CN113845588A - A kind of preparation method of anti-porcine rotavirus egg yolk antibody and use thereof - Google Patents
A kind of preparation method of anti-porcine rotavirus egg yolk antibody and use thereof Download PDFInfo
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- CN113845588A CN113845588A CN202110577755.8A CN202110577755A CN113845588A CN 113845588 A CN113845588 A CN 113845588A CN 202110577755 A CN202110577755 A CN 202110577755A CN 113845588 A CN113845588 A CN 113845588A
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- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
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Abstract
本发明公开了一种抗猪轮状病毒的卵黄抗体的制备方法，包括如下步骤：注射免疫本发明制备的RVA灭活疫苗，连续免疫4次，每次间隔2周，2免后，当抗体中和效价达到1：200以上时认为免疫合格，每天收集鸡蛋，做好标记，4℃保存备用，提取卵黄抗体IgY。本发明方法制备得到的卵黄抗体对仔猪RVA的预防、治疗均有一定的效果，且能显著的缩短病程，可用于制备检测、预防和/或治疗猪轮状病毒感染引起的相关疾病的产品。
The invention discloses a method for preparing egg yolk antibody against porcine rotavirus, comprising the following steps: injecting and immunizing the RVA inactivated vaccine prepared by the invention, immunizing 4 times in a row, with an interval of 2 weeks each time, after 2 immunizations, when the antibody is immunized When the neutralization titer reached 1:200 or more, the immunization was considered qualified. Eggs were collected every day, labeled, stored at 4°C for later use, and the egg yolk antibody IgY was extracted. The yolk antibody prepared by the method of the invention has certain effects on the prevention and treatment of RVA in piglets, and can significantly shorten the course of disease, and can be used to prepare products for detecting, preventing and/or treating related diseases caused by porcine rotavirus infection.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a preparation method and application of a yolk antibody for resisting porcine rotavirus.
Background
Group A Rotavirus (RVA) belongs to Reoviridae (Reoviridae), and Rotavirus (Rotavirus) is an unencapsulated double-stranded RNA virus, is an important zoonotic virus, is one of important pathogens causing diarrhea of infants and young animals worldwide, and causes great economic loss. While initially RVA was considered host-specific, increasing epidemiological data now indicate that RVA can be transmitted from animal to human species, and from one animal to another.
Porcine group A rotavirus is an important pathogen causing severe diarrhea of piglets, and the infected piglets mainly show symptoms of diarrhea, dehydration, vomiting, anorexia and the like. The high prevalence of RVA in pigs causes significant economic losses to the swine industry, and treatment of RVA infections can only be symptomatic by replenishing liquids and electrolytes, and there is no effective antiviral treatment. The RVA-specific passive maternal antibodies (local effects) obtained by ingesting colostrum (systemically absorbed and re-secreted into the gut) are also key factors for protecting piglets, but due to placental barrier and insufficient amount of maternal milk ingested by piglets, the method of obtaining maternal antibodies by immunizing sows to let the piglets suckle also cannot completely ensure that the piglets are free from infection. The existing market also has the advantage of using attenuated vaccine to prevent and treat porcine rotavirus, but the time of about one month is needed for generating specific antibody after the attenuated vaccine is immunized, and the death rate of the piglet infected with rotavirus is higher in the period.
Therefore, there is a need to develop an antibody that can rapidly act to control the spread of porcine rotavirus, and reduce the mortality of pigs.
Disclosure of Invention
The invention aims to provide a preparation method and application of a yolk antibody for resisting porcine rotavirus, the yolk antibody prepared by the method has certain effect on preventing and treating piglet RVA, can obviously shorten the course of disease, and can be used for preparing products for detecting, preventing and/or treating related diseases caused by porcine rotavirus infection.
In order to achieve the purpose, the invention provides the technical scheme that:
the invention provides a method for preparing yolk antibody, which comprises injecting inactivated vaccine of G9P [23] RVA virus strain into laying hen for immunization, when the neutralizing titer of antibody reaches 1: collecting eggs when the egg yolk antibody content is more than 200 ℃, and extracting the yolk antibody IgY;
the G9P [23] RVA strain has the accession number V202132.
Chickens are of great interest as a source for the production of antibodies because Immunoglobulin Y (IgY) is deposited in large amounts in the egg yolk, which makes chickens a good source for obtaining large amounts of specific polyclonal antibodies. The antibody yield from egg yolk is estimated to be almost 18 times that from rabbits according to the weight of the antibody produced by each animal, the egg yolk antibody is equivalent to human IgG, the antibody affinity is higher, the specific lgY inhibits the growth, movement and adhesion of pathogenic bacteria through the combination with the pathogenic bacteria, and a large number of experiments prove that the IgY plays an important role in reducing or preventing the degree of diarrhea of human, calves, mice, pigs and the like caused by RV infection. However, the titer and therapeutic effect of IgY antibodies are affected by various factors, such as the type and dosage of the antigen, whether or not an adjuvant is used, the route of immunization, the frequency of vaccination, the age of the layer, and the like. Aiming at the above influencing factors, firstly, the invention selects the inactivated G9P [23] RVA virus strain as antigen to immunize the laying hens, prepares and purifies the IgY of the anti-G9P [23] RVA, and the neutralization titer is as high as 1: 306.
further, the preparation method of the inactivated vaccine of the G9P [23] RVA virus strain comprises the following steps: the virus liquid obtained by recovering and culturing the G9P [23] RVA virus strain is mixed with an inactivator, an inactivated antigen is obtained after inactivation, and then the inactivated antigen is mixed with an adjuvant to obtain the antigen.
Further, the inactivator is selected from one or more of beta-propiolactone, formalin and diethylene imine, and in a specific embodiment of the invention, the inactivator is beta-propiolactone;
further, the mass volume fraction of the beta-propiolactone is 0.025%;
the inactivation conditions are as follows: the temperature is 25-39 ℃, and the inactivation time is 4-24 h;
preferably, the temperature is 37 ℃ and the inactivation time is 12 h.
Further, the inactivated antigen: the volume ratio of the adjuvant is 1: 0.5-2, and preferably 1: 1.
Further, the adjuvant is an oil adjuvant, preferably Freund's complete adjuvant and/or Freund's incomplete adjuvant; in a particular embodiment of the invention, the adjuvants are Freund's complete adjuvant and Freund's incomplete adjuvant.
Further, the immunization method comprises the following steps: 0.5-2 ml is injected for each immunization, 4 times of continuous immunization are carried out, 2 weeks are separated for each immunization, and after the second immunization, when the neutralizing titer of the antibody reaches 1: when the immune response is more than 200, the immune response is qualified.
Further, the laying hens are 100-120 days old.
The invention also provides a yolk antibody against porcine rotavirus, which is prepared by the preparation method.
The invention also provides application of the yolk antibody for resisting the porcine rotavirus in preparing products for detecting, preventing and/or treating related diseases caused by infection of the porcine rotavirus.
Further, the product is selected from one of a preparation, a medicament and a feed additive.
Research proves that (Lixiayu, 2008) IgY almost completely loses activity after being treated by pepsin at 37 ℃ for 1h under the condition of low pH, and the RVA-resistant yolk antibody prepared by the invention can effectively reduce the RVA infection of newborn piglets after being orally taken.
The invention has the following beneficial effects:
the yolk antibody prepared by the method has a neutralizing titer of 1: 306, can be used for the treatment of porcine rotavirus, and the test result shows that the anti-G9P < 23 > RVA IgY prepared by the method of the invention can effectively reduce the infection of RVA of newborn piglets and obviously reduce the morbidity and the fatality rate of the piglets by preventing the newborn piglets; the composition is used for treating the sick piglets aged for more than 7 days, can effectively reduce the fatality rate, can obviously shorten the course of disease for 1-1.3 days, and has better effects on the prevention and treatment of RVA of the piglets.
Drawings
FIG. 1 is a graph of the IgY neutralization titers as a function of immunization time;
FIG. 2 is a graph showing the control effect of anti-G9P [23] RVA IgY;
FIG. 3 is a short course of anti-G9P [23] RVA IgY.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the accompanying drawings, and it should be understood that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.
The experimental methods in the examples of the present invention are all conventional methods unless otherwise specified, and the experimental materials used therein are all purchased from conventional biochemical reagents, and the data referred to in the examples are average values.
Reagent and experimental animal
Reagent
MA-104 passage cell line, preserved by the animal medicine laboratory of the university of national southwest; porcine strain G9P [23]]RVA(RVA/Pig-tc/CHN/SCJY-13/2017/G9P[23]) The microorganism deposit number of (a) is: v202132; the classification nomenclature is: porcine reovirus RVA/Pig-tc/CHN/SCMY-A3/2017/G9P, Latin literature name is Rotavirus Group A rotaviruses Animal Virus, preservation time: 25/4/2021, depository: china center for type culture CollectionHiding the address: wuhan university in China; TCID50 is 105.5100 mu L; freund's Complete Adjuvant (FCA) and Freund's Incomplete Adjuvant (FIA) were purchased from Sigma.
Laboratory animal
120-feather 110 +/-3-day-old kalanchoe brown young laying hens which are purchased from a certain laying hen breeding company in Sichuan, and basic immunity is finished by the breeding company before purchase; RVA-positive diarrhea pig farm newborn piglets and diarrhea piglets.
Example 1 preparation of G9P [23] RVA inactivated vaccine
(1) And (3) culturing and inactivating viruses: the preserved porcine G9P [23] strain is recovered, cultured and collected, beta-propiolactone inactivator with final concentration of 0.025% is added into the virus liquid, and the virus liquid is inactivated in a shaking incubator at constant temperature of 37 ℃ for 12 h.
(2) Virus inactivation effect test: inoculating the inactivated virus antigen to MA-104 cells, culturing for 48-72 h in a constant-temperature incubator at 37 ℃ and 5% CO2, observing CPE, simultaneously setting up a negative control and an inactivated RVA positive control, if the positive control has typical RVA cytopathy, and the cells inoculated with the inactivated virus solution and the cells of the negative control group have no cytopathy, blindly transferring the cell culture inoculated with the inactivated virus solution for 2 generations, if the CPE is not yet found, indicating that the RVA virus solution is completely inactivated, after blindly transferring the RVA virus solution for 3 generations, having no cytopathy, and the cells inoculated with the inactivated RVA virus solution have typical cytopathic effects such as cell shedding, wire drawing, aggregation and the like.
(3) Mixing the RVA antigen which is completely inactivated by inspection with the FCA/FIA adjuvant with the same volume, and ultrasonically oscillating to form milky viscous water-in-oil emulsion, namely the prepared inactivated vaccine.
And (3) carrying out safety inspection on the prepared inactivated vaccine:
and (3) sterility detection: 100 mu L of the inactivated vaccine is taken and coated on an LB agar culture plate, and no bacteria grow after the inactivated vaccine is cultured for 24 hours at 37 ℃.
And (3) safety detection: the inactivated vaccine is warmed up at room temperature, SPF-grade BALB/c female mice of 6-8 weeks old are respectively inoculated in a mode of subcutaneous multipoint injection on the back, the inoculation dose is 1 mL/mouse, the inoculated BALB/c mice are full of spirit, the appetite is normal, the absorption of injection parts is good, and any adverse abnormal phenomena such as redness and swelling do not occur.
Example 2 preparation of yolk antibody
(1) Immunization of chickens and egg collection
After the laying hens are purchased, the laying hens are adapted for 1 week, 1mL of immune G9P [23] RVA inactivated vaccine is injected into leg and/or chest muscles, continuous immunization is carried out for 4 times, each time is separated by 2 weeks, and after 2-immunization, when the neutralizing titer of antibodies reaches 1: and (3) judging the immunity to be qualified when the temperature is over 200 ℃, collecting eggs every day, marking, and storing at 4 ℃ for later use.
(2) Determination of neutralizing potency of yolk antibody
Randomly collecting 5 eggs every 7d after 2 nd immunization, sterilizing in 0.1% benzalkonium bromide water solution at 42 deg.C for 15min, taking out, air drying, and separating egg yolk with egg white and egg yolk separator; putting the yolk into a sterile beaker, blowing and uniformly mixing, adding 1mL of uniformly mixed yolk into 9mL of deionized water, uniformly mixing, and adjusting the pH value of the yolk solution to 5.0-5.2; after the adjustment is finished, the mixture is placed in a refrigerator with the temperature of 4 ℃ for standing for 4h, supernatant fluid is taken and centrifuged at 6000rpm/min at the temperature of 4 ℃ for 25min, and the supernatant fluid is taken and stored for later use.
The supernatant of the yolk solution was diluted 2-fold in a gradient with DMEM (dilution factor: 2)1、22、23、......、 214) Add it to 2 wells of each dilution in 96 well cell plate, 60 μ Ι _ per well; then adding TPCK pancreatin with final concentration of 30 mug/mL and 5% CO at 37 DEG C2Activating in an incubator for 1h, then diluting the activated virus solution with DMEM to a final concentration of 100TCID50/100 muL virus solution, adding 60 muL virus solution into each well, fully mixing, and incubating in a 37 ℃ 5% CO2 incubator for 1 h; after removing the 96-well plate with the MA-104 cells forming a monolayer and washing 3 times with Hanks' solution, the incubated virus and yolk solution mixture was added to the plate (100. mu.L per well), and finally the cell maintenance solution (50. mu.L per well) was added, and the plate was incubated in a 5% CO2 incubator at 37 ℃ to repeat the experiment 3 times, observing to 6d daily, and recording the number of wells with cytopathic effect (CPE). The neutralization titer of the yolk liquid is calculated by a Reed-muech method, as shown in figure 1:
the specific IgY of G9P RVA can be detected in the first week after the second-time immunization, the IgY slowly rises after the third-time immunization, the IgY rapidly rises in the first week after the fourth-time immunization, and the neutralizing titer of the yolk antibody reaches 1: 210, neutralization titers increased to 1: 408.
(3) extraction and purification of IgY antibody
When the yolk antibody titer reaches 1: collecting 1000 immunized eggs and a small amount of non-immunized eggs, washing chicken manure and the like outside the eggs with distilled water respectively at 200 hours, soaking in 0.1% benzalkonium bromide water solution at 42 ℃ for disinfection for 15min, taking out and drying in the air. The neutralization titer of IgY after purification was determined to be 1: 306.
test example clinical application of yolk antibody
During the period from 10 months to 2021 months in 2020, piglets in 3-scale pig farms in northern Sichuan of Yangyang, Yanshan jiajiang and Meishan of Yangyang have diarrhea of piglets of different ages in different days, which causes certain loss, especially suckling piglets within 1 week of delivery room. Mianyang northern Sichuan SJ pig farm: the incidence rate of diarrhea of piglets within 2 weeks is 75% -100%, the piglets within 2-3 days begin to have diarrhea, and the excrement is yellow with foul smell; le shan jia jiang LS pig farm: in 10 months, the number of diarrhea piglets in a pig farm begins to increase, and the incidence becomes frequent from the usual incidence. Diarrhea occurs from 1 day old to weaning, diarrhea feces are yellow to gray, diarrhea piglets are emaciated, dehydrated and even dead, and vomiting occurs occasionally; meishan MH pig farm: the feed is a commercial pig farm, 240 piglets (12 piglets) of 25 days old are introduced at the beginning of 11 months in 2020, diarrhea begins to appear in two piglets at 8 days after arrival, excrement is gray and gradually presents a water sample, doxycycline and amoxicillin are added into the feed at the beginning, and no obvious effect is seen. The use of antibiotics for prevention and treatment is basically ineffective, and the diseases are slightly relieved by feeding oral rehydration salt and glucose solution, but the effect is slight. Fresh diarrhea stools were collected and tested to confirm RVA infection.
1. anti-G9P [23] RVA IgY for RVA prophylaxis
Mianyang north chuan SJ pig farm 15 litters (172): the anti-RVA IgY is orally taken 2 ml/head piglet before colostrum is sucked in 9 litters (104 heads) of the IgY treatment group, and 6 litters (68 heads) of the control group according to the original antibiotic prevention scheme of a pig farm;
le shan jiajiang LS pig farm 8 litters (78 heads): IgY treatment group 5 litters (48), anti-RVA IgY 2 ml/litter before colostrum intake, control group 3 litters (30), according to the original antibiotic prevention schedule in pig farm. Grouping and processing are shown in Table 1, and results are shown in Table 2.
TABLE 1 IgY RVA prevention test
TABLE 2 anti-RVA IgY for the prophylaxis of RVA in newborn piglets
Note: in the same farm, the right shoulder in the same row differed significantly (p < 0.05).
The results in table 2 show that the oral administration of 2 ml/head of anti-RVA IgY before the newborn piglets eat the colostrums can effectively prevent the diarrhea of the piglets, and remarkably reduce the fatality rate which is respectively 37.5 percent and 70.8 percent; the control group was as high as 72.1% and 100%, respectively.
2. anti-G9P [23] RVA IgY for the treatment of RVA
Treatment of diarrhea in suckling piglets
Mianyang north chuan SJ pig farm suckling piglet 17 litter (175): wherein, 9 litters (93 litters) are 3-7 days old, 5 litters (53 litters) are used in an IgY treatment group, and 4 litters (40 litters) are used in a control group; 8 litters (82) with 8-14 days of age, 4 litters (42) in an IgY treatment group and 4 litters (40) in a control group; the treatment method is the same as that in Table 1.
Le shan jia jiang LS pig farm: 8 litters (72) with age less than 7 days, 4 litters (37) in the IgY treatment group and 4 litters (35) in the control group; 8 litters (67) at 8-14 days of age, 5 litters (43) in IgY treated group and 3 litters (24) in control group. In the IgY treatment group, anti-RVA IgY (less than 7 days old, 2 mL/head; more than 7 days old, 3 mL/head) is injected into whole nest of muscles immediately if diarrhea occurs, and in the control group, all piglets are simultaneously orally taken with liquid supplementing salt according to the original antibiotics in a pig farm. The day of treatment initiation was recorded as day 0 (as in table 3) and the trial observed for continued weaning.
TABLE 3 treatment of RVA infection in suckling piglets
Treatment of diarrhea in weaned piglets
8 weaned (25-30 days old) piglets in an LS pig farm in Leshan mountain (95 heads): 73 heads (column 6) of the IgY treatment group are injected intramuscularly with anti-RVA IgY 4 ml/head, and 24 heads (column 2) of the control group are added with amoxicillin and vitamin complex (shown in table 3).
Brow MH pig farm 200 (10 columns): IgY treatment group 140 heads (column 7), neck intramuscular injection of anti-RVA IgY 4 ml/head; in the control group, 60 populus oral liquid (column 3) is orally taken by 4ml per head, and all pig feeds are added with compound multivitamins and amoxicillin (see table 4).
TABLE 4 treatment of RVA infection in weaned piglets
The treatment conditions of the diarrhea of the suckling piglets and the weaning piglets are shown in the table 5-7.
TABLE 5 therapy of anti-RVA IgY for RVA infection in < 7-day-old piglets
Note: in the same farm, the right shoulders in the same row are obviously different; differences were very significant.
TABLE 6 anti-RVA IgY for the treatment of RVA infection in piglets aged > 7 days
Note: in the same farm, the same row of right shoulders differed significantly.
TABLE 7 anti-RVA IgY for the treatment of RVA infection in weaned piglets
Note: in the same farm, the same row of right shoulders differed significantly.
The results in table 5 show that IgY treated sick piglets less than 7 days old slightly better than the control group; the results in tables 6 and 7 show that when the IgY is used for treating piglets aged for more than 7 days and sick piglets after weaning, the mortality rate can be reduced by the IgY treatment, and the course of disease can be obviously shortened by 1-1.7 days.
Three pig farm data were combined and analyzed, and the results showed: 2mL of anti-G9P [23] RVA IgY is orally taken for preventing RVA infection of newborn piglets, so that the fatality rate (p is less than 0.01) can be remarkably reduced, the average fatality rate of an IgY prevention group is 48.0 percent, and the fatality rate of an antibiotic control group is 80.6 percent (see figure 2); intramuscular injection of 2-4 mL of anti-G9P [23] RVA IgY is not effective in reducing the fatality rate of piglets less than 7 days old and weaned piglets, but can significantly (p < 0.05) reduce the fatality rate of sick piglets 7-14 days old (see FIG. 2); the treatment of intramuscular injection of anti-G9P [23] RVA IgY can remarkably shorten the course of 7-14 days old sick piglets and sick piglets after weaning for 1-1.3 days (see figure 3).
In conclusion, the IgY of anti-G9P [23] RVA prepared by the method has the neutralization potency of 1: 306; the anti-G9P [23] RVA IgY is used for preventing newborn piglets, can effectively reduce the infection of the newborn piglets to the RVA, remarkably reduce the morbidity and the fatality rate of the piglets, shorten the course of disease by 1-1.3 days, and has better effect on the prevention and treatment of the RVA of the piglets.
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all changes in equivalent flow or equivalent structure, which are made by using the description of the present invention and are directly or indirectly applied to other related technical fields should be covered by the scope of the present invention.
Claims (10)
1. A method for preparing yolk antibody, which is characterized in that G9P [23] RVA virus strain inactivated vaccine is injected into laying hens for immunization, and when the neutralizing titer of the antibody reaches 1: collecting eggs when the egg yolk antibody content is more than 200 ℃, and extracting the yolk antibody IgY;
the G9P [23] RVA strain has the accession number V202132.
2. The method according to claim 1, wherein the G9P [23] RVA inactivated vaccine is prepared by the method comprising: the virus liquid obtained by recovering and culturing the G9P [23] RVA virus strain is mixed with an inactivator, an inactivated antigen is obtained after inactivation, and then the inactivated antigen is mixed with an adjuvant to obtain the antigen.
3. The preparation method according to claim 2, wherein the inactivator is selected from one or more of beta-propiolactone, formalin, and divinyl imine, preferably beta-propiolactone;
further, the mass volume fraction of the beta-propiolactone is 0.025%;
the inactivation conditions are as follows: the temperature is 25-39 ℃, and the inactivation time is 4-24 h;
preferably, the temperature is 37 ℃ and the inactivation time is 12 h.
4. The method of claim 2, wherein the inactivated antigen: the volume ratio of the adjuvant is 1: 0.5-2, and preferably 1: 1.
5. The process according to claim 2 or 4, wherein the adjuvant is an oil adjuvant, preferably Freund's complete adjuvant and/or Freund's incomplete adjuvant; more preferably Freund's complete adjuvant and Freund's incomplete adjuvant.
6. The method of claim 1, wherein the immunization method comprises: 0.5-2 ml is injected for each immunization, 4 times of continuous immunization are carried out, 2 weeks are separated for each immunization, and after the second immunization, when the neutralizing titer of the antibody reaches 1: when the immunity is above 200, the immunity is qualified.
7. The preparation method according to claim 1, wherein the laying hens are 100-120 days old.
8. A yolk antibody against porcine rotavirus, which is prepared by the preparation method of any one of claims 1 to 7.
9. Use of the yolk antibody against porcine rotavirus of claim 8 in the preparation of products for detecting, preventing and/or treating diseases related to porcine rotavirus infection.
10. Use according to claim 9, wherein the product is selected from one of a formulation, a medicament, a feed additive.
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