CN113845588B - Preparation method and application of yolk antibody for resisting porcine rotavirus - Google Patents
Preparation method and application of yolk antibody for resisting porcine rotavirus Download PDFInfo
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- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
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Abstract
The invention discloses a preparation method of an anti-porcine rotavirus egg yolk antibody, which comprises the following steps: injection immunization the RVA inactivated vaccine prepared by the invention is continuously immunized for 4 times, each time is divided into 2 weeks, and after 2 days of immunization, when the neutralization titer of the antibody reaches 1: and when the immune is above 200, collecting eggs every day, marking, preserving at 4 ℃ for standby, and extracting the yolk antibody IgY. The yolk antibody prepared by the method has certain effects 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.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a preparation method and application of an anti-porcine rotavirus egg yolk antibody.
Background
Group a Rotavirus (Group A Rotavirus, RVA) belongs to Reoviridae (Rotavirus), rotavirus (Rotavirus) is a non-envelope 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. Initially RVA was considered host-specific, and now more and more epidemiological data suggest that RVA can be transmitted from animal to animal across species to humans, as well as from one animal across species to another.
Group A rotavirus is an important pathogen causing severe diarrhea in piglets, and infected piglets mainly show symptoms such as diarrhea, dehydration, vomiting, anorexia and the like. The high prevalence of RVA in pigs results in a major economic loss to the pig industry and the treatment of RVA infection can only be treated by supplementing liquid and electrolytes to the disease, and there is no effective antiviral therapeutic approach. RVA-specific passive maternal antibodies (local action) obtained by taking in colostrum (systemic absorption and re-secretion to the intestinal tract) are also key factors for protecting piglets, but the method of obtaining maternal antibodies by immunizing piglets through lactation by the sows cannot completely guarantee the piglets to avoid infection due to placenta barrier, insufficient breast milk intake of piglets and the like. At present, the attenuated vaccine is used for preventing and treating the porcine rotavirus in the market, but the generation of the specific antibody after the immunization of the attenuated vaccine takes about one month, and during the period, the death rate of piglets infected with the rotavirus is higher.
Therefore, there is a need to develop an antibody that can act rapidly to control the spread of porcine rotavirus and reduce mortality in pigs.
Disclosure of Invention
The invention aims to provide a preparation method and application of an anti-porcine rotavirus egg yolk antibody, and the egg yolk antibody prepared by the method has a 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 above purpose, the technical scheme provided by the invention is as follows:
the invention provides a preparation method of yolk antibody, which is to inject the inactivated vaccine of G9P 23 RVA virus strain into the body of the laying hen for immunization, when the neutralizing titer of the antibody reaches 1: when the temperature is above 200, collecting eggs and extracting yolk antibody IgY;
the G9P 23 RVA virus strain has the preservation number of V202132.
Chickens are of great interest as a source for the production of antibodies, because Immunoglobulin Y (IgY) is deposited in large amounts on egg yolk, which makes chickens a good source for obtaining large amounts of specific polyclonal antibodies. According to the weight estimation of the antibody produced by each animal, the yield of the antibody from yolk is almost 18 times that of the antibody from rabbits, the yolk antibody is equivalent to human IgG, the affinity of the antibody 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 diarrhea degree of people, calves, mice, pigs and the like caused by RV infection. However, igY antibody titers, therapeutic effects are affected by a number of factors, such as antigen type and dose, whether adjuvants are used, the immunization route, the frequency of vaccination, the age of the layer, etc. Aiming at the influencing factors, firstly, the invention selects the inactivated G9P 23 RVA virus strain as antigen to immunize the laying hen, and the neutralizing titer of the IgY which is obtained after the preparation and purification and is resistant to the G9P 23 RVA 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: and mixing the virus liquid obtained after resuscitating and culturing G9P 23 RVA virus strain with inactivating agent to obtain inactivated antigen, and mixing with adjuvant to obtain the final product.
Further, the inactivating agent is selected from one or more of beta-propiolactone, formalin and diethyl imine, and in the specific embodiment of the present invention, the inactivating agent 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 hours;
preferably at 37℃for a period of 12 hours.
Further, the inactivating antigen: the volume ratio of the adjuvant is 1:0.5-2, preferably 1:1.
Further, the adjuvant is an oil adjuvant, preferably Freund's complete adjuvant and/or Freund's incomplete adjuvant; in a specific embodiment of the invention, the adjuvants are Freund's complete adjuvant and Freund's incomplete adjuvant.
Further, the immunization method comprises the following steps: each time of immunization is injected with 0.5-2 ml, the immunization is continuously carried out for 4 times, each time is separated by 2 weeks, and after the second immunization, when the neutralization titer of the antibody reaches 1: above 200, the immunization was considered acceptable.
Further, the laying hen is 100-120 days old.
The invention also provides an anti-porcine rotavirus yolk antibody, which is prepared by the preparation method.
The invention also provides application of the yolk antibody against the porcine rotavirus in preparing products for detecting, preventing and/or treating related diseases caused by porcine rotavirus infection.
Further, the product is selected from one of a preparation, a drug and a feed additive.
Researches prove that (Li Xiaoyu, 2008) IgY is treated for 1h by adding pepsin at 37 ℃ under the condition of low pH, and almost completely loses activity, and the anti-RVA egg yolk antibody prepared by the invention can effectively reduce the infection of newborn piglets with RVA after being orally taken.
The invention has the following beneficial effects:
the neutralization titer of the egg yolk antibody prepared by the method is 1:306, can be used for treating pig rotavirus, the test result shows that the G9P 23 RVA IgY prepared by the method can prevent newborn piglets, effectively reduce the infection RVA of newborn piglets, and obviously reduce the morbidity and mortality of piglets; the composition is used for treating the ill piglets of age greater than 7 days, can effectively reduce the death rate of the ill piglets, can obviously shorten the course of the illness by 1 to 1.3 days, and has better effects on preventing and treating the RVA of the piglets.
Drawings
FIG. 1 is a graph showing the change of IgY neutralization titers with immunization time;
FIG. 2 is a graph showing the control effect of the anti-G9P 23 RVA IgY;
figure 3 shows a shortened disease course of anti-G9P 23 RVA IgY.
Detailed Description
The following description of the embodiments of the present invention, taken in conjunction with the accompanying drawings, will be clearly and fully described in terms of the embodiments of the present invention, which are to be considered in part, but not in all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The experimental methods in the examples of the present invention, unless specified, were all conventional methods, and the experimental materials used, unless specified, were all purchased from conventional biochemical reagent companies, and the data in the examples were all average values.
Reagent and experimental animal
Reagent(s)
The MA-104 passage cell line is preserved by animal medicine laboratory of southwest national university; pig-derived strain G9P 23]RVA(RVA/Pig-tc/CHN/SCJY-13/2017/G9P[23]) The microorganism deposit number of (2) is: v202132; the classification nomenclature is: porcine reovirus RVA/Pig-tc/CHN/SCMY-A3/2017/G9P, latin Wen Xueming is Rotavirus Group A rotaviruses Animal Virus, preservation time: 2021, 4, 25, collection: china center for type culture collection, preservation address: university of chinese armed chinese; TCID50 of 10 5.5 100. Mu.L; freund's Complete Adjuvant (FCA), freund's Incomplete Adjuvant (FIA) were purchased from Sigma company.
Experimental animal
The young sea orchid laying hens with 120 feather of 110+/-3 days old are purchased from a certain Sichuan laying hen breeding company, and basic immunity is completed by the breeding company before purchase; RVA positive diarrhea pig farm primary piglets and diarrhea piglets.
EXAMPLE 1 preparation of a G9P 23 RVA inactivated vaccine
(1) Virus culture and inactivation: recovering and culturing the stored pig source G9P 23 strain, collecting virus liquid, adding beta-propiolactone inactivating agent with final concentration of 0.025% into the virus liquid, and inactivating in shaking incubator at 37 deg.C for 12 hr.
(2) And (3) virus inactivation effect test: inoculating the inactivated virus antigen to MA-104 cells, culturing in a constant temperature incubator with 5% CO2 at 37 ℃ for 48-72 hours, observing CPE, setting negative control and non-inactivated RVA positive control at the same time, if the positive control shows typical RVA cytopathy, but the cells inoculated with the inactivated virus liquid and the cells of the negative control group do not show cytopathy, carrying out blind transmission on the cell culture inoculated with the inactivated virus liquid for 2 generations, if the CPE is still not seen, indicating that the RVA virus liquid is completely inactivated, and after blind transmission of the inactivated RVA virus liquid for 3 generations, no cytopathy is seen, and the cells inoculated with the non-inactivated RVA virus liquid show typical cytopathy such as cell shedding, wiredrawing, aggregation and the like.
(3) And mixing the RVA antigen completely inactivated by inspection with an equal volume of FCA/FIA adjuvant, and performing ultrasonic vibration to obtain a milky viscous water-in-oil emulsion, namely the prepared inactivated vaccine.
Safety inspection is carried out on the prepared inactivated vaccine:
and (3) sterile detection: 100 mu L of the inactivated vaccine is taken and coated on an LB agar plate, and no bacteria grow after being cultured for 24 hours at 37 ℃.
And (3) safety detection: the inactivated vaccine is warmed at room temperature, and is inoculated into SPF-grade BALB/c female mice with 6-8 weeks of age by subcutaneous multipoint injection at the back, the inoculation dosage is 1 mL/mouse, and the inoculated BALB/c mice are full in spirit, normal in appetite, good in absorption at injection sites and free from any adverse and abnormal phenomena such as redness and swelling.
EXAMPLE 2 preparation of egg yolk antibody
(1) Immunization of chickens and collection of eggs
Laying hens are adapted for 1 week after purchase, 1mL of G9P 23 RVA inactivated vaccine is immunized by intramuscular injection of legs and/or chest, 4 times of continuous immunization are carried out, each time is separated by 2 weeks, and after 2 days of immunization, when the neutralization titer of the antibody reaches 1: 200. the immunization is considered to be qualified, eggs are collected every day, marked and stored at 4 ℃ for standby.
(2) Determination of yolk antibody neutralization titers
Randomly collecting 5 eggs every 7d of immune group and non-immune group after the 2 nd immunization, sterilizing in 0.1% benzalkonium bromide aqueous solution at 42 ℃ for 15min, taking out, air drying, and separating yolk with an egg white yolk separator; putting yolk into a sterile beaker, blowing and mixing uniformly, taking 1mL of the uniformly mixed yolk, adding into 9mL of deionized water, mixing uniformly again, and adjusting the pH value of the yolk solution to 5.0-5.2; after the adjustment, placing the mixture into a refrigerator at 4 ℃ for standing for 4 hours, taking supernatant, centrifuging at 6000rpm/min at 4 ℃ for 25 minutes, and taking the supernatant for storage for later use.
The yolk liquid supernatant was subjected to 2-fold gradient dilution with DMEM (dilution ratio: 2) 1 、2 2 、2 3 、......、 2 14 ) Add it to each dilution 2 well of a 96 well cell plate, 60 μl per well; then adding TPCK pancreatin with a final concentration of 30 mug/mL and 5% CO at 37 DEG C 2 Activating in incubator for 1 hr, diluting activated virus solution with DMEM to final concentration of 100TCID50/100 μl, adding 60 μl per well, mixing thoroughly, and incubating in 5% CO2 incubator at 37deg.C for 1 hr; after taking out 96-well cell plates forming monolayer MA-104 cells, washing 3 times with Hanks solution, adding the incubated virus and yolk liquid mixture to the cell plates (100. Mu.L per well), finally adding cell maintenance liquid (50. Mu.L per well), placing the cell maintenance liquid in a 5% CO2 incubator at 37 ℃ for culture, repeating the experiment 3 times, observing 6d every day, and recording the number of cytopathic effect (CPE) holes. The neutralization titers of the yolk liquid were calculated by Reed-muech method as shown in FIG. 1:
the specific IgY resisting G9P 23 RVA can be detected in the first week after the second, the rising slowly starts after the third, the rising rapidly starts in the first week after the fourth, and the neutralization titer of the yolk antibody reaches 1 when the fourth is: 210, the neutralization titer rises to 1 in the third week after four days: 408.
(3) Extraction and purification of IgY antibodies
When the yolk antibody titer reached 1: at 200, 1000 immunized eggs and a small amount of non-immunized chicken eggs are collected, the external chicken manure and the like of the eggs are respectively washed by distilled water, soaked in a 0.1% benzalkonium chloride aqueous solution at 42 ℃ for sterilization for 15min, taken out and dried. The neutralization potency of the purified IgY after IgY purification was determined to be 1 by the method reported in the reference (Akita et al, 1993; li Xiaoyu, 2008): 306.
clinical application of test example egg yolk antibody
During the period from 10 months in 2020 to 3 months in 2021, piglets in 3 scale pig farms of Miyang North Sichuan, leshan Zhijiang and Mirabilis have diarrhea at different ages, causing certain loss, especially suckling piglets in 1 week in the delivery room. Mianyang North Sichuan SJ pig farm: the occurrence rate of diarrhea of piglets within 2 weeks is 75-100%, the diarrhea of piglets within 2-3 days begins to appear, and the feces are yellow with malodor; leshan Zhijiang LS pig farm: the number of diarrhea piglets in the pig farm starts to increase after 10 months, and the diarrhea piglets become frequent from usual occasional. Diarrhea is caused from 1 day old to weaning, diarrhea feces are from yellow to gray, diarrhea piglets are emaciated, dehydrated and even die, and vomiting is caused occasionally; eyebrow mountain MH pig farm: for a commercial pig raising farm, 240 piglets (12 columns) of 25 days old are introduced in the early 11 months of 2020, diarrhea starts to appear in the two columns on the 8 th day after the period, the feces are gray and gradually water-sample, and doxycycline and amoxicillin are added into the feed initially, so that no obvious effect is seen. The antibiotics are basically ineffective in preventing and treating, and the oral rehydration salt and glucose solution are fed to slightly relieve symptoms, but have little effect. Fresh diarrhea stool was collected for detection and confirmed as RVA infection.
1. anti-G9P 23 RVA IgY for RVA prophylaxis
Mianyang northern Sichuan SJ pig farm 15 nest (172 heads): the IgY treatment group has 9 fosters (104 heads), the anti-RVA IgY 2 ml/first piglet is orally taken before sucking the colostrum, the control group has 6 fosters (68 heads), and the original antibiotic prevention scheme of a pig farm is adopted;
8 fossa (78 head) of the Leshan Zhijiang LS pig farm: the IgY treatment group has 5 fosters (48 heads), and the oral administration of the anti-RVA IgY 2 ml/head before sucking the colostrum, and the control group has 3 fosters (30 heads), according to the original antibiotic prevention scheme of a pig farm. The grouping and processing are shown in Table 1 and the 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 that: the right shoulder of the same column differed significantly (p < 0.05) in the same farm.
The results in Table 2 show that 2 ml/head anti-RVA IgY can effectively prevent diarrhea of piglets by oral administration before eating colostrum, and the death rate is obviously reduced to 37.5% and 70.8% respectively; the control group was up to 72.1% and 100%, respectively.
2. anti-G9P 23 RVA IgY for RVA treatment
Treatment of diarrhea in piglets
17 litter (175 heads) of the suckling piglets in Mianyang North Sichuan SJ pig farm: wherein, the 3-7 days old 9 fosters (93 heads), the IgY treatment group 5 fosters (53 heads), the control group 4 fosters (40 heads); 8-14 days old 8 fos (82 head), igY treatment group 4 fos (42 head) and control group 4 fos (40 head); the treatment method is the same as in table 1.
Leshan Zhijiang LS pig farm: less than 7 day old 8 litters (72 heads), igY treated group 4 litters (37 heads), control group 4 litters (35 heads); 8-14 days old 8 fosters (67 heads), igY-treated 5 fosters (43 heads) and control 3 fosters (24 heads). The IgY treatment group is immediately and integrally injected with RVA IgY (less than 7 days old, 2 mL/head; more than 7 days old, 3 mL/head) for treating diarrhea, and the control group is orally taken with liquid supplementing salt according to the original antibiotics of pig farm, and all piglets are simultaneously taken. Treatment was initiated when the day was 0 (as in table 3), and trial observations continued weaning.
TABLE 3 treatment of piglet RVA infection
Treatment of diarrhea in weaned piglets
8 columns (95 heads) of weaned piglets (25-30 days old) in LS pig farm of Leshan Zhijiang: the anti-RVA IgY 4 ml/head intramuscular injection of 73 heads (column 6) of the IgY treatment group and the amoxicillin and compound vitamin (as shown in Table 3) of the 24 heads (column 2) feed of the control group.
Eyebrow mountain MH pig farm 200 head (10 columns): igY treatment group 140 heads (7 columns), neck intramuscular injection of anti-RVA IgY 4 ml/head; control group 60 (3 columns) oral poplar flower oral liquid 4 ml/head, all pig feeds added compound multidimensional and amoxicillin (see Table 4).
Table 4 weaned piglet RVA infection treatment
The treatment conditions of diarrhea of the suckling piglets and weaned piglets are shown in tables 5-7.
TABLE 5 anti-RVA IgY for the treatment of < 7 day-old piglet infection RVA
Note that: in the same farm, the right shoulders in the same row have significant differences; * The difference was very significant.
TABLE 6 anti-RVA IgY for the treatment of > 7 day-old piglets infected with RVA
Note that: the right shoulder of the same row in the same farm varies significantly.
TABLE 7 anti-RVA IgY for the treatment of weaned piglet infection RVA
Note that: the right shoulder of the same row in the same farm varies significantly.
The results in Table 5 show that IgY treats less than 7 days old diseased piglets slightly better than the control group; the results in tables 6 and 7 show that IgY treatment can reduce mortality and significantly shorten the course of disease by 1 to 1.7 days when IgY is used for treating piglets which are older than 7 days and have developed after weaning.
The three pig farm data were combined and analyzed and the results showed: 2mL of anti-G9P 23 RVA IgY is orally taken for newborn piglets to prevent RVA infection, the death rate of illness (P is less than 0.01) can be remarkably reduced, the average death rate of IgY prevention group is 48.0%, and the death rate of illness of antibiotic control group is 80.6% (see figure 2); intramuscular injection of 2-4 mL anti-G9P 23 RVA IgY does not effectively reduce the mortality of piglets less than 7 days of age and postweaning piglets, but can significantly (P < 0.05) reduce the mortality of 7-14 days of age-onset piglets (see figure 2); intramuscular injection of G9P 23 RVA IgY can obviously shorten the disease course of 7-14 days old disease-onset piglet and postweaning disease-onset piglet by 1-1.3 days (see figure 3).
In summary, the neutralizing titer of the IgY against G9P 23 RVA prepared by the method of the invention is 1:306. The anti-G9P 23 RVA IgY can be used for preventing newborn piglets, effectively reducing the infection RVA of newborn piglets, obviously reducing the morbidity and the mortality of piglets and shortening the course of disease by 1-1.3 days, and has better effect on preventing and treating the RVA of the piglets.
The foregoing description is only exemplary embodiments of the present invention and is not intended to limit the scope of the present invention, and any equivalent processes or modifications using the present invention, or direct or indirect application in other related technical fields, should be considered as included in the scope of the present invention.
Claims (12)
1. The preparation process of egg yolk antibody features that the inactivated vaccine of G9P 23 RVA strain is injected into egg chicken for immunization and the neutralizing titer of the antibody reaches 1: when the temperature is above 200, collecting eggs and extracting yolk antibody IgY;
the G9P 23 RVA virus strain has the preservation number of V202132.
2. The method of claim 1, wherein the method of preparing the inactivated vaccine for the G9P 23 RVA strain comprises: and mixing the virus liquid obtained after resuscitating and culturing G9P 23 RVA virus strain with inactivating agent to obtain inactivated antigen, and mixing with adjuvant to obtain the final product.
3. The preparation method according to claim 2, wherein the inactivating agent is selected from one or more of β -propiolactone, formalin, and polyethyleneimine;
the inactivation conditions are as follows: the temperature is 25-39 ℃ and the inactivation time is 4-24 h.
4. A method of preparation according to claim 3, wherein the inactivating agent is β -propiolactone;
the mass volume fraction of the beta-propiolactone is 0.025%; the inactivation conditions are as follows: the temperature is 37 ℃ and the inactivation time is 12 hours.
5. The method of claim 2, wherein the inactivating antigen: the volume ratio of the adjuvant is 1:0.5-2.
6. The method of preparation according to claim 2 or 5, wherein the adjuvant is an oil adjuvant.
7. The method according to claim 6, wherein the adjuvant is Freund's complete adjuvant and/or Freund's incomplete adjuvant.
8. The method of claim 1, wherein the immunization method is: each time of immunization is injected with 0.5-2 ml, the immunization is continuously carried out for 4 times, each time is separated by 2 weeks, and after the second immunization, when the neutralization titer of the antibody reaches 1: and the immunization is qualified when the temperature is above 200.
9. The method of claim 1, wherein the layer is 100 to 120 days old.
10. An anti-porcine rotavirus egg yolk antibody prepared by the method of any one of claims 1 to 9.
11. Use of the yolk antibody against porcine rotavirus according to claim 10 for the preparation of a product for detecting, preventing and/or treating diseases associated with porcine rotavirus infection.
12. The use according to claim 11, wherein the product is selected from one of a formulation, a medicament, a feed additive.
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