CN109851672B - Egg yolk antibody composite preparation for resisting Chinese bee sacbrood virus and preparation method thereof - Google Patents
Egg yolk antibody composite preparation for resisting Chinese bee sacbrood virus and preparation method thereof Download PDFInfo
- Publication number
- CN109851672B CN109851672B CN201910142191.8A CN201910142191A CN109851672B CN 109851672 B CN109851672 B CN 109851672B CN 201910142191 A CN201910142191 A CN 201910142191A CN 109851672 B CN109851672 B CN 109851672B
- Authority
- CN
- China
- Prior art keywords
- virus
- yolk antibody
- chinese bee
- chitosan
- bee sacbrood
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Landscapes
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention belongs to the technical field of animal biological products, and particularly relates to a yolk antibody composite preparation for resisting Chinese bee sacbrood virus and a preparation method thereof. Mixing and stirring a graphene oxide turbid liquid and a chitosan solution to obtain a graphene oxide/chitosan adjuvant; adding a Chinese bee sacbrood virus particle solution into the graphene oxide/chitosan adjuvant to obtain a graphene oxide/chitosan virus vaccine; immunizing a laying hen by using the vaccine as an immunizing antigen, collecting eggs laid by the immunized laying hen, separating egg white and egg yolk, extracting a yolk antibody, and purifying to obtain the yolk antibody for resisting the Chinese bee sacbrood virus; mixing the algae extract with the yolk antibody to obtain the yolk antibody composite preparation for resisting the Chinese bee sacbrood virus. The yolk antibody composite preparation has the advantages of high biological value, good prevention and treatment effects, no toxic or side residues, strong specificity, quick action, long lasting action and the like.
Description
Technical Field
The invention belongs to the technical field of animal biological products, and particularly relates to a yolk antibody composite preparation for resisting Chinese bee sacbrood virus and a preparation method thereof.
Background
The Chinese honeybee is used as a precious resource to make great contribution to honey production and global grain production, and promotes the development of global economy to a certain extent. The bee also plays an extremely important role in maintaining the ecological balance of animals and plants. As Chinese Bee Sacbrood disease Virus (CSBV) mainly infects 1-3 days old larvae, the infection rate and death rate of the larvae are extremely high, and large-area death of Bee colonies is finally caused, thus bringing huge economic loss to the Bee industry. CSBV is a virus of infectious soft rot, has a diameter of 26-30 nm, and is an icosahedral, non-enveloped and positive-strand RNA virus particle. The colour of the larvae infected with CSBV changes from milky to pale yellow, gradually becoming dry and dark brown shortly after death. CSBV was first discovered in the guangdong in 1972 and reappeared in 2008 in liaoning, after a large area of bee colonies infected, has led to a devastating hit in agriculture and natural ecosystem.
Currently, the control of the disease has been attempted using various methods. Such as feeding heterologous royal jelly, dsRNA, recombinant expressed virus structural protein and the like. The above methods have been limited in practical use due to such or other disadvantages. In order to overcome the defects of the method, the method for preventing and treating the viral diseases by using the egg yolk antibody is one of the more common methods, and has good effect and simple preparation. Therefore, the invention successfully prepares the yolk antibody composite preparation for resisting the Chinese bee sacbrood virus based on the background condition, and is applied to preventing and treating the CSBV infection of Chinese bees.
Disclosure of Invention
In order to overcome the defects and shortcomings of the prior art, the invention mainly aims to provide a preparation method of a yolk antibody compound preparation for resisting Chinese bee sacbrood virus.
The invention also aims to provide the yolk antibody compound preparation for resisting the Chinese bee sacbrood virus, which is prepared by the preparation method.
The invention further aims to provide application of the egg yolk antibody composite preparation for resisting the Chinese bee sacbrood virus.
The purpose of the invention is realized by the following technical scheme:
a preparation method of a yolk antibody compound preparation for resisting Chinese bee sacbrood virus comprises the following steps:
(1) dissolving Chitosan (CS) in an acetic acid solution with the mass fraction of 0.5-2% to obtain a chitosan solution with the mass fraction of 2.5-10%;
(2) carrying out ultrasonic treatment on 0.25-1.25 mg/ml Graphene Oxide (GO) suspension for the first time at the temperature of 20-40 ℃ to uniformly disperse the system; then mixing the chitosan solution with the same volume of the chitosan solution prepared in the step (1), and then carrying out ultrasonic treatment again to uniformly disperse the system; stirring for 0.5-3.5 h at 20-40 ℃ to obtain a graphene oxide/chitosan (GO/CS) adjuvant;
(3) slowly adding a Chinese bee sacbrood virus (CSBV) particle solution into the graphene oxide/chitosan adjuvant prepared in the step (2) drop by drop, fully and uniformly mixing, and then stirring for 0.5-2.5 h at 20-40 ℃ to obtain a graphene oxide/chitosan virus vaccine;
(4) taking the graphene oxide/chitosan virus vaccine prepared in the step (3) as an immune antigen to immunize a laying hen, collecting eggs laid by the immunized laying hen, separating egg white and egg yolk, extracting egg yolk antibodies, and purifying to obtain the egg yolk antibodies for resisting the Chinese bee sacbrood virus;
(5) mixing the algae extract with the yolk antibody prepared in the step (4) to obtain a yolk antibody composite preparation for resisting Chinese bee sacbrood virus, wherein the using amount of the algae extract is 0.1-10% of the mass of the yolk antibody;
the condition of the first ultrasonic treatment in the step (2) is preferably 200W, and 40kHz ultrasonic treatment is carried out for 5-15 min, so that the system is uniformly dispersed and has good dispersion degree;
the secondary ultrasonic treatment in the step (2) is preferably carried out under 200W and 40kHz ultrasonic treatment for 5-20 min, so that the system is uniformly dispersed and has good dispersion degree;
the concentration of the virus particles in the graphene oxide/chitosan virus vaccine in the step (3) is preferably 0.20-5 mg/ml;
the Chinese bee sacbrood virus particles in the step (3) are preferably at least two of the following virus strains: jiangxi strain, Beijing strain, Shaanxi strain (such as CSBV-SXYL strain), Liaoning strain (such as CSBV-LN/2009), Fujian strain, Henan strain, Yunnan strain, Sichuan strain, etc.;
the preparation method of the Chinese bee sacbrood virus particle solution in the step (3) comprises the following steps:
artificial propagation of Chinese bee sacbrood virus: respectively infecting the bee larvae with different Chinese bee sacbrood virus strains, and mixing the infected larvae in equal mass; homogenizing 5-15 g of bee larvae infected with Chinese bee sacbrood virus, centrifuging, and collecting supernatant containing the virus; infecting 80-480 healthy bee larvae with the virus-containing supernate, and collecting the bee larvae infected with Chinese bee sacbrood virus;
purifying Chinese bee sacbrood virus: homogenizing the bee larvae infected with the Chinese bee sacbrood virus collected in the step (i), centrifuging, and collecting the supernatant containing the virus; resuspending the pellet with 0.2M PBS buffer, homogenizing again, centrifuging, collecting virus-containing supernatant, and combining the two collected virus-containing supernatants; mixing the supernatant containing the virus with chloroform with the same volume, homogenizing and homogenizing again, then carrying out differential centrifugation, and removing the precipitate to obtain the supernatant; performing ultracentrifugation on the supernatant, removing the supernatant, and taking a precipitate to obtain virus particles; diluting the virus particles with PBS buffer solution to obtain Chinese bee sacbrood virus (CSBV) particle solution;
the homogenate is preferably homogenized by a glass homogenizer until the homogenate is completely homogenized;
the homogenization condition is preferably to homogenize for 5-30 min on an ice bath;
the conditions of the differential centrifugation are preferably as follows: centrifuging at 1000rpm for 10min, 5000rpm for 30min, and 15000rpm for 30 min;
the condition of ultracentrifugation is preferably 35000rpm centrifugation for 60 min;
the laying hens in the step (4) are preferably laying hens aged 2 months;
the graphene oxide/chitosan virus vaccine used in the step (4) as an antigen for immunizing the laying hens preferably comprises the following steps:
taking the graphene oxide/chitosan virus vaccine as an antigen, and injecting the laying hen by adopting a mode of subcutaneous multipoint injection combined with intramuscular injection; after primary immunization, the immunization is strengthened once every 10 days, and multiple times of immunization are carried out;
the subcutaneous multi-point injection part is preferably the neck, the roots of the double wings, the back, the pectoralis major muscle part and the like;
the intramuscular injection part is preferably the most abundant part of the muscle on the outer thigh of one side of the chicken;
the dosage of the injection is preferably 1 ml/chicken and 0.2 ml/site, and the primary immunization dosage is the same as the subsequent boosting immunization dosage;
the multiple immunization is preferably carried out until the antibody titer reaches 25The above;
the specific operations of extracting and purifying the yolk antibody in the eggs in the step (4) are preferably as follows:
separating egg yolk from egg white, mixing PBS buffer solution with the egg yolk according to the volume ratio of (2-10) to 1, stirring uniformly, and adding PEG 6000 reagent to enable the final concentration to be 2.0-5.5 wt%; stirring the mixture at 20-40 ℃ for 20-60 min, centrifuging at 2000-8000 rpm for 10-60 min, and collecting supernatant to obtain a yolk antibody;
the algae in the step (5) is preferably at least one of Dunaliella salina, Chlorella vulgaris, Spirulina and Haematococcus pluvialis;
the method for preparing the algae extract liquid in the step (5) is preferably:
adding fresh water into algae to adjust the wet weight of cells to 200g/L, then crushing the algae cells under the high-pressure condition of 80-120 MPa, centrifuging at low speed, and collecting supernatant to obtain an algae extracting solution;
a yolk antibody compound preparation for resisting Chinese bee sacbrood virus is prepared by the preparation method;
the egg yolk antibody compound preparation for resisting Chinese bee sacbrood virus is applied to preparing products for preventing and treating Chinese bee sacbrood virus infection;
compared with the prior art, the invention has the following advantages and effects:
(1) the invention compares the effects of three immunologic adjuvants, and compares different immunization interval time and times, and the result shows that the antibody titer is highest when graphene oxide/chitosan (GO/CS) adjuvant is used for immunization at an interval of 10 days in the three immunologic adjuvants, so that a laying hen immunization program for efficiently preparing the anti-CSBV infection yolk antibody is successfully established.
(2) According to the invention, the oxidized graphene/chitosan virus vaccine is prepared by adopting Chinese bee sacbrood virus strains from different regional sources and is used as an immune antigen to immunize the laying hens, and the prepared egg yolk antibody overcomes the problems of poor antibody effect, short duration and the like caused by regional differences.
(3) The algae extract is added into the prepared egg yolk antibody, so that the growth and development speed and the disease resistance of bee larvae are improved, the palatability of bees to the egg yolk antibody is improved, and the administration time is shortened. And the addition of the algae extract also improves the stability and the storage period of the yolk antibody, and is beneficial to the transportation and popularization of products.
(4) The yolk antibody composite preparation with high antibody titer is prepared by the invention, and the analysis and detection of the antibody have the advantages of high biological titer, strong specificity, quick action and long lasting action.
(5) Through a plurality of tests in actual production and breeding, the invention discovers that the protective effect of the yolk antibody on the prevention and treatment of the bee against CSBV infection reaches 100 percent, and the protective effect is very obvious.
(6) The egg yolk antibody composite preparation prepared by the invention can last for more than 4 months for the protection time of bee infection resistance, and the preparation process is simple to operate, low in cost and convenient for large-scale industrial preparation.
Drawings
FIG. 1 is an electron micrograph of differentially centrifuged purified CSBV particles according to the present invention.
FIG. 2 is a pictorial representation of a yolk and yolk antibodies produced by the present invention; wherein, A: egg yolk before separation; b, yolk antibody after separation.
FIG. 3 is a graph showing the results of the experiment of the complex formulation prepared in example 1 in preventing bees from being infected with CSBV; wherein, A: larvae on day 4; b: day 3 after challenge; c: first dose (observed on day 9); d: medication 2 (day 11 observation); e: medication 3 (13 days observation); f: the results were observed on day 15.
FIG. 4 is a graph showing the results of the experiment of the complex formulation prepared in example 1 in treating bees against CSBV infection; wherein, A: the first dose (day 3, day four observation); b: medication 2 (fifth day, sixth day observation); c: the drug was attacked once from 7 days and observed on day 9; d: the results were observed after 12 days.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
Jiangxi strain of Chinese bee sacbrood virus is disclosed in reference literature (Li Yong, Zeng Zhi, Wu wavelet, et al. cloning and sequence analysis of capsid protein vp1 gene of Jiangxi strain of Chinese bee sacbrood virus [ J ]. Heilongjiang animal husbandry, 2015 (7): 175-;
the Chinese bee sacbrood virus strain Beijing strain (CSBV-BJ/2010) is disclosed in the reference literature (Xushu, Zhongting, Huying, et al. Chinese bee sacbrood virus CSBV-BJ/2010 gene cloning and sequence analysis [ J ]. Shanghai university of traffic reports (agricultural science edition), 2011, 29 (5): 44-48);
the Chinese bee sacbrood virus strain Shaanxi strain (CSBV-SXYL strain) has been disclosed in the literature reference (Li Hui. Chinese bee sacbrood virus biology characteristics analysis and its replication research in larva primary cells [ D ]. 2010);
chinese bee sacbrood virus strain Liaoning strain (LNQY-2008 strain) has been disclosed in the reference (Huiyang Chinese sacbrood virus genotyping and comparison of biological properties of representative strains thereof [ D ]. university of California medical, 2017.);
the Chinese bee sacbrood virus strain Fujian strain is disclosed in the reference literature (Xia Xiao Cui, Zhou Bing Feng, Wei Tai Yun. preparation and application of the antibody of the non-structural protein of the Chinese bee sacbrood virus [ J ]. Fujian agriculture and forestry university report (Nature science edition), 2014, 43 (5): 499 and 503);
example 1
(1) Artificial propagation and purification of CSBV
Artificial propagation of CSBV: respectively infecting five Chinese bee sacbrood virus strains of Jiangxi strain, Beijing strain, Shaanxi strain, Liaoning strain and Fujian strain to bee larvae, and mixing the infected larvae in equal mass; then 5g of bee larvae infected with Chinese bee sacbrood virus are taken, homogenized by a glass homogenizer until the mixture is completely homogenized, centrifuged, and then the supernatant containing the virus is collected; infecting 1 batch of healthy bee larva (80 larvae of 2 days old) with the virus-containing supernatant, and collecting the diseased larva;
purifying CSBV: homogenating the collected bee larvae infected with the Chinese bee sacbrood virus on an ice bath for 30min until the bee larvae are completely homogenized, centrifuging, and collecting the supernatant containing the virus; resuspending the precipitate with 0.2M PBS buffer, homogenizing in ice bath for 30min, centrifuging, collecting the supernatant containing virus, and mixing the two supernatants; mixing the supernatant containing the virus with chloroform with the same volume, homogenizing for 30min on an ice bath so as to better extract and purify the virus, and performing differential centrifugation by adopting the following parameters, wherein the differential centrifugation is performed at 1000rpm multiplied by 10min, 5000rpm multiplied by 30min and 15000rpm multiplied by 30min, and then the precipitate is discarded to obtain the supernatant; ultracentrifuging the supernatant at 35000rpm for 60min, discarding the supernatant, collecting precipitate, adding PBS buffer solution into the precipitate, diluting to obtain Chinese bee sacbrood disease virus (CSBV) particle solution (figure 1), and collecting 5 μ l of the solution, and observing with electron microscope to obtain figure 1;
(2) dissolving Chitosan (CS) in an acetic acid solution with the mass fraction of 1% to obtain a chitosan solution, wherein the mass fraction of the chitosan is 5%;
(3) carrying out ultrasonic treatment (200W, 40kHz) on GO turbid liquid with the concentration of 0.5mg/ml for 10min at 25 ℃ to uniformly disperse graphene oxide; mixing the chitosan/acetic acid solution with the same volume as the chitosan/acetic acid solution prepared in the step (1), and then carrying out ultrasonic treatment (200W, 40kHz) for 20min to fully and uniformly mix the chitosan/acetic acid solution and the chitosan/acetic acid solution; then stirring for 2h at 25 ℃ to obtain a graphene oxide/chitosan (GO/CS) adjuvant;
(4) slowly adding the solution of Chinese bee sacbrood virus (CSBV) particles prepared in the step (1) into the graphene oxide/chitosan adjuvant prepared in the step (3) drop by drop, fully and uniformly mixing, and then stirring for 30min at 25 ℃ to obtain a graphene oxide/chitosan virus vaccine, wherein the final concentration of virus protein is 0.25 mg/ml;
(5) immunizing the laying hens and collecting eggs: taking the graphene oxide/chitosan virus vaccine prepared in the step (4) as an antigen, performing subcutaneous injection on the neck, the root of each double wing, the back and the pectoralis major muscle of a laying hen with the age of 2 months of birth day respectively, and performing intramuscular injection at the most abundant muscle of the outer thigh of one side of the laying hen at the same time, wherein in order to compare the influence of the immunization interval time on the immunization effect, after the initial immunization, the boosting interval time in the embodiment is divided into 4 groups which are 7 days, 10 days, 14 days and 19 days respectively, in addition, 10 hens injected with PBS buffer solution are used as negative control, the injection dosage is 1 ml/laying hen, 0.2 ml/site, the initial immunization dosage and the subsequent boosting dosage are the same, and the boosting immunization is counted for 3 times;
(6) separation and purification of yolk antibody: separating yolk from egg white by using an egg white separator, mixing PBS buffer solution and the yolk according to the volume ratio of 2:1, adding PEG 6000 reagent after uniformly stirring to ensure that the final concentration is 2.0 wt%; the mixture was stirred at 25 ℃ for 30min, centrifuged at 4000rpm for 30min, and the supernatant was collected to give egg yolk antibodies (FIG. 2), which were stored at 4 ℃ until use.
(7) Inoculating the dunaliella salina into a natural seawater culture pond to culture and grow to a stable period (the dunaliella salina cells are bright yellow brown), then filtering step by step to remove impurities and other organisms, then precipitating the dunaliella salina by using a natural precipitation method, washing twice by fresh water, and centrifugally collecting the dunaliella salina; adding fresh water into the collected dunaliella salina to adjust the wet weight of the cells to 200g/L, then crushing the dunaliella salina cells by a high-pressure crushing method under the high-pressure condition of 800MPa, centrifuging at low speed, and collecting the supernatant to obtain a dunaliella salina extracting solution;
(8) and (3) mixing the dunaliella salina extract prepared in the step (7) with the egg yolk antibody prepared in the step (6) to obtain the egg yolk antibody composite preparation for resisting the Chinese bee sacbrood virus, wherein the using amount of the algae extract is 0.1 percent of the mass of the egg yolk antibody.
Example 2
(1) Artificial propagation and purification of CSBV
Artificial propagation of CSBV: respectively infecting five Chinese bee sacbrood virus strains of Jiangxi strain, Beijing strain, Shaanxi strain, Liaoning strain and Fujian strain to bee larvae, and mixing the infected larvae in equal mass; then taking 10g of bee larvae infected with Chinese bee sacbrood virus, homogenizing the bee larvae by using a glass homogenizer until the bee larvae are completely homogenized, centrifuging, and then collecting supernate containing the virus; infecting 3 batches (240 larvae of 2 days old) of healthy bee larvae with the virus-containing supernatant, and collecting the diseased larvae;
purifying CSBV: homogenating the collected bee larvae infected with the Chinese bee sacbrood virus on an ice bath for 5min until the bee larvae are completely homogenized, centrifuging, and collecting the supernatant containing the virus; resuspending the pellet with 0.2M PBS buffer, homogenizing again for 15min on ice bath, centrifuging, collecting the supernatant containing virus, and combining the two collected supernatants containing virus; mixing the supernatant containing the virus with chloroform with the same volume, homogenizing for 5min on an ice bath so as to better extract and purify the virus, and performing differential centrifugation by adopting the following parameters, wherein the differential centrifugation is performed at 1000rpm multiplied by 10min, 5000rpm multiplied by 30min and 15000rpm multiplied by 30min, and then the precipitate is discarded to obtain the supernatant; ultracentrifuging the supernatant at 35000rpm for 60min, removing the supernatant, collecting precipitate, and adding PBS buffer solution into the precipitate to dilute until the concentration of viral protein is 5mg/ml, to obtain Chinese bee sacbrood virus (CSBV) particle solution;
(2) dissolving Chitosan (CS) in an acetic acid solution with the mass fraction of 0.5% to obtain a chitosan solution, wherein the mass fraction of the chitosan is 2.5%;
(3) carrying out ultrasonic treatment (200W, 40kHz) on GO turbid liquid with the concentration of 0.25mg/ml for 5min at the temperature of 20 ℃ to uniformly disperse graphene oxide; mixing the chitosan/acetic acid solution with the same volume as the chitosan/acetic acid solution prepared in the step (1), and then carrying out ultrasonic treatment (200W, 40kHz) for 15min to fully and uniformly mix the chitosan/acetic acid solution and the chitosan/acetic acid solution; then stirring for 3.5h at 20 ℃ to obtain a graphene oxide/chitosan (GO/CS) adjuvant;
(4) slowly adding the solution of Chinese bee sacbrood virus (CSBV) particles prepared in the step (1) into the graphene oxide/chitosan adjuvant prepared in the step (3) drop by drop, fully and uniformly mixing, and then stirring for 2.5 hours at 20 ℃ to obtain a graphene oxide/chitosan virus vaccine, wherein the final concentration of virus protein is 0.5 mg/ml;
(5) immunizing the laying hens and collecting eggs: taking the graphene oxide/chitosan virus vaccine prepared in the step (4) as an antigen, performing subcutaneous injection on the neck, the roots of the double wings, the back and the pectoralis major muscle of a laying hen aged for 2 months, and performing intramuscular injection at the most abundant muscle of the outer thigh of one side of the laying hen, wherein after primary immunization, the primary immunization is performed once every 10 days, the injection dosage is 1ml per laying hen and 0.2ml per site, and the primary immunization dosage is the same as the subsequent immunization dosage; after multiple immunizations, the titer of the antibody is determined by an indirect ELISA method, and when the titer reaches 25Collecting eggs, marking correspondingly, storing the collected eggs in a refrigerator at 4 ℃ for separating yolk antibody;
(6) separation and purification of yolk antibody: separating yolk from egg white by using an egg white separator, mixing PBS buffer solution and the yolk according to the volume ratio of 8:1, adding PEG 6000 reagent after uniformly stirring to ensure that the final concentration is 5.5 wt%; stirring the mixture at 40 deg.C for 20min, centrifuging at 2000rpm for 60min, collecting supernatant to obtain yolk antibody, and storing at 4 deg.C;
(7) inoculating the dunaliella salina into a natural seawater culture pond to culture and grow to a stable period (the dunaliella salina cells are bright yellow brown), then filtering step by step to remove impurities and other organisms, then precipitating the dunaliella salina by using a natural precipitation method, washing twice by fresh water, and centrifugally collecting the dunaliella salina; adding fresh water into the collected dunaliella salina to adjust the wet weight of the cells to 200g/L, then crushing the dunaliella salina cells by a high-pressure crushing method under the high-pressure condition of 100MPa, centrifuging at low speed, and collecting the supernatant to obtain a dunaliella salina extracting solution;
(8) and (3) mixing the dunaliella salina extracting solution prepared in the step (7) with the egg yolk antibody prepared in the step (6) to obtain the egg yolk antibody composite preparation for resisting the Chinese bee sacbrood virus, wherein the using amount of the algae extracting solution is 2% of the mass of the egg yolk antibody.
Example 3
(1) Artificial propagation and purification of CSBV
Artificial propagation of CSBV: respectively infecting five Chinese bee sacbrood virus strains of Jiangxi strain, Beijing strain, Shaanxi strain, Liaoning strain and Fujian strain to bee larvae, and mixing the infected larvae in equal mass; then taking 15g of bee larvae infected with Chinese bee sacbrood virus, homogenizing the bee larvae by using a glass homogenizer until the bee larvae are completely homogenized, centrifuging, and then collecting supernate containing the virus; infecting 6 batches (480 larvae of 2 days old) of healthy bee larvae with the virus-containing supernatant, and collecting the diseased larvae;
purifying CSBV: homogenizing the collected bee larvae infected with the Chinese bee sacbrood virus on an ice bath for 20min until the bee larvae are completely homogenized, centrifuging, and collecting the supernatant containing the virus; resuspending the pellet with 0.2M PBS buffer, homogenizing again for 5min on ice bath, centrifuging, collecting the supernatant containing virus, and combining the two collected supernatants containing virus; mixing the supernatant containing the virus with chloroform with the same volume, homogenizing for 10min on an ice bath so as to better extract and purify the virus, and performing differential centrifugation by adopting the following parameters, wherein the differential centrifugation is performed at 1000rpm multiplied by 10min, 5000rpm multiplied by 30min and 15000rpm multiplied by 30min, and then the precipitate is discarded to obtain the supernatant; ultracentrifuging the supernatant at 35000rpm for 60min, removing the supernatant, collecting precipitate, and adding PBS buffer solution into the precipitate to dilute until the concentration of viral protein is 10mg/ml, to obtain Chinese bee sacbrood virus (CSBV) particle solution;
(2) dissolving Chitosan (CS) in an acetic acid solution with the mass fraction of 2% to obtain a chitosan solution, wherein the mass fraction of the chitosan is 10%;
(3) carrying out ultrasonic treatment (200W, 40kHz) on GO turbid liquid with the concentration of 1.25mg/ml for 15min at 40 ℃ to uniformly disperse graphene oxide; mixing the chitosan/acetic acid solution with the same volume as the chitosan/acetic acid solution prepared in the step (1), and then carrying out ultrasonic treatment (200W, 40kHz) for 5min to fully and uniformly mix the chitosan/acetic acid solution and the chitosan/acetic acid solution; then stirring for 3.5h at 40 ℃ to obtain a graphene oxide/chitosan (GO/CS) adjuvant;
(4) slowly adding the solution of Chinese bee sacbrood virus (CSBV) particles prepared in the step (1) into the graphene oxide/chitosan adjuvant prepared in the step (3) drop by drop, fully and uniformly mixing, and then stirring for 0.5h at 40 ℃ to obtain a graphene oxide/chitosan virus vaccine, wherein the final concentration of virus protein is 5 mg/ml;
(5) immunizing the laying hens and collecting eggs: taking the graphene oxide/chitosan virus vaccine prepared in the step (4) as an antigen, performing subcutaneous injection on the neck, the roots of the double wings, the back and the pectoralis major muscle of a laying hen aged for 2 months, and performing intramuscular injection at the most abundant muscle of the outer thigh of one side of the laying hen, wherein after primary immunization, the primary immunization is performed once every 10 days, the injection dosage is 1ml per laying hen and 0.2ml per site, and the primary immunization dosage is the same as the subsequent immunization dosage; after multiple immunizations, the titer of the antibody is determined by an indirect ELISA method, and when the titer reaches 25Collecting eggs, marking correspondingly, storing the collected eggs in a refrigerator at 4 ℃ for separating yolk antibody;
(6) separation and purification of yolk antibody: separating yolk from egg white by using an egg white separator, mixing PBS buffer solution and the yolk according to the volume ratio of 10:1, adding PEG 6000 reagent after uniformly stirring to ensure that the final concentration is 3.0 wt%; stirring the mixture at 20 deg.C for 60min, centrifuging at 8000rpm for 10min, collecting supernatant to obtain yolk antibody, and storing at 4 deg.C;
(7) inoculating the dunaliella salina into a natural seawater culture pond to culture and grow to a stable period (the dunaliella salina cells are bright yellow brown), then filtering step by step to remove impurities and other organisms, then precipitating the dunaliella salina by using a natural precipitation method, washing twice by fresh water, and centrifugally collecting the dunaliella salina; adding fresh water into the collected dunaliella salina to adjust the wet weight of the cells to 200g/L, then crushing the dunaliella salina cells by a high-pressure crushing method under the high-pressure condition of 120MPa, centrifuging at low speed, and collecting the supernatant to obtain a dunaliella salina extracting solution;
(8) and (3) mixing the dunaliella salina extract prepared in the step (7) with the yolk antibody prepared in the step (6) to obtain the yolk antibody composite preparation for resisting the Chinese bee sacbrood virus, wherein the using amount of the algae extract is 10% of the mass of the yolk antibody.
Comparative example 1 Freund group vaccine
(1) Artificial propagation and purification of CSBV: the specific operation was the same as in example 1;
(2) preparation of Freund's adjuvant: adding 2g of lanolin and 4ml of liquid paraffin into a sterile mortar, and fully grinding to obtain Freund's Incomplete Adjuvant (FIA). Then adding 1ml of BCG into FIA, fully grinding into water-in-oil state to obtain Freund's Complete Adjuvant (FCA);
(3) adding the solution of Chinese bee sacbrood virus (CSBV) particles prepared in the step (1) into the FCA and FIA prepared in the step (2), and fully and uniformly mixing to obtain FCA and FIA containing virus ions, wherein the final concentration of virus protein is 0.2 mg/ml;
(4) immunizing the laying hens: taking the FCA and FIA containing the virus ions prepared in the step (3) as antigens, performing subcutaneous injection under the neck, the lower part of the double-wing root, the back and the breast muscle respectively, and performing intramuscular injection at the most abundant part of the muscle at the outer side of the thigh at one side of the chicken, wherein the FCA containing the virus ions is used for the first immunization, the FIA containing the virus ions is used for the subsequent boosting immunization, the boosting immunization is performed once every 14 days after the first immunization, the injection dosage is 1 ml/chicken and 0.2 ml/site, the initial immunization dosage is the same as the subsequent boosting immunization dosage, and the boosting immunization is performed for 3 times in total;
(5) separation and purification of yolk antibody: the same as in example 1.
(6) Preparation of Dunaliella extract was the same as in example 1;
(7) and (3) mixing the brine alga extracting solution prepared in the step (6) with the egg yolk antibody prepared in the step (5) to obtain the egg yolk antibody composite preparation for resisting the Chinese bee sacbrood virus, wherein the using amount of the alga extracting solution is 0.1 percent of the mass of the egg yolk antibody.
Comparative example 2 white oil group vaccine
(1) Artificial propagation and purification of CSBV: the specific operation was the same as in example 1;
(2) preparation of white oil adjuvant: mixing Span-80 and white oil according to a volume ratio of 1:15, and shaking at 50 ℃ for 10min to obtain a transparent homogeneous state to obtain an oil phase; mixing Tween-80 with the Chinese bee sacbrood virus (CSBV) particle solution prepared in the step (1) according to the volume ratio of 1:20, and fully and uniformly mixing to obtain a water phase; mixing the water phase and the oil phase to obtain a white oil vaccine, wherein the final concentration of the viral protein is 0.2 mg/ml;
(3) immunizing the laying hens: taking the white oil group vaccine prepared in the step (3) as an antigen, performing subcutaneous injection under the neck, the double-wing root, the back and the breast muscle respectively, and performing intramuscular injection at the most abundant muscle of the thigh of one side of the chicken, wherein after primary immunization, the boosting immunity is performed once every 14 days, the injection dosage is 1 ml/chicken and 0.2 ml/site, the primary immunization dosage is the same as the subsequent boosting immunity dosage, and the boosting immunity is performed for 3 times in total;
(4) separation and purification of yolk antibody: the same as in example 1.
(5) Preparation of Dunaliella extract was the same as in example 1;
(6) and (3) mixing the brine alga extracting solution prepared in the step (5) with the egg yolk antibody prepared in the step (4) to obtain the egg yolk antibody composite preparation for resisting the Chinese bee sacbrood virus, wherein the using amount of the alga extracting solution is 0.1 percent of the mass of the egg yolk antibody.
Comparative example 3
The yolk antibodies obtained in the steps (1) - (6) are stored at 4 ℃ for later use as in example 1;
(7) and (3) mixing water with the yolk antibody prepared in the step (6) according to the mass ratio of 1: 0.008, and obtaining the yolk antibody preparation for resisting the Chinese bee sacbrood virus.
Effects of the embodiment
(1) In order to evaluate the influence of the immunization interval time on the immunization effect, the yolk antibody prepared in the step (6) in the example 1 is subjected to antibody titer by adopting an ELISA method, and the ELISA operation process is carried out according to standard steps. And after the color development is finished, measuring the light absorption value at 450nm by using a full-automatic enzyme standard instrument. The main parameters are as follows:
(1) sample dilution: using 0.01M PBS solution (0.01M Na)2HPO4，0.01M NaH2PO4) The sample is diluted. Diluting CSBV antigen (0.2mg/ml) according to a ratio of 1:2000, wherein the prepared yolk antibody dilution gradient is 1:500, and the digoxin labeled CSBV virus is used as a secondary antibody, and the dilution ratio is 1: 3200;
(2) sample adding: adding 100 mul of 1:2000 antigen diluent into a micropore plate, then adding 100 mul of coating solution, sealing by a preservative film, and coating for 1.5h at 37 ℃;
(3) spin-drying: and throwing off the coating liquid, and patting dry on absorbent paper.
(4) And (3) sealing: adding 100 μ l of blocking solution into each well, using the blocking solution in situ, i.e. PBS buffer solution containing 0.5% Tween-20(W/T) and 1% BSA (W/T), and blocking in a constant temperature cabinet at 37 ℃ for 1.5 h;
(5) spin-drying: throwing off confining liquid, and patting dry on absorbent paper;
(6) adding enzyme-labeled primary antibody: adding 100 μ l of 1:500 yolk antibody diluent (prepared in example 1 and comparative examples 1 and 2) into a microplate, repeating each treatment three times, and incubating at 37 ℃ for 1.5h under the sealing of preservative film;
(7) washing the plate: discarding the supernatant, adding PBST (PBS, 0.5% Tween-20), washing for 3 times, and patting dry;
(8) adding enzyme-labeled secondary antibody: taking a labeled secondary antibody diluted by 1:3200 times, adding 100 mu l of the labeled secondary antibody into each hole, and sealing the hole in a constant temperature box at 37 ℃ for 1.5 hours;
(9) color development: add 1 drop of substrate buffer, and protect from light at 37 ℃ for 15 min. Note that: adding a substrate buffer solution, and then adding the substrate solution;
(10) and (4) terminating: adding 50 mul of stop solution into each hole;
(11) and (3) light absorption value determination: the ELISA operation process is carried out according to standard steps, after color development is terminated, a full-automatic enzyme standard instrument is used for measuring a light absorption value at 450nm, and the egg yolk antibody titer is evaluated according to the OD value.
Table 1 shows the ELISA detection results after different immunization interval treatments, and it can be seen from the table that the graphene oxide/chitosan virus vaccine prepared in the example has the highest antibody titer after multiple immunizations at intervals of 10 days by multi-point injection. While the OD values measured in the freund vaccine group (optimal interval of 14 days) and the white oil vaccine group (optimal interval of 14 days) were lower than those of the graphene oxide/chitosan virus vaccine group, there was no significant difference between the two groups (table 2).
TABLE 1 results of ELISA detection of yolk antibody titer after different immunization interval treatment
| Interval of time | Repeat 1 (OD)450nmValue) | Repeat 2 (OD)450nmValue) | Repeat group (OD)450nmValue) |
| 7 days | 2.960 | 2.711 | 2.863 |
| 10 days | 3.888 | 3.842 | 3.875 |
| 14 days | 3.851 | 3.719 | 3.725 |
| 19 days | 3.603 | 3.741 | 3.685 |
TABLE 2 results of ELISA detection of yolk antibody titer after immunization with different adjuvants
| Group of | Interval of time | Repeat group 1 | Repeat group 2 | Repeat group 3 |
| Freund group | 14 days | 3.239 | 2.978 | 3.014 |
| White oil group | 14 days | 2.960 | 2.229 | 2.870 |
| GS-CO group | 10 days | 3.888 | 3.842 | 3.875 |
Practical application of yolk antibody composite preparation in preventing and treating honeybee anti-CSBV infection
(1) The bees were randomly divided into 7 groups, i.e., a prevention group 1, a prevention group 2, a prevention group 3, a treatment group 1, a treatment group 2, a treatment group 3, and a control group. Each group is provided with 3 batches of repetition so as to ensure the accuracy and the scientificity of experimental data. The preventive groups 1, 2 and 3 were fed 15ml 1 times with the complex formulation prepared in comparative example 1, the complex formulation prepared in comparative example 2 and the complex formulation prepared in example 1, respectively, and were fed 1 time on day 2, and bees were infected with 0.5ml of purified CSBV on day 3, respectively. Thereafter, the time to onset and mortality of the bees were recorded daily. The treatment groups 1, 2 and 3 were fed 1 time with 0.5ml of purified CSBV to bees, and the combination preparation prepared in comparative example 1, the combination preparation prepared in comparative example 2 and the combination preparation prepared in example 1 were fed on day 2 and day 3, respectively, to give 15ml of each. Thereafter, the time to onset and mortality of the bees were recorded daily. Control group: bees were first fed 1 time with 0.5ml of purified CSBV, and then 15ml of PBS was fed on day 2 and day 3, respectively. Thereafter, the time to onset and mortality of the bees were recorded daily.
The results show that: the control group bees all die, and in the 3 prevention groups, the egg yolk antibody compound preparation protection rate of the GO-CS group is the highest and can reach 100 percent (figure 3), while the prevention protection rates of the Freund group and the white oil group reach 96.6 percent and 9.41 percent. In the 3 treatment groups, the GO-CS group egg yolk antibody compound preparation has the highest protection rate which can reach 100 percent (figure 4), and the Freund group and white oil group have the treatment protection rates of 94.7 percent and 92.8 percent. Therefore, the GO-CS group egg yolk antibody has 100 percent of protection effect in preventing and treating the CSBV infection of bees.
(2) Bees were randomly divided into 2 groups, each group was provided with 3 replicates to ensure the accuracy and scientificity of experimental data, and the composite preparation prepared in comparative example 3 and the composite preparation prepared in example 1 were fed for 3 days continuously 1 time a day, 15ml each time.
The results show that: the bee of the compound preparation prepared in comparative example 3 needs 60 minutes to eat, while the bee of the compound preparation prepared in example 1 can eat within 20-30 minutes, and the honey yield of the bee fed with the compound preparation prepared in example 1 is 1-5% higher than that of the bee fed with the compound preparation prepared in comparative example 3.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (8)
1. A preparation method of a yolk antibody compound preparation for resisting Chinese bee sacbrood virus is characterized by comprising the following steps:
(1) dissolving chitosan in 0.5-2% of acetic acid solution by mass fraction to obtain 2.5-10% of chitosan solution by mass fraction;
(2) carrying out ultrasonic treatment on 0.25-1.25 mg/ml graphene oxide suspension for the first time at the temperature of 20-40 ℃ to uniformly disperse the system; then mixing the chitosan solution with the same volume of the chitosan solution prepared in the step (1), and then carrying out ultrasonic treatment again to uniformly disperse the system; then stirring for 0.5-3.5 h at 20-40 ℃ to obtain a graphene oxide/chitosan adjuvant;
(3) slowly adding the Chinese bee sacbrood virus particle solution into the graphene oxide/chitosan adjuvant prepared in the step (2) drop by drop, fully and uniformly mixing, and then stirring for 0.5-2.5 h at 20-40 ℃ to obtain a graphene oxide/chitosan virus vaccine;
(4) taking the graphene oxide/chitosan virus vaccine prepared in the step (3) as an immune antigen to immunize a laying hen, collecting eggs laid by the immunized laying hen, separating egg white and egg yolk, extracting egg yolk antibodies, and purifying to obtain the egg yolk antibodies for resisting the Chinese bee sacbrood virus;
(5) mixing the dunaliella salina extract with the yolk antibody prepared in the step (4) to obtain a yolk antibody composite preparation for resisting the Chinese bee sacbrood virus, wherein the using amount of the dunaliella salina extract is 0.1-10% of the mass of the yolk antibody;
the preparation method of the Chinese bee sacbrood virus particle solution in the step (3) comprises the following steps:
artificial propagation of Chinese bee sacbrood virus: respectively infecting the bee larvae with different Chinese bee sacbrood virus strains, and mixing the infected larvae in equal mass; homogenizing 5-15 g of bee larvae infected with Chinese bee sacbrood virus, centrifuging, and collecting supernatant containing the virus; infecting 80-480 healthy bee larvae with the virus-containing supernate, and collecting the bee larvae infected with Chinese bee sacbrood virus;
purifying Chinese bee sacbrood virus: homogenizing the bee larvae infected with the Chinese bee sacbrood virus collected in the step (i), centrifuging, and collecting the supernatant containing the virus; resuspending the pellet with 0.2M PBS buffer, homogenizing again, centrifuging, collecting virus-containing supernatant, and combining the two collected virus-containing supernatants; mixing the supernatant containing the virus with chloroform with the same volume, homogenizing and homogenizing again, then carrying out differential centrifugation, and removing the precipitate to obtain the supernatant; performing ultracentrifugation on the supernatant, removing the supernatant, and taking a precipitate to obtain virus particles; diluting the virus particles with PBS buffer solution to obtain Chinese bee sacbrood virus particle solution;
the graphene oxide/chitosan virus vaccine used as the antigen to immunize the laying hens in the step (4) specifically comprises the following steps:
taking the graphene oxide/chitosan virus vaccine as an antigen, and injecting the laying hen by adopting a mode of subcutaneous multipoint injection combined with intramuscular injection; after the primary immunization, the immunization is boosted once every 10 days, and multiple times of immunization are carried out.
2. The method for preparing a yolk antibody complex preparation against Chinese bee sacbrood virus according to claim 1, wherein the yolk antibody complex preparation comprises:
carrying out ultrasonic treatment on the first ultrasonic treatment in the step (2) for 5-15 min under the condition of 200W and 40 kHz;
and (3) carrying out ultrasonic treatment on the strips subjected to ultrasonic treatment again in the step (2) for 5-20 min at 40 kHz.
3. The method for preparing a yolk antibody complex preparation against Chinese bee sacbrood virus according to claim 1, wherein the yolk antibody complex preparation comprises:
the concentration of virus particles in the graphene oxide/chitosan virus vaccine in the step (3) is 0.20-5 mg/ml.
4. The method for preparing a yolk antibody complex preparation against Chinese bee sacbrood virus according to claim 1, wherein the yolk antibody complex preparation comprises:
the Chinese bee sacbrood virus particles in the step (3) are at least two of the following virus strains: jiangxi, Beijing, Shaanxi, Liaoning, Fujian, Henan, Yunnan and Sichuan.
5. The method for preparing a yolk antibody complex preparation against Chinese bee sacbrood virus according to claim 1, wherein the yolk antibody complex preparation comprises:
the laying hens in the step (4) adopt laying hens of which the initial laying day age is 2 months.
6. The method for preparing a yolk antibody complex preparation against Chinese bee sacbrood virus according to claim 1, wherein the yolk antibody complex preparation comprises:
the parts for subcutaneous multi-point injection are neck, roots of double wings, back and pectoralis major muscle parts;
the intramuscular injection part is the most abundant part of the muscle on the outer thigh of one side of the chicken.
7. A yolk antibody complex preparation for resisting Chinese bee sacbrood virus, which is prepared by the preparation method of any one of claims 1-6.
8. The use of the yolk antibody complex preparation for resisting Chinese bee sacbrood virus of claim 7 in the preparation of products for preventing and treating Chinese bee sacbrood virus infection.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910142191.8A CN109851672B (en) | 2019-02-26 | 2019-02-26 | Egg yolk antibody composite preparation for resisting Chinese bee sacbrood virus and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910142191.8A CN109851672B (en) | 2019-02-26 | 2019-02-26 | Egg yolk antibody composite preparation for resisting Chinese bee sacbrood virus and preparation method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109851672A CN109851672A (en) | 2019-06-07 |
| CN109851672B true CN109851672B (en) | 2021-03-23 |
Family
ID=66898901
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910142191.8A Active CN109851672B (en) | 2019-02-26 | 2019-02-26 | Egg yolk antibody composite preparation for resisting Chinese bee sacbrood virus and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109851672B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115736102A (en) * | 2022-12-29 | 2023-03-07 | 河南中医药大学 | Feed additive for preventing and treating bee paralysis and preparation and use methods thereof |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102504023B (en) * | 2011-11-03 | 2013-09-11 | 辽宁医学院 | Method for preparing egg yolk antibody, egg yolk antibody prepared by method and application of egg yolk antibody |
| CN103760344B (en) * | 2013-09-30 | 2015-10-14 | 辽宁医学院 | The ELISA detection method of Chinese bee sacbrood poison antibody |
| CN106520810A (en) * | 2016-11-03 | 2017-03-22 | 锦州医科大学 | Chinese bee cysticercus virus structure protein VP2 gene prokaryotic expression used for egg yolk antibody preparation |
| CN106872697B (en) * | 2017-02-10 | 2018-05-15 | 江西农业大学 | Apis cerana sac brood virus immunity colloidal gold strip |
-
2019
- 2019-02-26 CN CN201910142191.8A patent/CN109851672B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN109851672A (en) | 2019-06-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102086447B (en) | Duck virus hepatitis strains and inactivated vaccine | |
| CN106267195B (en) | Triple antigen-antibody complex for porcine viral diarrhea and preparation method thereof | |
| CN102258777B (en) | Method for preparing inactivated vaccine and combined vaccine by breeding infectious bursal disease virus (IBDV) by chicken embryo source cell line | |
| CN110251671B (en) | Preparation method of goose astrovirus egg yolk antigen-antibody complex | |
| CN111000993B (en) | Bivalent inactivated vaccine for duck viral hepatitis and duck reovirus disease and preparation method thereof | |
| CN111420042A (en) | Duck circovirus and adenovirus bivalent inactivated vaccine and preparation method of yolk antibody thereof | |
| CN103305474A (en) | Porcine pseudorabies virus strain as well as inactivated vaccine and applications thereof | |
| CN103820397A (en) | Muscovy duck parvovirus and application thereof | |
| CN102805864B (en) | Newcastle disease and H9N2 subtype avian influenza bivalent inactivated vaccine and preparation method thereof | |
| CN101113176A (en) | Method for preparing infectious chicken Fabricius bursa refined yolk cryodesiccation antibody | |
| CN104587460A (en) | Mink viral enteritis and canine distemper binary living vaccine as well as preparation method and application thereof | |
| CN108912227A (en) | A kind of Yolk antibody and its preparation method and application of anti-duck reovirus | |
| CN109851672B (en) | Egg yolk antibody composite preparation for resisting Chinese bee sacbrood virus and preparation method thereof | |
| CN109097340B (en) | Avian adenovirus, quadruple vaccine and preparation method thereof | |
| CN111349619A (en) | Duck circovirus tissue inactivated vaccine and preparation method of yolk antibody thereof | |
| CN109438575B (en) | Rhabdoviral egg yolk antibody of largemouth bass | |
| CN104087559A (en) | Infectious bursal disease virus, inactivated vaccine for infectious bursal disease virus and preparation method of vaccine | |
| CN104069489B (en) | Newcastle disease and infectious bursa of Fabricius bivalent inactivated vaccine and preparation method thereof | |
| CN103122336B (en) | Goose parvovirus H-strain and application thereof in preventing and treating gosling plague | |
| CN102965344A (en) | Production of infectious bronchitis virus and vaccine from cell line | |
| CN106563125B (en) | Duck hepatitis A virus III type compound live vaccine and preparation method thereof | |
| CN103834620B (en) | NDV, ewcastle disease inactivated vaccine and preparation method thereof | |
| CN112063596A (en) | Pigeon paramyxovirus type 1 PPMV-1/BJ-C strain and application thereof | |
| CN107982532B (en) | Duck hepatitis A virus antigen-antibody complex vaccine and preparation method thereof | |
| CN104357402B (en) | Monoclonal antibody with prevention and treatment gosling plague effect |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20210226 Address after: 471400 No.17, lizashu village, Baihe Township, Song County, Luoyang City, Henan Province Applicant after: Luoyang fengzaokang Biotechnology Co.,Ltd. Address before: 471000 No. 48, Xiyuan Road, Jianxi District, Henan, Luoyang Applicant before: HENAN University OF SCIENCE AND TECHNOLOGY |
|
| TA01 | Transfer of patent application right | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |