can be respectively inoculated;

(2) and (3) virus culture: and (4) performing virus inoculation operation after the cell confluency in the cell factory reaches more than 90%. Before virus inoculation, a cell factory is selected for cell counting, and according to a counting result, the number of virus infection is calculated according to 0.005-0.2, so that the required virus amount is obtained; activating the virus by using trypsin, wherein the activation condition is that the temperature is 37 ℃, the concentration of the trypsin is 10-20 mu g/ml, and the activation is 15-60 min; inoculating the activated viruses into a cell factory, and culturing each inoculated serotype virus at 35-37 ℃ for 2-5 days;

(3) and (3) harvesting viruses: collecting the liquid in each cell factory in a container when the cytopathic effect is complete (the cell shedding reaches more than 75 percent), and carrying out downstream treatment;

(4) downstream operations: firstly, carrying out cell disruption treatment, disrupting cells by adopting a freeze-thaw or high-pressure homogenization mode, wherein the freeze-thaw temperature is-30-37 ℃, the high-pressure homogenization pressure range is 100-1000bar, after the high-pressure homogenization treatment is finished, carrying out cell debris clarification filtration operation, adopting two-stage filtration, wherein the first stage is a 1.0-0.5 mu m deep filter, the second stage is a 0.2 mu m sterilization filter, after the clarification filtration is finished, carrying out ultrafiltration concentration on virus liquid, adopting a 300K pore diameter ultrafiltration membrane, and carrying out 10-time concentration on the treated virus harvest liquid to obtain the monovalent serotype vaccine stock solution.

The invention also relates to the application of the hexavalent reassortant oral rotavirus vaccine in preparing the oral rotavirus vaccine.

Detailed Description

Materials and methods

1. The main materials are as follows:

1.1 Vero cell derived from ATCC (American type culture collection American Standard culture Collection), 123 generations, ATCC number: TL-CCL-81.4;

1.2 human bovine reassortant rotavirus virus seed from NIH (National Institutes of Health American National Institutes of Health)

MVS-BRV-1 human-bovine rotavirus G1 serotype

MVS-BRV-2 human-bovine rotavirus G2 serotype

MVS-BRV-3: human-bovine rotavirus G3 serotype

MVS-BRV-4: human-bovine rotavirus G4 serotype

1290 XUK: human-bovine rotavirus G8 serotype

MVS-BRV-10: human-bovine rotavirus G9 serotype

1.3 rotavirus working vaccine generation strain: the rotavirus working generation is that each serotype vaccine strain is prepared and produced by Wuhan biological products research institute Limited liability company (obtained by amplification subculture on the basis of NIH virus species).

1.4 cell factory (cell factory cat # 173240) from Corning

1.5 cell culture VP-SFM and Virus culture DMEM from GIBCO

Example 1 preparation of stocks of respective serotypes of viruses

Cell passage: the culture medium of each serotype virus is Vero cells, Vero cell working seeds are firstly recovered, after being cultured for 3-5 days at 37 ℃, subculture is carried out according to a certain proportion, culture containers are respectively a T25, a T75, a T175 cell culture bottle, a 10-layer cell factory and a 40-layer cell factory, and G1, G2, G3, G4, G8 and G9 serotype rotaviruses are respectively inoculated after being cultured for 3-5 generations in the 40-layer cell factory.

And (3) virus culture: after the confluence degree of cells in the cell factory reaches more than 90 percent, the virus inoculation operation can be carried out. Before virus inoculation, a cell factory was selected for cell counting, and based on the counting result, the required amount of virus was calculated in terms of MOI (multiplicity of viral infection) 0.005-0.2, and the virus was activated with trypsin. The activation condition is that the trypsin concentration is 10-20 μ g/ml at 37 deg.C, and the activation time is 15-60 min. The activated virus is inoculated into a cell factory, and each serotype virus after inoculation is cultured for 2-5 days at 35-37 ℃.

And (3) harvesting viruses: after the cytopathic effect is completed (cell shedding reaches over 75%), the liquid in each cell factory is collected in a container for downstream processing.

Downstream operations: firstly, carrying out cell disruption treatment, namely disrupting cells by adopting a freeze-thaw mode or a high-pressure homogenization mode, wherein the freeze-thaw temperature is-30-37 ℃, the high-pressure homogenization pressure range is 100-1000bar, and after the high-pressure homogenization treatment is finished, carrying out cell debris clarification filtration operation by adopting two-stage filtration, wherein the first stage is a 1.0 mu m +0.5 mu m deep filter, and the second stage is a 0.2 mu m sterilizing filter. And after the clarification and filtration are finished, carrying out ultrafiltration concentration on the virus liquid, and concentrating the treated virus harvest liquid by 10 times by adopting an ultrafiltration membrane with the aperture of 300K to obtain the monovalent serotype vaccine stock solution.

Example 2 development of vaccine protection Agents

(1) Determination of the acid resistance of the Virus

The lowest pH value that a hexavalent rotavirus vaccine can tolerate is determined through experiments.

Mixing the sexavalent serotype virus mixed vaccine (mixing sexavalent vaccine stock solution with equal volume) with 1% sodium citrate solution with equal volume.

(2) Preparing 7 disposable sterile 50ml centrifuge tubes, wherein 6 centrifuge tubes are respectively added with 2ml of the hexavalent seedlings mixed in the step (1), and the other 1 centrifuge tube is added with 1ml of the hexavalent mixed seedlings and 1ml of DMEM for comparison.

(3) The pH was adjusted, and 0.1M hydrochloric acid (0 ml, 0.4ml, 0.6ml, 0.7ml, 0.8ml, 1.2 ml) was added to 6 centrifuge tubes, respectively, to obtain corresponding pH values of 7.84, 5.02, 4.00, 3.50, 3.05, 2.00.

(4) The 7 centrifugal tubes were incubated in a 37 ℃ water bath for 2 h.

(5) At the end of the incubation period, DMEM was added to 20ml in each centrifuge tube.

(6) And (3) subpackaging the test samples into freezing storage tubes, and storing in a refrigerator at the temperature of-70 ℃ for detection.

(7) And repeatedly detecting the virus titer (FFA) for three times by using a sample corresponding to each pH value, and comparing the virus titer (FFA) with a control group, wherein the lowest pH value corresponding to the group without obvious change of the titer is the lowest pH value which can be tolerated by the bovine human reassorted rotavirus attenuated vaccine strain. Table 1 shows the results of three titer determinations for the six serotypes.

TABLE 1 results of three titre tests for six serotypes (unit: lgFFU/ml)

It can be seen that at pH above 4.0, there was no significant change in titer compared to the control group, whereas at pH below 4.0, there was a significant decrease in viral titer. Thereby determining that the lowest pH value which can be tolerated by the bovine human reassortant rotavirus attenuated vaccine strain is 4.0.

(2) Optimal antacid buffer pair selection

Four common acids (1M in concentration) were titrated with 1M sodium hydroxide by acid-base titration, and the buffer capacity of the four acids between pH4-7 was determined based on the amount of sodium hydroxide consumed. Between pH4 and 7, the buffer capacity is: citric acid (consuming sodium hydroxide 17ml), malic acid (consuming sodium hydroxide 10ml), phosphoric acid (consuming sodium hydroxide 9ml), acetic acid (consuming sodium hydroxide 8ml), namely determining the buffer pair with stronger buffer capacity as sodium hydroxide pair citric acid.

(3) Determination of antacid buffering versus buffering capacity

According to the reference, the gastric acid content in the infant endocrine for 20 minutes is in the range of 0.8-1.0mM, three 50ml centrifuge tubes are taken, hydrochloric acid is added in the amount of 0.8mM, 0.9mM and 1.0mM respectively, 0.1M sodium citrate is added into the three centrifuge tubes respectively, the pH is monitored simultaneously, the addition is stopped when the pH rises to 4.0, and the results are shown in Table 2.

TABLE 2 buffer to buffer capacity measurement results

6-7.5ml of 0.1M sodium citrate is needed for neutralizing 0.8mM-1.0mM hydrochloric acid, and the conversion mass is as follows: 0.1548g to 0.1935g, namely 2ml of formula contains sodium citrate in the following amount: 0.1548g to 0.1935g, wherein the required amount of sodium citrate per 100ml is as follows: 7.74g-9.67 g.

(4) Antacid capacity identification of antacid

Four sets of experiments were set in parallel, four 15ml centrifuge tubes were taken, and set 1, set 2, set 3, and set 4 were labeled respectively, wherein:

group 1: mixing 1ml of hexavalent mixed seedlings with 1m of DMEM in equal volume;

group 2: preparing an antacid with the concentration of 2 times in advance according to the calculated amount of the antacid, and taking 1ml to mix with the hexavalent mixed seedlings in equal volume;

group 3: mixing the hexavalent mixed seedlings with 1ml of antacid with concentration of 2 times, and adding 1ml (1.0mM) of 1M hydrochloric acid;

group 4: mixing the hexavalent mixed seedlings and 1ml of 1M hydrochloric acid respectively in equal volume;

(2) four centrifuge tubes were placed in a 37 ℃ water bath and incubated for 2 h.

(3) After the incubation time was complete, 1ml of each of group 1, group 2 and group 4 was quickly added to the prepared centrifuge tubes and supplemented with 9ml of DMEM, 1.3ml of DMEM and 8.7ml of DMEM in group 3, respectively, to stop the hydrochloric acid effect.

(4) And (4) subpackaging the four groups of samples into a freezing storage tube, labeling, and storing in a refrigerator at the temperature of-70 ℃ for detection.

(5) The titer test (FFA test) is carried out on each group of samples, and the antacid effect of the antacid is determined by the difference of the titers of each group.

The results are shown in Table 3.

TABLE 3 antacid buffer capacity and Effect on Titavale of hexavalent Miao (unit: lgFFU/ml)

It can be seen that there was essentially no difference in the titer of each serotype in group 1, group 2, and group 3, while the drop in titer in group 4 was significant, indicating that the antacid selected was sufficient to neutralize 1.0mM hydrochloric acid and that the antacid itself had no effect on the virus.

EXAMPLE 3 screening of different formulations

The contents of the components of the 5 vaccine protective agents are shown in Table 4

TABLE 4 contents of the various components of the various protective agents

Each formulation was mixed with the most temperature sensitive serotype G9 virus and subjected to accelerated stability testing, the results of which are shown in Table 5

TABLE 5 Virus stability tests for different protection formulations (all data are mean of three test results in lgFFU/ml)

It can be seen that the five formulations all meet the requirements for the protection of G9, wherein the titer of G9 is lowest when F5 is placed at 37 ℃ for 7 days, the G9 is the best protection, and F5 is the optimal protective agent formulation of rotavirus vaccine.

Example 4 vaccine formulation

(1) Preparation of protective agent

Firstly, preparing 1.5 times of protective agent mother liquor according to the components and contents of the developed final formula

(2) Mixing stock solutions of hexavalent vaccine

Calculating the amount of hexavalent stock solution according to the prepared target titer (6.2lgFFU/ml), sequentially adding each stock solution into the same container sterilized in advance, mixing thoroughly, sterilizing by 0.2 μm, and filtering into another container

(3) Mixing 1.5 times of the mother liquor of the protective agent and the prepared hexavalent mixed solution according to the volume ratio of 2:1 to obtain a semi-finished product

(4) And (4) conveying the semi-finished product to a subpackaging room, subpackaging according to the specification of 2 ml/bottle, labeling and outsourcing to obtain the finished product.

The results of the detection of the titer of each serotype of monovalent virus on the vaccine preparations are shown in Table 6 below

TABLE 6 titer test results for each serotype of the finished vaccine

Serotype	Finished product (20111103)
		G1	1.36×106FFU/ml
G2	7.28×105FFU/ml
		G3	1.53×106FFU/ml
G4	1.21×106FFU/ml
		G8	1.09×106FFU/ml
G9	9.85×105FFU/ml

From the results, the titer of each serotype drifts less and can satisfy each bloodClear type is more than 5.00 multiplied by 10⁵FFU/ml requirements.

Finally, it should be noted that the above embodiments are only used to help those skilled in the art understand the essence of the present invention, and are not used to limit the protection scope of the present invention.