CN111471658A - Virus purification method and bivalent inactivated vaccine prepared by same - Google Patents
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Abstract
The invention relates to a virus purification method and a bivalent inactivated vaccine prepared by the method, wherein the virus purification method comprises the steps of centrifuging and filtering virus liquid of viruses to obtain supernate serving as a sample to be concentrated; concentrating the sample to be concentrated through a 100KD membrane package to obtain a virus concentrated solution; and purifying the virus concentrated solution by using a 4FF gel chromatography column to obtain a purified virus solution. According to the application, virus liquid is purified by membrane-packed ultrafiltration concentration and purification treatment combined with 4FF gel chromatography to obtain the high-purity porcine epidemic diarrhea virus and porcine transmissible gastroenteritis virus, the removal rate of foreign proteins reaches 91%, and the effective antigen content reaches 95%. After the purified virus is matched with 201 adjuvant to prepare the bivalent vaccine for inoculating piglets, the body temperature fluctuation is obviously reduced, meanwhile, the antibody titer is respectively improved by 69.5 percent and 139 percent after immunization for 28 days compared with that before purification, and the safety and the immunization effect are effectively improved.
Description
Technical Field
The invention relates to the technical field of veterinary vaccines, in particular to a virus purification method and a bivalent inactivated vaccine prepared by the method.
Background
Porcine Epidemic Diarrheal (PED) is an acute, highly-contact intestinal infectious disease caused by Porcine Epidemic Diarrhea Virus (PEDV) and characterized by watery diarrhea, vomiting and decreased appetite. Pigs at all ages are susceptible to the disease, especially piglets are the most serious, and the death rate is up to 100%. PED is reported for the first time in 1982 in China and continues to be popular in China, and until 2010, variant strain PEDV begins to be popular in China, which causes great economic loss to the pig industry in China. In recent years, the popularity of PED is more and more extensive, new variant strains continuously appear, the antigenicity of commercial vaccines and epidemic strains is not matched, and the immune effect is poor, so that the healthy development of the pig industry in China is seriously influenced.
PEDV belongs to the genus coronaviruses of the family coronaviridae, is an unfractionated, single-stranded, positive-stranded RNA virus (ssRNA) with an envelope that has a genome of approximately 28.5kb in length. The virus particles of PEDV are polymorphic, have a roughly spherical shape with an average diameter of 95-190nm, and have petaloid fibrils with a length of about 18nm on the surface, radially distributed from the core to the periphery. PEDV is not acid and alkali resistant, is sensitive to ethanol and chloroform, can be killed by a common disinfectant, is resistant to low temperature and high temperature, is relatively stable at 4-50 ℃, and loses the infection capacity after being treated for 30min at the temperature of more than 60 ℃.
Porcine Transmissible Gastroenteritis (TGE) is an acute and highly contact infectious disease of the digestive tract of pigs caused by Transmissible gastroenteritis virus (TGEV), and has the main clinical symptoms of vomiting, severe diarrhea and dehydration, and is often mixed with other diseases, pigs of all breeds and ages are susceptible to the virus, newborn piglets are particularly severe, and the death rate gradually decreases with the increase of the age of days. It is often mixed with pathogens such as Porcine rotavirus (PoRV), Porcine Epidemic Diarrheal Virus (PEDV), pathogenic escherichia coli (Enteropathogenic e.coli) and the like, is a main cause of diarrheal death of piglets in large-scale pig farms, and causes serious economic loss to the pig industry in China.
TGEV belongs to family Coronaviridae, genus α coronavirus, is observed under electron microscope after purifying, TGEV particle form is various, mostly circular, ellipse, the diameter is about 90 nm-200 nm, the surface has double-layer membrane structure, the outermost layer is the tunica vaginalis formed by double-layer lipid layer, there are petal-like projections of 8 nm-24 nm on the tunica vaginalis, TGEV is relatively stable under the low temperature condition, it is low to the heat resistance.
Specific therapeutic drugs for PED and TGE are not available at present, clinically, the vaccine immunization is an effective measure for pre-controlling PED and TGE, and has the remarkable characteristics of acute morbidity, high mortality and great harm. At present, attenuated vaccines, inactivated vaccines, subunit vaccines and the like of PED and TGE are developed, the vaccines can reduce serious hazards caused by PED and TGE to a certain extent, but the vaccines have weak reaction specificity and low level of neutralizing antibodies, and because PEDV and TGEV particles are fragile and low in tolerance, high-purity and complete-structure virus antigens are urgently needed, and a new process is developed to obtain high-quality viruses. The safety and effectiveness of vaccines are mainly influenced by factors such as virus titer and antigen purity. Vaccines produced by conventional processes have certain side effects in clinical applications, mainly related to the heterologous components contained in the viral antigens. These heterologous components not only tend to cause immune side effects, which can cause problems such as sow miscarriage, but also compromise the efficacy of the vaccine. Since a virus cultured in a cell culture as an antigen has a certain nonspecific reaction in clinical applications, further improvement of the virus titer and the antigen purity is urgently required in order to improve the purity, safety and effectiveness of a vaccine.
The membrane-packed concentration technology is that feed liquid circulates on the upper surface of a membrane at a certain flow rate, substances smaller than the pore diameter of the membrane can permeate the membrane to a permeation end, and substances larger than the pore diameter of the membrane can be intercepted by the membrane, so that the concentration of target substances and the fractional separation of different substances are realized. The membrane-packed ultrafiltration belongs to a tangential flow filtration technology, liquid tangentially flows through the surface of a membrane, a part of solution is pressed into a filtration membrane by transmembrane pressure generated by fluid, a intercepted part circularly flows back in a system, the liquid continuously flows through the surface of the filtration membrane at a certain speed in the whole process, the surface of the filtration membrane is washed while filtering, and a gel layer can not be formed on the surface of the membrane, so that particles in feed liquid can not block the filtration membrane quickly, stable filtration speed is kept, lower shearing force is realized, mild operation can effectively prevent glycoprotein on the surface of virus from falling off and protein from gathering, and meanwhile, permeation and removal of foreign proteins are facilitated.
The molecular sieve gel chromatography is a porous substance without surface charge, when a sample with various components moves in the gel, the substance with high molecular weight can not enter the gel pores and almost vertically moves downwards between the gels because of different molecular weights when the sample is eluted by a buffer solution, and the substance with low molecular weight enters the gel pores to perform 'detour' operation, so that the substance can sequentially flow out of the gel column according to the size of the molecular weight to achieve the purpose of separation. The separation and purification method of gel chromatography is widely applied to the production of human vaccines and veterinary vaccines, and can effectively separate target proteins and foreign proteins to obtain pure target proteins.
Disclosure of Invention
The invention aims to provide a virus purification method and a bivalent inactivated vaccine prepared by the method, which are used for overcoming the technical problems of poor vaccine safety and poor immune effect caused by low virus purity in the prior art.
In a first aspect, the present invention provides a method of virus purification comprising:
(1) centrifuging and filtering the virus liquid of the virus to obtain supernatant serving as a sample to be concentrated;
(2) concentrating the sample to be concentrated through a 100KD membrane package to obtain a virus concentrated solution;
(3) purifying the virus concentrated solution by using a 4FF gel chromatography column to obtain a purified virus solution;
the virus is porcine epidemic diarrhea virus or porcine transmissible gastroenteritis virus.
Further, in step (1), the porcine epidemic diarrhea virus is cultured by using an adherent Vero cell, and/or the porcine transmissible gastroenteritis virus is cultured by using an adherent ST cell.
Further, the concentration in the step (2) is 10-15 times of the concentration of the sample to be concentrated.
Further, the method of step (2) is as follows:
moving the sample to be concentrated into a sample treatment tank, circulating for 5-10 min, keeping the pressure at the liquid inlet end at 0.05-0.1 MPa, opening the permeation end to continuously permeate a 100KD membrane package, replenishing medical normal saline solution with the same volume as the residual volume to carry out washing filtration when the sample volume is concentrated to 1/10-1/40 of the sample volume, circulating for 5-10 min, and opening the permeation end to continuously permeate; repeating the washing and filtering process for multiple times, and circulating for 2-5 min to obtain the virus concentrated solution.
The circulation process of each part is preferably high pump speed circulation for 2min, the pressure of the liquid inlet end is preferably 0.1MPa, and the high pump speed is 1.5-2.0L/min.
In membrane-packed concentration, when the flow rate is high, the concentration efficiency can be increased, but the protein recovery rate in the sample is reduced, the integrity of virus particles is not facilitated, and when the flow rate is low, the concentration of virus liquid is not facilitated, and the concentration efficiency is also poor. The invention adopts a 100KD membrane package to carry out concentration by a specific concentration process, can realize the highest recovery rate and ensure the integrity of virus particles.
Further, the step (1) is to take the supernatant as the sample to be concentrated after the virus liquid is subjected to continuous flow centrifugation and membrane filtration.
Further, the filler of the 4FF gel chromatographic column in the step (3) is Sepharose 4FF agarose gel chromatographic filler.
Further, the purification conditions in step (3) are: the linear flow rate of the virus concentrated solution is 30-80 cm/h, the pressure is less than 2.5bar, the sample loading amount is controlled at 10-15% of the column volume, and medical normal saline solution is adopted for elution.
Further, the method of step (3) is:
cleaning and disinfecting the molecular sieve gel chromatographic column by using 0.5-1.0 mol/L NaOH solution, washing the molecular sieve gel chromatographic column by using sterile water until the pH value is 7.0-7.5, balancing the molecular sieve gel chromatographic column by using medical normal saline solution until the conductivity of the molecular sieve gel chromatographic column is consistent after the molecular sieve gel chromatographic column reaches the front column, stabilizing the pH value at 7.0, and detecting UV by using ultraviolet280The baseline is stable, and finally the virus concentrated solution obtained in the step (2) is loaded and eluted by medical normal saline solution until UV is reached280When the value begins to rise, collecting the first protein peak, namely the purified virus liquid.
In a second aspect, the present invention provides a bivalent inactivated vaccine comprising inactivated porcine epidemic diarrhea virus and porcine transmissible gastroenteritis virus purified by the above method and 201 adjuvant.
Furthermore, the 201 adjuvant belongs to an oil adjuvant, consists of corresponding injectable oil and a surfactant, has a strong immune enhancement effect and small side effect compared with the traditional Freund adjuvant, belongs to a classical oil emulsion adjuvant, can specifically induce lymphocyte proliferation reaction and neutralizing antibody reaction, has high reaction generation speed and high strength, and is superior to other adjuvants. Further, the ratio of the inactivated porcine epidemic diarrhea virus, the inactivated porcine transmissible gastroenteritis virus and the 201 adjuvant is 2: 1: 3.
the invention provides a virus purification method and a bivalent inactivated vaccine prepared by the method, which have the following beneficial effects:
(1) the process of the invention is not only suitable for porcine epidemic diarrhea virus, but also suitable for porcine transmissible gastroenteritis virus, and only one virus purification method is available in the existing method. The method disclosed by the invention is simple to operate, good in repeatability, low in cost, high in treatment efficiency and suitable for industrial large-scale production. The porcine epidemic diarrhea virus and the porcine transmissible gastroenteritis virus obtained by purification have high purity, the use amount of the antigen is reduced in the later vaccine preparation process, the cost is saved, the problem of side reaction of the vaccine is fundamentally solved, and the method has good popularization prospect.
(2) The invention solves the technical problem that the porcine epidemic diarrhea virus and the porcine transmissible gastroenteritis virus are difficult to realize high purity, realizes the preparation of the high purity porcine epidemic diarrhea virus and the porcine transmissible gastroenteritis virus by membrane-packed ultrafiltration concentration purification treatment and Sepharose 4FF molecular sieve gel chromatography purification treatment, obtains complete virus particles, obviously improves compared with the traditional process, and is the fundamental guarantee of the vaccine quality.
(3) The invention uses 100KD membrane pack to hyperfiltrate and concentrate, Sepharose 4FF gel chromatography purification process, two purification steps are closely connected and can not be divided. The sample loading amount of gel chromatography purification directly limits the amount and efficiency of large-scale production, so that the concentration step can remove part of protein, and the volume of later-stage antigen processing is reduced, thereby being a key step for realizing large-scale high-efficiency production.
(4) According to the process, the removal rate of the foreign protein is up to 91 percent, the content of the effective antigen is up to 95 percent, the side reaction of the vaccine caused by the foreign protein is reduced to be lower, the safety of the vaccine is improved, the good immunogenicity is kept, and a foundation is laid for the prevention and control of the porcine epidemic diarrhea and the porcine transmissible gastroenteritis.
(5) The technical parameters and the production scale of the invention can be linearly enlarged, and the production scale can be enlarged to 500L, 1000L, 2000L, 3000L and 5000L, and all the production scales have operability.
Drawings
FIG. 1 is a 4FF gel chromatography purification map of porcine epidemic diarrhea virus provided in example 4 of the present invention;
FIG. 2 is a 4FF gel chromatography purification map of porcine transmissible gastroenteritis virus provided in example 4 of the present invention;
FIG. 3 is the electron microscope observation result of porcine epidemic diarrhea virus provided in example 4 of the present invention;
FIG. 4 is an electron microscope observation result of transmissible gastroenteritis virus provided in example 4 of the present invention;
FIG. 5 shows the body temperature change after vaccination with and without purified vaccine provided in Experimental example 2 of the present invention.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
In the following examples, the apparatus used is as follows:
the types of microfiltration membranes used for clarification filtration are: sidoris 0.65 um.
The membrane package types used for ultrafiltration concentration were: 3M 21446807E-SW.
The molecular sieve gel chromatographic column model is as follows: BPG200/1000 Column.
The molecular sieve gel chromatographic column filler is as follows: sepharose 4FF gel chromatography packing.
The virus solution was as follows: culturing porcine epidemic diarrhea virus by rotary bottle culture and suspension sheet carrier with toxin value not less than 107.0TCID50Perml, spinner flask culture and suspension plate vehicle culture of porcine transmissible gastroenteritis virus are commercially available.
Medical normal saline formulation (1L) 0.79g disodium hydrogen phosphate, 0.43g potassium dihydrogen phosphate, and 5.8g sodium chloride.
For each step obtainedThe products are respectively passed through A280Detecting the protein content and passing the TCID50And (5) detecting the virus titer.
Formula v of flow rate 1.56 pi r2In ml/min, r represents the radius of the column.
EXAMPLE 1 Membrane-pack Ultrafiltration concentration Process
1 System Pre-processing
1.1 respectively installing 30KD, 100KD and 300KD membrane packages into an ultrafiltration control device, connecting corresponding pipelines, and circularly washing a hollow fiber column for 10min by using sterile water for injection after assembly.
1.2 System integrity detection
Pressure maintenance measures the integrity of the system.
1.3 processing of the System
Cleaning and sterilizing, namely circularly sterilizing the system for 30min by using a sterile 1.0 mol/L NaOH solution, cleaning the system by using sterile water for injection, washing off residual alkali solution until the pH value is 7.0, and finally balancing a membrane and a pipeline by using a sterile medical physiological saline solution.
2 Virus Ultrafiltration concentration Process
Adopting continuous flow centrifugal treatment, filtering the porcine epidemic diarrhea virus and the porcine transmissible gastroenteritis virus which are treated by a 0.65um filter membrane into a sample treatment tank to obtain an antigen 15L to be concentrated, circulating for 2min at a high pump speed, keeping the pressure of a liquid inlet end at 0.1MPa, continuously permeating the permeation end in a non-pressure state, supplementing medical physiological saline with the same volume as the rest volume when the sample volume is concentrated to 1/10 of the sample volume, washing and filtering for the first time, circulating for 2min at the high pump speed, opening the permeation end for continuous permeation, repeating the above operations for washing and filtering for four times, finally circulating the concentrated solution for 2min, and collecting the concentrated solution 1.5L.
TABLE 1 evaluation of the Effect of concentration by ultrafiltration of porcine epidemic diarrhea Virus
TABLE 2 evaluation of the Effect of the porcine transmissible gastroenteritis Virus by ultrafiltration concentration
The results in tables 1 and 2 show that the membrane packs with different pore diameters have obvious effect difference on the ultrafiltration concentration of the porcine epidemic diarrhea and the porcine transmissible gastroenteritis virus. The removal effect of the 300KD membrane-coated hybrid protein is the best, but the virus recovery rate is lower; the recovery of virus was best with a membrane package with a pore size of 30kD, but the treatment efficiency was the worst. The three indexes of comprehensive treatment efficiency, foreign protein removal rate and virus recovery rate, and a membrane package with the aperture of 100KD is the most suitable aperture in ultrafiltration concentration.
Example 2
This example provides a 100KD membrane-packed ultrafiltration concentration process, which has the same specific steps as example 1, except that step 2 of the virus concentration process is adjusted to:
adopting continuous flow centrifugal treatment, filtering the porcine epidemic diarrhea virus and the porcine transmissible gastroenteritis virus which are treated by a 0.65um filter membrane into a sample treatment tank to obtain an antigen 15L to be concentrated, circulating for 2min at a high pump speed, keeping the pressure of a liquid inlet end at 0.04MPa, continuously permeating the permeation end in a non-pressure state, supplementing medical physiological saline with the same volume as the rest volume when the sample volume is concentrated to 1/10 of the sample volume, washing and filtering for the first time, circulating for 2min at the high pump speed, opening the permeation end for continuous permeation, repeating the above operations for washing and filtering for four times, finally circulating the concentrated solution for 2min, and collecting the concentrated solution 1.5L.
Example 3
This example provides a 100KD membrane-packed ultrafiltration concentration process, which includes the following steps:
adopting continuous flow centrifugal treatment, filtering the porcine epidemic diarrhea virus and the porcine transmissible gastroenteritis virus which are treated by a 0.65um filter membrane into a sample treatment tank to obtain an antigen 15L to be concentrated, circulating for 2min at a high pump speed, keeping the pressure of a liquid inlet end at 0.1MPa, continuously permeating the permeation end in a non-pressure state, supplementing medical physiological saline with the same volume as the rest volume when the sample volume is concentrated to 1/10 of the sample volume, washing and filtering for the first time, circulating for 2min at the high pump speed, opening the permeation end for continuous permeation, repeating the above operations for washing and filtering for four times, finally circulating the concentrated solution for 2min, and collecting the concentrated solution 1.5L.
TABLE 3 evaluation of the concentration of porcine epidemic diarrhea virus by ultrafiltration
TABLE 4 evaluation of the Effect of the porcine transmissible gastroenteritis Virus by concentration by ultrafiltration
In the ultrafiltration concentration process using the 100KD membrane, the concentration effect is compared when the pressure at the liquid inlet end is 0.04MPa and 0.1MPa, and the results in tables 3 and 4 show that when the pressure at the liquid inlet end is 0.1MPa, the removal effect of the impurity protein is the best, the treatment efficiency is the highest, but the virus recovery rate is low. But the pressure of the liquid inlet end is kept to be 0.1MPa, which is the most suitable pressure in the ultrafiltration concentration.
EXAMPLE 4 molecular Sieve gel chromatography Process
1 System Pre-processing
1.1 installing the 4FF molecular sieve chromatographic column in place and measuring the column effect.
1.2 treating molecular sieve gel chromatographic column with sterile 0.5 mol/L NaOH for 2 Column Volumes (CV), washing with sterile injection water to pH7.0, balancing ion exchange column with medical normal saline solution until the conductivity column is consistent, pH is stabilized at 7.0, and ultraviolet UV is used280The baseline was stable.
2 purification Process
And (3) loading the concentrated virus sample, setting the linear flow rate of the loaded sample to be 30cm/h, and controlling the pressure to be less than 2.0 bar. Loading amount of 5%, 10% and 15% of column volume, eluting protein with medical normal saline solution after loading is finished, and waiting for UV280Collecting eluted samples when the peak value begins to start, and finishing UV when the peak value of the target protein is finished280Value ofStopping collecting the elution sample of the first elution peak when the value is reduced to the minimum or the value begins to increase, and obtaining the sample after virus purification.
TABLE 5 evaluation of purification Effect of porcine epidemic diarrhea Virus
TABLE 6 evaluation of purification Effect of porcine transmissible gastroenteritis Virus
Detection of protein concentration and TCID by A280 method50And (5) detecting the virus titer.
Fig. 1 and fig. 2 are 4FF gel chromatography purification profiles of porcine epidemic diarrhea virus and porcine transmissible gastroenteritis virus provided in this example.
FIGS. 3 and 4 show the results of the electron microscope observation of porcine epidemic diarrhea virus and transmissible gastroenteritis virus purified at 15% loading according to this example. The purified virus was negatively stained and observed by electron microscopy to check the integrity of the virions. Figure 3 shows that the PEDV particles are more intact, with an intact "corona"; figure 4 shows that TGEV particles are more intact, with intact "crowns" visible.
The results in the table show that the removal rate of the 5% sample amount of the hybrid protein is high, and the virus recovery rate is also high. The removal rate of the impurity protein is reduced when the sample amount is increased, and the recovery rate of the virus is simultaneously reduced. The sample loading amount of 10-15% is more suitable in large-scale production.
EXAMPLE 5 purification Process of porcine epidemic diarrhea Virus and porcine transmissible gastroenteritis Virus by spinner bottle culture
(1) Taking Vero cells and ST cells subjected to adherent culture in a roller bottle at 37 ℃, respectively inoculating porcine epidemic diarrhea virus and porcine transmissible gastroenteritis virus, and performing continuous flow centrifugation on the obtained virus liquid, wherein the Vero cells and the ST cells are respectively treated by a 3+0.8um filter membrane or a 0.65um filter membrane to obtain a sample to be concentrated;
(2) performing ultrafiltration concentration by using a membrane, namely treating a 15L sample to be concentrated by using a 100KD membrane to obtain a concentrated sample 1.5L in the same way as in the example 1;
(3) molecular sieve gel chromatography as in example 4, sample 1.5L after concentration was loaded at 10% column volume (15L) and the first peak eluted was collected.
EXAMPLE 6 purification of porcine epidemic diarrhea Virus and porcine transmissible gastroenteritis Virus by suspension of sheet Carrier
(1) Taking Vero cells and ST cells which are subjected to suspension culture at 37 ℃, respectively inoculating porcine epidemic diarrhea virus and porcine transmissible gastroenteritis virus, and obtaining virus liquid, carrying out continuous flow centrifugation on the virus liquid, and treating the virus liquid by using a 0.65um filter membrane to obtain a sample to be concentrated;
(2) performing ultrafiltration concentration by using a membrane, namely treating a 15L sample to be concentrated by using a 100KD membrane to obtain a concentrated sample 1.5L in the same way as in the example 1;
(3) molecular sieve gel chromatography as in example 4, sample 1.5L after concentration was loaded at 10% column volume (15L) and the first peak eluted was collected.
Table 7 evaluation of the Effect of porcine epidemic diarrhea virus purified in example 5 and example 6
TABLE 8 evaluation of Effect of porcine transmissible gastroenteritis Virus obtained by purification in examples 5 and 6
The results of the above table show: the combination of 100KD membrane-packed ultrafiltration concentration and 4FF gel chromatography purification process is not only suitable for porcine epidemic diarrhea virus and porcine transmissible gastroenteritis virus cultured by spinner bottles, but also suitable for porcine epidemic diarrhea virus and porcine transmissible gastroenteritis virus cultured by suspension sheet carriers, and the virus recovery rate of the two culture modes can reach 92%, and the removal rate of foreign protein is as high as 94%.
Example 7 Large Scale production of high purity porcine epidemic diarrhea Virus and porcine transmissible gastroenteritis Virus
(1) Centrifuging the continuous antigen flow, taking supernatant 300L, and treating with 0.65um filter membrane to obtain sample to be concentrated;
(2) concentrating by membrane-packed ultrafiltration, which is the same as in example 1, and treating a sample to be concentrated of 300L with a 100KD membrane pack to obtain a concentrated sample of 30L;
(3) molecular sieve gel chromatography as in example 4, concentrated sample 30L was loaded at 15% column volume (15L) and the first peak eluted was collected and the purification was completed in two runs.
Example 8 Large Scale production of high purity porcine epidemic diarrhea Virus and porcine transmissible gastroenteritis Virus
(1) Centrifuging the continuous antigen flow, taking supernatant 500L, and treating with 0.65um filter membrane to obtain sample to be concentrated;
(2) concentrating by membrane filtration, namely treating a sample to be concentrated of 300L by a membrane with the pressure of 100KD to obtain a concentrated sample 45L in the same way as in the example 1;
(3) molecular sieve gel chromatography as in example 4, concentrated sample 45L was loaded at 15% column volume (15L) and the first peak eluted was collected and three additional runs were required to complete the purification.
Table 9 evaluation of the Effect of porcine epidemic diarrhea virus purified in example 7 and example 8
TABLE 10 evaluation of Effect of porcine transmissible gastroenteritis Virus obtained by purification in example 7 and example 8
Example 9 production of a porcine epidemic diarrhea Virus, porcine transmissible gastroenteritis Virus, bivalent inactivated vaccine
On the basis of examples 1-8, the optimal production process parameters are determined, and the production scale can be enlarged to 2000L, 3000L and 5000L by adopting a linear amplification principle and matching corresponding tanks and pipelines.
In this example, the virus solution for vaccine testing was purified using the determined optimal production process parameters, and the porcine epidemic diarrhea virus solution and the purified virus solution, the porcine transmissible gastroenteritis virus solution and the purified virus solution were inactivated with 0.2% formaldehyde for 48 hours, respectively. The inactivated virus liquid is detected by a cell method to completely inactivate the virus. The 201 adjuvant is adopted to be matched with the porcine epidemic diarrhea virus and the porcine transmissible gastroenteritis virus according to the proportion of 3:2:1 to obtain the bivalent inactivated vaccine.
Test example 1 adjuvant comparative test
In this test example, the high-purity porcine epidemic diarrhea virus and porcine transmissible gastroenteritis virus prepared in example 7 were inactivated with 0.2% formaldehyde for 48 hours, respectively. The inactivated virus liquid is detected by a cell method to completely inactivate the virus. Emulsifying the 201 adjuvant and the CPC adjuvant with porcine epidemic diarrhea virus and porcine transmissible gastroenteritis virus respectively to prepare the bivalent inactivated vaccine. Wherein the 201 adjuvant is matched with porcine epidemic diarrhea virus and porcine transmissible gastroenteritis virus according to the proportion of 3:2: 1; the CPC adjuvant is matched with porcine epidemic diarrhea virus and porcine transmissible gastroenteritis virus according to the proportion of 3:4:8 (the proportion of the CPC adjuvant is 20%). The two bivalent inactivated vaccines are subjected to vaccine neutralizing antibody detection, and the adjuvant effect is compared. The method comprises the following specific steps:
5 healthy weaned piglets of 4 weeks old are selected per group, 12 healthy weaned piglets are selected, and PEDV and TGEV neutralizing antibodies in serum are detected to be negative before immunization. The injection is carried out by adopting 2ml of immunization dose per head on a posthai acupoint. Animals were observed for local and systemic responses after vaccination and blood was collected for PEDV, TGEV neutralizing antibody levels at 14 days, 28 days.
The results shown in the following table were obtained:
TABLE 11201 evaluation of the Effect of adjuvant and CPC adjuvant
The neutralizing antibody level of the piglets is continuously monitored 14 days and 28 days after the first immunization, and the detection result shows that: the titer of the TGEV and PEDV neutralizing antibody rapidly rises from 14 days to 28 days after primary immunization, but the antibody level of the bivalent inactivated vaccine prepared by the 201 adjuvant is obviously higher than that of the CPC adjuvant after immunization, so the 201 adjuvant is preferred.
Test example 2 vaccine safety test
In this test example, the porcine epidemic diarrhea virus and transmissible gastroenteritis virus bivalent inactivated vaccine prepared in example 9 was used to immunize newborn piglets, and the specific steps were as follows:
and selecting 5 newborn piglets with the age of 3 days for 10 piglets in each group, and detecting PEDV and TGEV neutralizing antibodies in serum to be negative before immunization. Two-fold doses (4 ml/head) were used for the post-acupuncture injection. And (4) observing whether the animals have local reaction and systemic reaction after the vaccination, and researching the safety of the vaccine. The specific results show that the body temperature, drinking water intake and diarrhea symptoms of the newborn piglets are observed.
Specifically, the results shown in fig. 5 were obtained, and the results showed that the body temperature fluctuation of the newborn piglets inoculated with the purified vaccine was significantly smaller than that of the newborn piglets inoculated with the unpurified vaccine. The newborn piglets are observed to have normal drinking and eating, no local and systemic reaction and no diarrhea symptom. The safety of the purified vaccine is obviously improved compared with the unpurified vaccine, and the purification is fully reflected to effectively reduce the side reaction of the vaccine.
Test example 3 detection of vaccine neutralizing antibody
In this test example, the porcine epidemic diarrhea virus and transmissible gastroenteritis virus bivalent inactivated vaccine prepared in example 9 is subjected to vaccine neutralizing antibody detection, and the specific steps are as follows:
5 healthy weaned piglets of 4 weeks old are selected per group, 12 healthy weaned piglets are selected, and PEDV and TGEV neutralizing antibodies in serum are detected to be negative before immunization. The injection is carried out by adopting 2ml of immunization dose per head on a posthai acupoint. Animals were observed for local and systemic responses after vaccination and blood was collected for PEDV, TGEV neutralizing antibody levels at 14 days, 28 days.
The results shown in the following table were obtained
TABLE 12 detection results of neutralizing antibodies of the vaccine obtained in example 9
The neutralizing antibody level of the piglets is continuously monitored 14 days and 28 days after the first immunization, and the detection result shows that: the TGEV neutralizing antibody titer is quickly increased from 14 days to 28 days after primary immunization, and the 14-day antibody titer 39 is increased to 21-day antibody titer 398 after unpurified vaccine immunization; the antibody titer increased from day 14 to day 28 after immunization of the purified vaccine to antibody titer 675. The titer of the PEDV neutralizing antibody is quickly increased from 14 days to 28 days after the primary immunization, and the titer of the antibody is increased from 24 days to 79 days after the immunization of unpurified vaccine; the antibody titer increased from 34 days to 189 days after immunization of the purified vaccine;
the antibody titer of the same vaccine preparation standard purified vaccine is obviously higher than that of the unpurified vaccine, which shows that the finished vaccine prepared from the purified antigen has better immune effect.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (10)
1. A method of virus purification, comprising:
(1) centrifuging and filtering the virus liquid of the virus to obtain supernatant serving as a sample to be concentrated;
(2) concentrating the sample to be concentrated through a 100KD membrane package to obtain a virus concentrated solution;
(3) purifying the virus concentrated solution by using a 4FF gel chromatography column to obtain a purified virus solution;
the virus is porcine epidemic diarrhea virus or porcine transmissible gastroenteritis virus.
2. The method of claim 1, wherein in step (1), the porcine epidemic diarrhea virus is cultured using adherent Vero cells and/or the porcine transmissible gastroenteritis virus is cultured using adherent ST cells.
3. The method according to claim 1 or 2, wherein the concentration in step (2) is 10-15 times that of the sample to be concentrated.
4. The method of claim 3, wherein the method of step (2) is as follows:
moving the sample to be concentrated into a sample treatment tank, circulating for 5-10 min, keeping the pressure at the liquid inlet end at 0.05-0.1 MPa, opening the permeation end to continuously permeate a 100KD membrane package, replenishing medical normal saline solution with the same volume as the residual volume to carry out washing filtration when the sample volume is concentrated to 1/10-1/40 of the sample volume, circulating for 5-10 min, and opening the permeation end to continuously permeate the 100KD membrane package; repeating the washing and filtering process for multiple times, and circulating for 2-5 min to obtain the virus concentrated solution.
5. The method according to any one of claims 1 to 4, wherein step (1) is continuous flow centrifugation of the virus fluid, membrane filtration and supernatant extraction as the sample to be concentrated.
6. The method of any one of claims 1-5, wherein the filler of the 4FF gel chromatography column in step (3) is Sepharose 4FF Sepharose chromatography filler.
7. The method according to claim 6, wherein the purification conditions in step (3) are: the linear flow rate of the virus concentrated solution is 30-80 cm/h, the pressure is less than 2.5bar, the sample loading amount is controlled at 10-15% of the column volume, and medical normal saline solution is adopted for elution.
8. The method of claim 7, wherein the method of step (3) is:
cleaning and disinfecting the molecular sieve gel chromatographic column by using 0.5-1.0 mol/L NaOH solution, washing the molecular sieve gel chromatographic column by using sterile water until the pH value is 7.0-7.5, balancing the molecular sieve gel chromatographic column by using medical normal saline solution until the conductivity of the molecular sieve gel chromatographic column is consistent after the molecular sieve gel chromatographic column reaches the front column, stabilizing the pH value at 7.0, and detecting UV by using ultraviolet280The baseline is stable, and finally the virus concentrated solution obtained in the step (2) is loaded and eluted by medical normal saline solution until UV is reached280When the value begins to rise, collecting the first protein peak, namely the purified virus liquid.
9. A bivalent inactivated vaccine comprising an inactivated porcine epidemic diarrhea virus purified by the method of any one of claims 1 to 9, an inactivated porcine transmissible gastroenteritis virus purified by the method of any one of claims 1 to 9, and an adjuvant 201.
10. The inactivated bivalent vaccine according to claim 9, wherein the inactivated porcine epidemic diarrhea virus purified by the method of any one of claims 1 to 9, the inactivated porcine transmissible gastroenteritis virus purified by the method of any one of claims 1 to 9, and the adjuvant 201 are mixed in a ratio of 2: 1: 3.
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