CN112979796A - Horse anti-A H5N1 tiger source influenza virus immunoglobulin and specific immunoglobulin and refining method thereof - Google Patents
Horse anti-A H5N1 tiger source influenza virus immunoglobulin and specific immunoglobulin and refining method thereof Download PDFInfo
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Abstract
The invention discloses a horse anti-A type H5N1 tiger source influenza virus immunoglobulin and a specific immunoglobulin and a refining method thereof, wherein the horse anti-A type H5N1 tiger source influenza virus immunoglobulin is prepared by constructing and expressing an A type tiger source influenza virus antigen through reverse genetics, obtaining whole blood after immunizing a horse, obtaining IgG and F (ab') 2 immunoglobulin from serum separated from the whole blood through precipitation, enzyme digestion and chromatography, and refining the original HA protein and the horse anti-A type tiger source influenza virus immunoglobulin in a combined manner. The horse anti-influenza A H5N1 virus immunoglobulin and the specific immunoglobulin and the refining method thereof firstly utilize the mutated tiger-derived influenza A virus as immunogen to refine the horse anti-influenza A virus immunoglobulin and the specific immunoglobulin, show good prevention and treatment effects and provide practical benefits for preventing and controlling the spread of highly pathogenic influenza virus infection in endangered wild animals in the Tiger family.
Description
Technical Field
The invention belongs to the technical field of gene medicines, and relates to equine anti-H5N 1 tiger source influenza virus immunoglobulin and specific immunoglobulin and a refining method thereof.
Background
Influenza a viruses belong to the orthomyxoviridae family, and are found in a wide range of hosts, including humans, mammals and birds, and can cause seasonal influenza and avian influenza. The influenza A virus H5N1 is a highly pathogenic subtype influenza A virus, and is firstly separated and found from domestic geese in Guangdong China in 1996. Human infection with influenza a H5N1 virus was reported in hong kong in 1997. The influenza A H5N1 virus has strong transmission, is frequently endemic and even pandemic in people and animal groups, and causes great loss to human health and social economy.
In recent years, cases infected with highly pathogenic influenza virus have been found in canines, felines and ferrets, and the virus has a strong transmitting power, and various animals often die because of not being cured in time and effectively. Wherein, the endangered wild animals of the family Tiger are susceptible to the influenza A H5N1 virus, China discovers that the tigers are infected with the influenza A H5N1 virus in 2002, the sick tigers have the symptoms of high fever, convulsion, pneumonia and the like in clinic, the death rate is up to more than 40%, and the significant resource and economic loss is caused to the country. Thus, establishing an immune barrier is an effective way to protect the wild animals endangered in the family of tigers from highly pathogenic influenza virus infections.
Currently, in the anti-influenza research field, serum therapy is widely concerned due to quick effect and remarkable effect, and related antiviral serum is also used for treating animals, but a horse antiserum product aiming at tiger-derived H5N1 influenza A is lacked, so that the specificity is poor, the treatment effect is general, and the treatment period is long. Therefore, the research provides a method for refining the immunoglobulin and the specific immunoglobulin aiming at the tiger-derived influenza A H5N1, and provides help for preventing and controlling the transmission of highly pathogenic influenza virus infection in the endangered wild animals of the family Tidae.
Disclosure of Invention
In order to achieve the aim, the invention provides a horse anti-H5N 1 tiger source influenza virus immunoglobulin and a specific immunoglobulin and a refining method thereof, wherein the mutated tiger source H5N1 tiger source influenza virus is used as immunogen to refine the horse anti-H5N 1 tiger source influenza virus immunoglobulin and the specific immunoglobulin for the first time, so that the horse anti-H5N 1 tiger source influenza virus immunoglobulin and the specific immunoglobulin have good prevention and treatment effects, provide practical benefits for preventing and controlling the spread of highly pathogenic influenza virus infection in endangered wild animals of the family of the Tiger, and solve the problems that the horse antiserum product aiming at the tiger source H5N1 influenza is lacked in the prior art, the specificity is poor, the treatment effect is general, and the treatment period is long.
The technical scheme adopted by the invention is that the refining method of the horse anti-influenza A H5N1 tiger source immunoglobulin expresses influenza A H5N1 tiger source antigen through reverse genetic construction, the horse is immunized by the antigen to obtain whole blood of the horse, and the purified IgG immunoglobulin and F (ab') 2 immunoglobulin are obtained by performing ammonium sulfate precipitation, enzyme digestion and protein A column affinity chromatography on serum separated from the whole blood.
Further, the expression of the tiger source influenza virus antigen H5N1 is constructed by reverse genetics, and the specific process is as follows:
HA protein and NA protein exist on the surface of H5 subtype influenza 2.3.4.4 pedigree A/Tiger/Heilongjiang/HDHZ/2016 virus, wherein a nucleotide sequence for coding HA protein is shown as SEQ ID NO.1, a nucleotide sequence for coding NA protein is shown as SEQ ID NO.2, a basic cleavage site of a nucleotide sequence aaagaggact for expressing-R-R-R-K-R-amino acid in HA protein is mutated into a nucleotide sequence aagacggact for expressing-R-R-R-K-D-amino acid, so that mutated HA protein is formed, and a gene sequence of the mutated HA protein is shown as SEQ ID NO. 3;
the mutated HA protein and the original NA protein are respectively connected with a pHH21 carrier after enzyme digestion, a restriction enzyme adopted during enzyme digestion of the pHH21 carrier is BmbI, then JM109 competent cells are transformed to obtain recombinant positive plasmids, the recombinant positive plasmids transfect MDCK cells, supernatants after the MDCK cells are transfected are inoculated to chicken embryos, allantoic fluid of the chicken embryos is collected to obtain rescued H5N1 tiger source influenza viruses, the rescued H5N1 tiger source influenza viruses are subjected to amplification culture in a chicken embryo culture mode, the amplification cultured H5N1 tiger source influenza viruses are inactivated and then purified to obtain purified H5N1 tiger source influenza viruses as antigens.
Further, the whole blood of the horse is obtained after the horse is immunized by the antigen, and the serum separated from the whole blood is subjected to ammonium sulfate precipitation, enzyme digestion and proteinA A column affinity chromatography to obtain purified IgG immunoglobulin and F (ab') 2 immunoglobulin, which specifically comprise:
using the purified influenza virus A H5N1 as an antigen, quantitatively determining the concentration of the purified influenza virus A H5N1 by protein, and adding an adjuvant in a ratio of 1: 1 volume ratio, injecting the mixture into a horse, respectively immunizing the horse at intervals of 7-14 days, collecting whole blood of the horse before each immunization, centrifugally separating the whole blood into serum and precipitate, collecting the serum, collecting a large amount of blood when the HI antibody titer in the serum reaches at least 1:5120, refining the obtained whole blood of the horse, aseptically preparing hyperimmune serum, separating IgG immunoglobulin in the hyperimmune serum by an ammonium sulfate precipitation method, carrying out enzyme digestion and chromatography on the IgG immunoglobulin, and obtaining F (ab') 2 immunoglobulin.
Furthermore, the method for separating IgG immunoglobulin in hyperimmune serum by ammonium sulfate precipitation specifically comprises the following steps:
step a: mixing 10ml of the obtained hyperimmune serum with physiological saline with one time volume, uniformly mixing, dripping saturated ammonium sulfate solution with the volume 0.5 time that of the hyperimmune serum into the mixture to ensure that the final concentration of the serum volume is 20 percent, uniformly mixing, standing the mixture at room temperature for 30min, centrifuging the mixture at the rotating speed of 5000rpm at the temperature of 4 ℃ for 20min, retaining the supernatant, and discarding the precipitate;
step b: dripping saturated ammonium sulfate solution with volume 1.5 times of the supernatant into the retained supernatant to make the final volume concentration of the supernatant 50%, mixing, standing at room temperature for 30min, centrifuging at 4 deg.C at 5000rpm for 20min, discarding the supernatant, and retaining the precipitate;
step c: adding 1 time of physiological saline into the reserved precipitate to fully dissolve the precipitate, then dripping 0.5 time of saturated ammonium sulfate solution into the reserved precipitate to ensure that the final concentration of the reserved precipitate is 33 percent, uniformly mixing, standing at room temperature for 30min, centrifuging at the rotating speed of 5000rpm at 4 ℃ for 20min, discarding supernatant, reserving the precipitate, repeating the step c for 3 times, adding 0.5 time of physiological saline into the obtained precipitate to fully dissolve the precipitate to obtain protein liquid, filling the protein liquid into a dialysis bag, dialyzing for 24 hours, changing the liquid once every 8 hours, and collecting the IgG immunoglobulin.
Further, the IgG immunoglobulin is subjected to enzyme digestion and chromatography to pass through a column to obtain the F (ab') 2 immunoglobulin, and the operation is specifically as follows: adding 0.0099g of pepsin activated by sodium acetate into each milliliter of IgG immunoglobulin solution, diluting the immunoglobulin solution to double volume by deionized water, adjusting the pH of the diluted immunoglobulin solution to 3.3, carrying out enzyme digestion treatment on the immunoglobulin solution with the pH adjusted under the water bath condition of the temperature of 30 ℃ for 3 hours, and adjusting the pH of the immunoglobulin solution to 7.2 after the enzyme digestion is finished to obtain the immunoglobulin after the enzyme digestion;
separating and purifying the F (ab') 2 immunoglobulin from the enzyme-digested immunoglobulin by using a ProteinA column affinity chromatography, adjusting a constant flow pump and a computer ultraviolet monitor, taking out air in the chromatographic column by using an injector, connecting equipment, connecting the ProteinA column to the constant flow pump, adjusting the T value of the computer ultraviolet monitor to be 100, adjusting the A value to be 0, adding 10ml of enzyme-digested immunoglobulin solution into PB solution with twice volume, diluting and sampling; repeatedly passing through the column for 3 times; continuously washing by using 30mM PB solution, and collecting the effluent until the value on the ultraviolet detector is not changed; cleaning the column bed with 0.1M glycine until the value of the ultraviolet detector is not changed; washing the column bed by using 30mM PB solution, and collecting eluent until the numerical value of the ultraviolet detector is not changed; adjusting the pH value of the collected eluent to 7.2; the eluate is the F (ab') 2 immunoglobulin.
Another object of the present invention is to provide equine influenza A H5N1 immunoglobulin purified by the above purification method.
Still another object of the present invention is to provide a equine anti-H5N 1 Tiger-derived influenza virus specific immunoglobulin refined from the above equine anti-H5N 1 Tiger-derived influenza virus immunoglobulin, which is obtained by binding the original HA protein of H5 subtype influenza 2.3.4.4 lineage A/Tiger/Heilongjiang/HDHZ/2016 with the equine anti-H5N 1 Tiger-derived influenza virus immunoglobulin.
Further, the original HA protein of H5 subtype influenza 2.3.4.4 lineage A/Tiger/Heilongjiang/HDHZ/2016 was specifically prepared as follows: HA protein and NA protein exist on the surface of H5 subtype influenza 2.3.4.4 lineage A/Tiger/Heilongjiang/HDHZ/2016 virus, wherein the nucleotide sequence for coding HA protein is shown in SEQ ID NO.1, an insect baculovirus expression system is used for constructing and expressing HA protein with His labels, a P-FsatBac1 vector containing HA protein is constructed, DH10Bac competent cells are transformed, shuttle plasmids are constructed, insect SF-9 cells are transfected and transmitted for 3 generations, the P3 generation of cells containing baculovirus and cell supernatant are obtained, the cells are centrifuged at 3000rpm and 4 ℃, cells are collected, after the cells are lysed by lysate, the cells are centrifuged at 12000rpm and the supernatant is collected and purified by a Ni + column, and the original HA protein of the H5 subtype influenza 2.3.4.4 lineage A/Tiger/Heilongjiang/HDHZ/2016 virus is obtained.
The invention also aims to provide a refining method of the equine anti-influenza A H5N1 Tiger source specific immunoglobulin, wherein the original HA protein of an H5 subtype influenza 2.3.4.4 pedigree A/Tiger/Heilongjiang/HDHZ/2016 and the equine anti-influenza A H5N1 source influenza virus immunoglobulin pass through an affinity chromatography column together, 10 mg-15 mg of the H5N1 original HA protein are combined with 1ml of the equine anti-influenza A H5N1 source influenza virus immunoglobulin, eluent is collected and is subjected to ultrafiltration concentration, and the equine anti-influenza A H5N1 Tiger source specific immunoglobulin is obtained.
The invention has the beneficial effects that: the invention firstly utilizes the mutated tiger-derived influenza A virus H5N1 pHH21-HDHZ-HA-RRRK-D as immunogen to refine horse anti-influenza A H5N1 tiger-derived influenza virus immunoglobulin and specific immunoglobulin, the mutated immunogen reduces pathogenicity, the titer after propagation in chicken embryo is higher, the invention is more favorable for expanded culture in a chicken embryo culture mode, the culture quantity is improved, the production cost is reduced, the horse immunization and refinement of IgG and F (ab ') 2 immunoglobulin are more favorable, the specificity is increased by combining the H5N1 tiger-derived influenza virus HA protein with the IgG and F (ab') 2 immunoglobulin, good prevention and treatment effects are shown, and practical benefits are provided for preventing and controlling the transmission of high-pathogenicity influenza virus infection in endangered wild animals in the Tiger family.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 shows the PCR identification result of the recombinant plasmid of the present invention.
FIG. 2 is a titer assay for rescuing viruses in an embodiment of the present invention.
FIG. 3 is a standard curve for BCA quantification of purified virus concentration according to an embodiment of the present invention.
FIG. 4 shows SDS-PAGE electrophoretic purity characterization of purified viruses according to an embodiment of the present invention.
FIG. 5 shows SDS-PAGE results of IgG and F (ab') 2 according to an embodiment of the present invention.
Fig. 6 is a graph of challenge protection against wild strains of tiger influenza in accordance with an embodiment of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
A refining method of horse anti-A H5N1 tiger source influenza virus immunoglobulin is characterized in that an expression A H5N1 tiger source influenza virus antigen is constructed through reverse genetics, horse whole blood is obtained after the horse is immunized by the antigen, serum separated from the whole blood is subjected to ammonium sulfate precipitation, enzyme digestion and protein A column affinity chromatography, and purified IgG immunoglobulin and F (ab') 2 immunoglobulin are obtained. The original HA protein of H5 subtype influenza 2.3.4.4 pedigree A/Tiger/Heilongjiang/HDHZ/2016 is expressed through a baculovirus expression system, and specific IgG immunoglobulin and F (ab ') 2 immunoglobulin are obtained after the original HA protein is combined with the prepared IgG immunoglobulin and F (ab') 2 immunoglobulin.
The influenza virus source of the A type H5N1 Tiger is H5 subtype influenza 2.3.4.4 lineage A/Tiger/Heilongjiang/HDHZ/2016, HA protein and NA protein exist on the surface of H5 subtype influenza 2.3.4.4 lineage A/Tiger/Heilongjiang/HDHZ/2016 virus, wherein the nucleotide sequence of the encoded HA protein is shown as SEQ ID NO.1, the nucleotide sequence of the encoded NA protein is shown as SEQ ID NO.2, the alkaline cleavage site of the nucleotide sequence aaagaggact expressing-R-R-R-K-R-amino acid in the HA protein is mutated into the nucleotide sequence aagacggact expressing-R-R-R-K-D-amino acid to form the mutated HA protein, so that the HA protein HAs low pathogenicity and is convenient to reproduce in chick embryos, and the gene sequence of the mutated HA protein is shown as SEQ ID NO. 3; the mutated HA protein and the original NA protein are respectively connected with a pHH21 vector (BmbI is a restriction enzyme adopted during enzyme digestion of a pHH21 vector), then JM109 competent cells are transformed to obtain recombinant positive plasmids, the recombinant positive plasmids transfect MDCK cells, supernatant after the MDCK cells are transfected is inoculated to chicken embryos, allantoic fluid of the chicken embryos is collected to obtain rescued H5N1 tiger source influenza viruses, the rescued H5N1 tiger source influenza viruses are subjected to amplification culture in a chicken embryo culture mode, the amplification cultured H5N1 tiger source influenza viruses are inactivated and then purified to obtain purified H5N1 tiger source influenza viruses pHH 21-HDHZ-RRHA-RRRK-D.
The purified influenza virus A H5N1 tiger source is used as an antigen, and the antigen and an adjuvant (the conventional adjuvant can be both) are mixed according to the ratio of 1: 1 volume ratio, injecting horses to obtain hyperimmune serum, and specifically, quantitatively determining the concentration of purified influenza A H5N1 tiger virus by protein, and mixing with adjuvant at a ratio of 1: 1 volume ratio, injecting the mixture into a horse, carrying out immunization on the horse at intervals of 7-14 days, collecting whole blood of the horse before each immunization, carrying out centrifugal separation to obtain serum and sediment, collecting the serum, removing the sediment, measuring HI antibody titer by adopting a trace hemagglutination inhibition test method, collecting a large amount of blood according to 16ml/kg when the HI antibody titer in the serum reaches at least 1:5120, optimally selecting the immune needle for 5 times, collecting the whole blood of the horse after 5 times, refining the obtained whole blood of the horse, aseptically preparing hyperimmune serum, separating IgG immunoglobulin in the hyperimmune serum by an ammonium sulfate precipitation method, carrying out enzyme digestion and chromatography on the IgG immunoglobulin, and obtaining F (ab') 2 immunoglobulin.
Separating IgG immunoglobulin in hyperimmune serum by an ammonium sulfate precipitation method, which comprises the following specific steps:
a. mixing 10ml of the obtained hyperimmune serum with physiological saline with a volume which is one time (10ml), uniformly mixing, dropwise adding saturated ammonium sulfate solution with a volume which is 0.5 time (5ml) of the volume of the hyperimmune serum to make the serum occupy 20% of the volume, uniformly mixing, standing at room temperature for 30min, centrifuging at the rotation speed of 5000rpm at 4 ℃ for 20min, keeping the supernatant, and discarding the precipitate;
b. dropwise adding saturated ammonium sulfate solution 1.5 times the volume of the supernatant into the supernatant to make the supernatant occupy 50% of the volume, mixing, standing at room temperature for 30min, centrifuging at 4 deg.C at 5000rpm for 20min, discarding the supernatant, and retaining the precipitate;
c. adding 1 time of physiological saline into the reserved precipitate to fully dissolve the precipitate, then dropwise adding 0.5 time of saturated ammonium sulfate solution into the reserved precipitate to ensure that the final concentration of the reserved precipitate is 33%, uniformly mixing, standing at room temperature for 30min, centrifuging at 4 ℃ at 5000rpm for 20min, discarding supernatant, reserving the precipitate, repeating the step c for 3 times, adding 0.5 time of physiological saline into the obtained precipitate to fully dissolve the precipitate to obtain protein liquid, filling the protein liquid into a dialysis bag, dialyzing with PBS for 24 hours, changing the liquid once every 8 hours, and collecting IgG immunoglobulin.
Performing enzyme digestion and chromatography on the obtained IgG immunoglobulin to pass through a column to obtain F (ab') 2 immunoglobulin, wherein the operation is specifically as follows: activating pepsin by using a sodium acetate solution; 0.0099g of pepsin activated by sodium acetate is added into each milliliter of IgG immunoglobulin solution, deionized water is used for diluting the IgG immunoglobulin solution to double volume, then hydrochloric acid solution is used for adjusting the pH of the diluted immunoglobulin solution to 3.3, then the immunoglobulin solution after the pH adjustment is subjected to enzyme digestion treatment for 3 hours under the water bath condition of 30 ℃, and after the enzyme digestion is finished, the pH of the immunoglobulin solution is adjusted to 7.2, so that the immunoglobulin after the enzyme digestion is obtained.
Separating and purifying the F (ab') 2 immunoglobulin by using protein A column affinity chromatography after enzyme digestion, which comprises the following steps: adjusting a constant flow pump and a computer ultraviolet monitor, taking out air in a chromatographic column by using an injector, connecting equipment, connecting a ProteinA column to the constant flow pump, washing a pipeline by using an ethanol solution with the volume fraction of 20% at the flow rate of 2ml/min, wherein the volume fraction is not less than 25ml, adjusting the T value of the computer ultraviolet monitor to be 100, adjusting the A value to be 0, adding a PB solution (phosphate buffer solution) with the volume twice that of 10ml of enzyme-digested immunoglobulin solution, diluting, and then loading at the flow rate of 1 ml/min; repeatedly passing through the column for 3 times; continuously washing with 30mM PB solution at the flow rate of 1ml/min, and collecting the effluent until the value on the ultraviolet detector is not changed; washing the column bed with 0.1M glycine at a flow rate of 1ml/min until the value of the ultraviolet detector does not change; washing the column bed with 30mM PB solution at a flow rate of 1ml/min, and collecting the eluate until the value of the ultraviolet detector is not changed; adjusting the pH of the collected eluent to 7.2 by using 1M Tris-HCl; the eluate is the F (ab') 2 immunoglobulin.
The application expresses and purifies the original HA protein of H5 subtype influenza 2.3.4.4 pedigree A/Tiger/Heilongjiang/HDHZ/2016 based on an insect baculovirus expression system, and specifically comprises the following steps: the research applies an insect baculovirus expression system to construct and express an original HA protein with a His label, constructs a p-FsatBac1 vector containing the original HA protein, then transforming DH10Bac competent cells, constructing shuttle plasmids, transfecting insect SF-9 cells, obtaining P3 generation of cells containing baculovirus and cell supernatant after 3 generations, centrifuging at 4 deg.C at 3000rpm, collecting cells, lysing the cells with lysis solution, centrifuging at 12000rpm at 4 deg.C, collecting supernatant, purifying HA protein with Ni + column, passing through affinity chromatography column together with purified horse anti-IgG immunoglobulin or F (ab ') 2 immunoglobulin, generally combining 10 mg-15 mg H5N1 original HA protein with 1ml horse anti-IgG immunoglobulin or F (ab') 2 immunoglobulin, collecting eluate, ultrafiltering and concentrating, thus obtaining the specific H5N1 subtype influenza virus horse anti-IgG immunoglobulin or F (ab') 2 immunoglobulin. The specific H5N1 subtype influenza virus horse anti-IgG immunoglobulin or F (ab ') 2 immunoglobulin is added with HA antigen under the action condition of basic antibody, so that the specific therapeutic action of the influenza virus horse anti-IgG immunoglobulin or F (ab') 2 immunoglobulin can be enhanced, the drug can quickly identify the action target point, and compared with the common IgG immunoglobulin or F (ab ') 2 immunoglobulin, the specific H5N1 subtype influenza virus horse anti-IgG immunoglobulin or F (ab') 2 immunoglobulin HAs quicker and more effective therapeutic effect.
Example 1
Expressing the tiger source influenza virus antigen of H5N 1A through reverse genetic construction:
materials: JM109 competent cells were purchased from Takara Bio; the influenza A H5N1 tiger virus A/Tigerheilengjiang/HDHZ/01/2016 is separated and preserved in a virus chamber of Changchun military animal research institute of military medicine academy of sciences; the pHH21 vector used for the reverse genetics construction was purchased from Invitrogen; technical services such as gene sequencing and primer synthesis are provided by Jinzhi corporation and Kumei organisms.
The method comprises the following steps:
gene synthesis: HA. The NA gene is synthesized by Cinzymes.
Virus rescue: the invention adopts the steps that the HA gene and the NA gene after mutation are respectively connected to a pHH21 vector, JM109 competent cells are transformed, and recombinant positive plasmids are extracted to transfect MDCK cells.
Inoculating chicken embryos with the supernatant of the transfected cells: inoculating the collected supernatant of the transfected MDCK cells into SPF (specific pathogen free) chick embryos of 9 days old, inoculating 100 mu l of allantoic cavities of each chick embryo, placing the chick embryos in an incubator at 37 ℃ for incubation, checking the death condition of the chick embryos after 24 hours, discarding the dead chick embryos, and measuring the blood coagulation titer of the allantoic fluid of the chick embryos after 48 hours to obtain the rescued H5N1 tiger source influenza virus.
Chicken embryo method for virus amplification culture: diluting virus liquid with DMEM medium 103And inoculating 9-11 day old chick embryos, inoculating 100 mu l of chick embryos in each chick embryo allantoic cavity. And (3) placing the inoculated chick embryos in a 37 ℃ incubator for incubation, observing whether the chick embryos die or not within 24 hours, discarding the dead chick embryos within 24 hours, and collecting chick embryo allantoic fluid after 48 hours. Centrifuging allantoic fluid at 3500rpm for 15 min, collecting supernatant to obtain large amount of cultured influenza virus A (H5N 1) and storing at-20 deg.C.
Inactivation of virus liquid: the obtained mass-cultured influenza virus A (H5N 1) fluid is inactivated by a formaldehyde method: to the obtained large amount of cultured influenza A H5N1 virus solution was added formaldehyde solution at a final concentration of 0.05%, mixed well and allowed to stand for 48 hours.
Antigen purification: the inactivated influenza A virus H5N1 is used as antigen for purification, and the antigen is purified from biological product of Changchun.
As a result:
PCR verification of the recombinant positive plasmid: the PCR verification result of the bacterial liquid of the recombinant positive plasmid shows that the mutant HA and the original NA in the recombinant positive plasmid are correctly constructed, as shown in FIG. 1, wherein A is the recombinant plasmid HA bacterial liquid verification, M is Marker, 1 and 2 are HA PCR results, B is the bacterial liquid verification of the NA recombinant plasmid, M is Marker, and 1 and 2 are the PCR results of NA.
Results of titer determination of rescued H5N1 tiger-derived influenza a virus: the result shows that the hemagglutination titer of the rescued H5N1 tiger influenza A virus is 26And the method can be used for the next experiment, and the result is shown in FIG. 2.
Rescued H5N1 tiger-derived influenza virus TCID50And EID50As a result: calculation of viral TCID Using Reed-Muench method50The TCID of rescued A-type H5N1 tiger influenza virus pHH21-HDHZ-HA-RRRK-D50Is 106.38EID of rescued influenza A H5N1 virus pHH21-HDHZ-HA-RRRK-D50Is 105.25/ml。
Example 2
Refining of horse anti-A H5N1 tiger source influenza virus immunoglobulin
Materials: the experimental horses were selected from the Lirioma vinpocetine group, rProtein A column purchased from GE.
The method comprises the following steps:
immunization of horses: two healthy adult horses are selected, and the obtained antigen is immunized 5 times, wherein the interval time of each immunization is 7-14 days, and the immunization mode is subcutaneous multi-point injection on the back.
Preparation of hyperimmune serum: after immunization according to the immunization program, blood is collected before each immunization, serum is separated, and HI antibody titer is measured by using a micro Hemagglutination Inhibition (HI) test method. When the HI antibody titer reaches more than 1:5120, a large amount of blood is collected according to 16ml/kg, and hyperimmune serum is prepared aseptically.
Separation and purification of IgG: separating and purifying IgG by using an ammonium acid precipitation method, mixing 10ml of hyperimmune serum with physiological saline with one time volume, uniformly mixing on a shaking table, dropwise adding 0.5 volume of saturated ammonium sulfate solution to make the final concentration of the saturated ammonium sulfate solution be 20%, uniformly mixing, and standing at room temperature for 30 min; centrifuging the mixed solution at 4 deg.C and 5000rpm for 20min, retaining supernatant, and discarding precipitate; dropwise adding 1.5 volume of saturated ammonium sulfate solution to a final concentration of 50%, uniformly mixing, and standing at room temperature for 30 min; centrifuging the mixed solution at 4 deg.C and 5000rpm for 20min, removing supernatant and retaining precipitate; adding 1 volume of physiological saline to fully dissolve the precipitate, dropwise adding 0.5 volume of saturated ammonium sulfate solution to make the final concentration of 33%, uniformly mixing, standing at room temperature for 30 min; centrifuging the mixed solution at 4 deg.C and 5000rpm for 20min, removing supernatant and retaining precipitate; repeating the previous step for 3 times; adding 0.5 volume of physiological saline to fully dissolve the precipitate, filling the protein solution into a dialysis bag, dialyzing for 24 hours by using PBS, and changing the solution every 8 hours to obtain IgG immunoglobulin.
Refining specific IgG: expressing and purifying H5N1 subtype influenza virus strain A/Tiger/Heilongjiang/HDHZ/2016 original HA protein based on insect baculovirus expression system: the research applies an insect baculovirus expression system to construct and express HA protein with a His label, construct a p-FsatBac1 vector containing HA, then transform DH10Bac competent cells to construct shuttle plasmids, then transfect insect SF9 cells, after 3 generations, centrifuge at 3000rpm at 4 ℃, collect cells, lyse the cells with lysate, centrifuge at 12000rpm at 4 ℃, collect supernatant, purify HA protein through a Ni + column, pass through an affinity chromatography column together with purified horse anti-IgG, generally 10-15mg H5N1 original HA protein is combined with 1ml horse anti-IgG, collect eluent, and obtain the specific H5N1 subtype influenza virus horse anti-IgG after ultrafiltration concentration.
Purification of specific F (ab') 2 immunoglobulins:
cleavage of first IgG: the obtained IgG was digested with pepsin to separate F (ab') 2 and Fc: 0.0099g of pepsin per ml of solution were added in an amount of 1: 3000 pepsin, sucking 1ml of 30mM sodium acetate solution and mixing with pepsin to activate pepsin; diluting immunoglobulin solution with deionized water to twice volume, and adjusting pH to 3.3 with hydrochloric acid; adding the mixed solution of pepsin and sodium acetate into the protein solution, placing the protein solution in a water bath kettle at the temperature of 30 ℃ for enzyme digestion for 3 hours, and reversing and uniformly mixing the mixture once every 20 minutes; the mixture was removed and adjusted to pH 7.2 with 1M Tris-HCl.
Affinity chromatography on the second part of Protein a column: separating and purifying the IgG immunoglobulin after enzyme digestion by using a ProteinA column affinity chromatography to obtain F (ab') 2; adjusting a constant flow pump and a computer ultraviolet monitor, taking out air in a chromatographic column by using an injector, connecting equipment, connecting a ProteinA column to the constant flow pump, washing a pipeline by using 20% ethanol solution at the flow rate of 2ml/min, wherein the flow rate is not less than 25ml, adjusting the T value of the computer ultraviolet monitor to be 100, adjusting the A value to be 0, adding 10ml of IgG immunoglobulin solution subjected to enzyme digestion into PB solution (phosphate buffer solution) with the volume twice as large as that of the IgG immunoglobulin solution, diluting the IgG immunoglobulin solution, and then loading the sample at the flow rate of 1 ml/min; repeatedly passing through the column for 3 times; continuously washing with 30mM PB solution at the flow rate of 1ml/min, and collecting the effluent until the value on the ultraviolet detector is not changed; washing the column bed with 0.1M glycine at a flow rate of 1ml/min until the value of the ultraviolet detector does not change; washing the column bed with 30mM PB solution at a flow rate of 1ml/min, and collecting the eluate until the value of the ultraviolet detector is not changed; adjusting the pH of the collected eluent to 7.2 by using 1M Tris-HCl; the eluate is F (ab') 2 immunoglobulin solution.
The third part expresses and purifies the H5N1 subtype influenza virus strain A/Tiger/Heilongjiang/HDHZ/2016 original HA protein based on an insect baculovirus expression system: the research applies an insect baculovirus expression system to construct and express HA protein with a His label, construct a p-FsatBac1 vector containing HA, then transform DH10Bac to construct shuttle plasmid, then transfect insect SF-9 cells, after 3 generations, centrifuge at 3000rpm at 4 ℃, collect cells, lyse the cells with lysate, centrifuge at 12000rpm at 4 ℃, collect supernatant, purify HA protein through a Ni + column, pass through an affinity chromatography column with F (ab ') 2 immunoglobulin and generally combine 10 mg-15 mg H5N1 original HA protein with 1ml horse anti F (ab ') 2 immunoglobulin, collect eluent, and obtain the horse anti F (ab ') 2 immunoglobulin of specific H5N1 subtype influenza virus after ultrafiltration and concentration.
Content purity detection and neutralization titer determination of F (ab') 2 immunoglobulin:
the content of F (ab') 2 immunoglobulin is determined by using a BCA quantification method; purity assays for F (ab') 2 immunoglobulins were performed using 10% SDS-PAGE; the method for measuring the neutralization titer comprises the following steps: taking a 96-well plate, adding 50 mu l of Opti-MEM containing 1 × antimicrobial-antitryotic into each well, and supplementing 40 mu l of Opti-MEM culture solution into each well of the 1 st column; adding 10-fold diluted F (ab') 2 immunoglobulin samples to the 1 st column of a 96-well plate, and repeating for 3 times for each sample; the liquid in column 1 of the 96-well plate was mixed, 50. mu.l of the liquid was aspirated into column 2 and mixed, and the same was done until column 10, and the remaining 50. mu.l of the liquid was discarded. Diluting the virus solution of H5N1 tiger-derived influenza A virus with Opti-MEM containing 1 × antimicrobial-antitomytic to 100TCID50/50 μ l, adding 50 μ l of the diluted solution to each well except for the negative control group, and placing the well in a 37 ℃ incubator for 1 hour; the mixture was added to MDCK plates and after incubation in an incubator at 37 ℃ for 48-72h, cytopathic status of each well in 96-well plates was observed and recorded and the hemagglutination titer of each well was determined, taking the highest dilution without cytopathic as neutralization titer.
As a result:
concentration determination of purified virus: the content of purified H5N 1A tiger source influenza virus was determined to be 2.5mg/ml by BCA quantification method, and the total amount of purification was 40 mg. The standard curve used for the BCA assay is shown in FIG. 3, R20.9947, can be used for the measurement of samples.
Purity identification of the purified virus: the SDS-PAGE results of purified H5N1 tiger-derived influenza virus are shown in FIG. 4, with the final M: marker, 1, 2: reduction, 3, 4: non-reducing results showed a single bright band, which led to the conclusion that the purified H5N 1A form of tiger source influenza virus was of better purity.
Horse serum neutralizing antibody titer test results:
the results of the detection of the neutralizing antibody titer of the horse serum are shown in table 1.
TABLE 1 horse serum neutralizing antibody titer test results
| Horse numbering | Pre-immune | First free rear 7d | Second-hand free rear 7d | 7d after three-free | Four-free rear 7d | Posterior five |
| 1 | 0 | 1:32 | 1:128 | 1：2048 | 1：4096 | 1:8192 |
| 2 | 0 | 1:32 | 1:256 | 1：1024 | 1：1024 | 1:2048 |
The results are shown in the above table, wherein the results of the antibody titer of horse No.1 are relatively good, so horse No.1 was selected for blood collection.
IgG immunoglobulin and F (ab') 2 immunoglobulin concentration assay results:
the protein standard curve is drawn after the standard protein of 2mg/ml is diluted, R2Has a value of 0.996 > 0.95, and can therefore be used to determine protein concentration. The total amount of IgG protein obtained was 106.5mg and F (ab') 2 was 49.7mg, as determined by the protein standard curve equation.
SDS-PAGE validation of IgG immunoglobulins and F (ab') 2 immunoglobulins:
as shown in FIG. 5, SDS-PAGE confirmed that purified IgG immunoglobulins had bands at about 55kDa and 25kDa, and purified F (ab') 2 had a single immunoglobulin at about 25 kDa.
Neutralization assay results for IgG immunoglobulins and F (ab') 2 immunoglobulins:
after detection, the titer level of the antibody is calculated by using a Reed-Muench method, the titer of the IgG is 1:4096,
example 3
Evaluation of therapeutic Effect of IgG immunoglobulin and F (ab') 2 immunoglobulin preparations
Materials: 6-8 week female Balb/c mice were purchased from Experimental animals, Inc. of Wei Tony, Beijing.
The method comprises the following steps:
H5N1 strain half lethal dose (MLD) in mice50) Measurement of
The virus was diluted to different doses with serum free DMEM medium. Each 50. mu.l was challenged by nasal drops and mice were observed for 14 consecutive days for mortality.
Evaluation of preventive and therapeutic effects in mice:
the mouse H5N1 tiger influenza wild strain challenge test is characterized in that a total of 56 mice is divided into seven groups, each group comprises 8 mice, three groups in the seven groups are IgG groups, three groups are F (ab') 2 groups, and one group is a control group.
IGg group: 400 mug of the first group of the drugs is taken 4h before the toxin is attacked, 400 mug of the second group of the drugs is taken 8h after the toxin is attacked, and 400 mug of the third group of the drugs is taken 24h after the toxin is attacked;
group F (ab') 2: 400 mug of medicine is taken in the first 4 hours of the fourth group, 400 mug of medicine is taken in the fifth group after toxin counteracting for 8 hours, and 400 mug of medicine is taken in the sixth group after toxin counteracting for 24 hours;
the seventh group was given PBS (phosphate buffered saline) as a control group.
Each mouse is administrated in the abdominal cavity, the interval time after the first administration is 24h, and the injection is carried out for 2 times at 5 times of MLD50And (3) counteracting the toxin by adopting a nasal drip mode, and measuring the weight and the survival rate of the mouse every day after counteracting the toxin.
All animal test conditions and procedures were in compliance with the ethical guidelines of the international society for pain research, and were approved by the national release military animal care and utility committee, and all experiments were conducted in the military veterinary institute biosafety tertiary laboratory (BSL-3).
As a result:
survival rate and weight change results of the mice in the prevention and treatment groups:
the virus control group and the experimental group are attacked by the H5N1 tiger influenza wild strain of 5MLD50, administration is respectively carried out 4 hours before challenge, 8 hours after challenge and 24 hours after challenge, the weight change and survival condition of the mice are observed every day, the results shown in the figure 6 are obtained, and A in the figure 6 is the weight change rate of the mice of each group after challenge; in fig. 6, B is the death change rate of mice in each group after challenge, the survival rate of prevention in IgG group was 100%, the survival rate of treatment after 8 hours of challenge was 60%, the survival rate of treatment after 24 hours of challenge was 0%, while the survival rate of prevention in F (ab') 2 group was 40%, the survival rate of treatment after 8 hours of challenge was 0%, and the survival rate of treatment after 24 hours of challenge was 0%.
From the results of the rate of change in body weight and survival rate, the IgG group had a very good preventive effect and an urgent therapeutic effect, and F (ab ') 2 also had a certain preventive effect but did not have a therapeutic effect, which may be related to a decrease in stability of F (ab') 2 after digestion, and thus the duration of action in vivo was greatly shortened, and thus the therapeutic effect was weak compared to IgG.
It is noted that, in the present application, relational terms such as first, second, and the like are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising an … …" does not exclude the presence of other identical elements in a process, method, article, or apparatus that comprises the element.
All the embodiments in the present specification are described in a related manner, and the same and similar parts among the embodiments may be referred to each other, and each embodiment focuses on the differences from the other embodiments.
The above description is only for the preferred embodiment of the present invention, and is not intended to limit the scope of the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention shall fall within the protection scope of the present invention.
SEQUENCE LISTING
<110> military medical institute of military sciences institute of military veterinary research institute
<120> equine anti-influenza A H5N1 tiger immunoglobulin and specific immunoglobulin and refining method thereof
<130> 20210426
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 1713
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 1
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aggatagatt tcttctggac aattttaaaa ccgaatgatg caatccactt tgagagtaat 780
ggaaatttca ttgctccaga atatgcatac aaaattgtca agaaaggaga ctccacaatt 840
atgagaagtg aagtggaata tggtaactgc aacaccaggt gtcagactcc ggtaggggcg 900
ataaactcta gtatgccatt ccataacata caccccctca ccatcggaga atgtcccaaa 960
tatgtgaaat cgaacaaatt agtccttgcg actggactca gaaatagtcc tcaaagagag 1020
agaagaagaa aaagaggact atttggagct atagcaggtt ttatagaggg aggatggcag 1080
ggaatggtag atggttggta tgggtaccac cacagcaatg agcaggggag tgggtacgct 1140
gcagacaaag gatctactca aaaggcaata gacggagtca ccaataaggt caactcgatc 1200
attgacaaaa tgaacactca gtttgaggcc gtaggaaggg aatttaatag cttagagagg 1260
agaatagaga atttaaacaa gaagatggaa gacggattcc tagatgtatg gacttataat 1320
gctgaacttc tggttctcat ggaaaatgaa agaactttag acttccatga ctcaaatgtc 1380
aagaaccttt acgataaggt cagactacaa cttaaggata atgcaaaaga gctgggcaac 1440
ggttgtttcg agttctatca caaatgtaat aatgaatgta tggaaagtgt aagaaacgga 1500
acgtatgact acccgcagta ttcagaagaa gcaagattaa aaagagagga aataagtgga 1560
gtaaaattgg agtcaatagg agtctaccaa atactgtcaa tttattcaac agtggcgagt 1620
tccctagtgc tgccaatcat gatggctggt ctatctttat ggatgtgttc caacgggtcg 1680
ttacagtgca gaatttgcat ttaagttgtg agc 1713
<210> 2
<211> 1330
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 2
taccattgga tcaatctgta tgataattgg aatagttagc ttaatgttgc agattgggaa 60
tatgatttca atattggtca gtcattcaat tcaaacaggg aatcaacacc aagctgaacc 120
aattagaaat actaattttc ttactaagaa cgctgtggct tcaataacat ttacgggcag 180
ctcatctctt tgtcccatta gaggatgggc tgtacacagt aaagacaaca gtataagaat 240
tggatccaag ggggatgtgt ttgtaattag agagccattc atctcatgct cccacatgga 300
atgcaggact ttctttttga ctcatggagc cttattgaat gacaagcact ccaacgggac 360
tgtcaaagac aggagccctc acagaacgct aatgagttgt cctgtgggtg aggctccctc 420
cccatataac tcgaggtttg agtctgttgc ttggtcagca agtgcttgcc atgatggcac 480
cagttggttg acaattggaa tttctggtcc agacaatggg gctgtggctg tgttgaaatc 540
aatggcataa taacagatac tatcaagagt tggaggaaca acatactgag gactcaagag 600
tctgaatgtg catgcgtaaa tggctcctgc tttactgtaa tgacagatgg gccaagtaat 660
ggacaggcat catataagat cttcaaaata gaaaaaggga aagtagttaa atcagtcgaa 720
ttgaatgctc ctaattatca ctatgaggaa tgctcctgtt atcctgattc tggcgaaata 780
atgtgtgtgt gcagggacaa ttggcatggc tcaaataggc cgtgggtgac tttcaatcag 840
aatttggagt atcaaatagg gtatatttgc agtggggttt tcggagacaa tccacggcca 900
aacgatggga cagggagttg tggtccaatg tccctcaacg gggcatatgg gataaagggg 960
ttttcattta aatacggtaa tggtgtttgg atcgggagaa ccaaaagcac taattccagg 1020
agcggttttg aaatgatttg ggatccaaat gggtggactg gaacggacag tgaattttca 1080
gtgaaacaag atatagtagc aataactgat tggtcaggat atagcgggag ttttgtccag 1140
catccggaac ttacaggatt agactgcata agaccttgct tctggattga gttaatcaga 1200
gggcggccaa aagagagcac gatttggact agtgggagca gcatatcttt ttgtggtgtg 1260
aatagtgaca ctgtgagttg gtcttggcca gacggtgctg agttgccatt caccatgaca 1320
agaagcgtca 1330
<210> 3
<211> 1713
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 3
atggagaaaa tagtgcttct ttttacaaca atcagtcttg ttaaaagcga tcatatttgc 60
attggatatc atgcaaataa ctcgacagag caggttgaca caataatgga gaagaacgtc 120
actgttacac atgcccaaga catactggaa aagacacaca acggaaagct ttgcgatcta 180
aatggggtga agcctctgat tttaaaagat tgtagtgtag caggatggct actcggaaac 240
ccatggtgtg acgaattcat caatgtacca gaatggtctt acatagtaga gaaggccaat 300
ccagtcaatg gcctctgtta cccagggaat ttcaacgatt atgaagaatt gaaacaccta 360
ttgagcagga taaaccattt tgagaaaata cagatcatcc ccaaagattc ttggtcagat 420
catgaagcct caatgggggt gagcgcagca tgttcatacc agggaaagtc ctccttcttc 480
agaaatgtag tgtggcttat caaaaagaac aatacatatc caacaataaa gaaagactac 540
aataatacca accgagaaga tctcttggta ctttggggga tccaccatcc taatgataag 600
gcagagcaaa tagcgctcta tcaaaaccca accacctata tttccattgg gacgtcaaca 660
ctaaaccagc gattggtacc aaaaatagcc actagatcca aaataaacgg gcaaagtggc 720
aggatagatt tcttctggac aattttaaaa ccgaatgatg caatccactt tgagagtaat 780
ggaaatttca ttgctccaga atatgcatac aaaattgtca agaaaggaga ctccacaatt 840
atgagaagtg aagtggaata tggtaactgc aacaccaggt gtcagactcc ggtaggggcg 900
ataaactcta gtatgccatt ccataacata caccccctca ccatcggaga atgtcccaaa 960
tatgtgaaat cgaacaaatt agtccttgcg actggactca gaaatagtcc tcaaagagag 1020
agaagaagaa aagacggact atttggagct atagcaggtt ttatagaggg aggatggcag 1080
ggaatggtag atggttggta tgggtaccac cacagcaatg agcaggggag tgggtacgct 1140
gcagacaaag gatctactca aaaggcaata gacggagtca ccaataaggt caactcgatc 1200
attgacaaaa tgaacactca gtttgaggcc gtaggaaggg aatttaatag cttagagagg 1260
agaatagaga atttaaacaa gaagatggaa gacggattcc tagatgtatg gacttataat 1320
gctgaacttc tggttctcat ggaaaatgaa agaactttag acttccatga ctcaaatgtc 1380
aagaaccttt acgataaggt cagactacaa cttaaggata atgcaaaaga gctgggcaac 1440
ggttgtttcg agttctatca caaatgtaat aatgaatgta tggaaagtgt aagaaacgga 1500
acgtatgact acccgcagta ttcagaagaa gcaagattaa aaagagagga aataagtgga 1560
gtaaaattgg agtcaatagg agtctaccaa atactgtcaa tttattcaac agtggcgagt 1620
tccctagtgc tgccaatcat gatggctggt ctatctttat ggatgtgttc caacgggtcg 1680
ttacagtgca gaatttgcat ttaagttgtg agc 1713
Claims (9)
1. The refining method of horse anti-influenza A H5N1 tiger source immunoglobulin is characterized in that the method comprises the steps of expressing influenza A H5N1 tiger source antigen through reverse genetic construction, immunizing horses with the antigen to obtain whole blood of horses, and performing ammonium sulfate precipitation, enzyme digestion and protein A column affinity chromatography on serum separated from the whole blood to obtain purified IgG immunoglobulin and F (ab') 2 immunoglobulin.
2. The method for refining equine anti-H5N 1 tiger-derived influenza virus immunoglobulin of claim 1, wherein said expression of H5N1 tiger-derived influenza virus antigen is performed by reverse genetics:
HA protein and NA protein exist on the surface of H5 subtype influenza 2.3.4.4 pedigree A/Tiger/Heilongjiang/HDHZ/2016 virus, wherein a nucleotide sequence for coding HA protein is shown as SEQ ID NO.1, a nucleotide sequence for coding NA protein is shown as SEQ ID NO.2, a basic cleavage site of a nucleotide sequence aaagaggact for expressing-R-R-R-K-R-amino acid in HA protein is mutated into a nucleotide sequence aagacggact for expressing-R-R-R-K-D-amino acid, so that mutated HA protein is formed, and a gene sequence of the mutated HA protein is shown as SEQ ID NO. 3;
the mutated HA protein and the original NA protein are respectively connected with a pHH21 carrier after enzyme digestion, a restriction enzyme adopted during enzyme digestion of the pHH21 carrier is BmbI, then JM109 competent cells are transformed to obtain recombinant positive plasmids, the recombinant positive plasmids transfect MDCK cells, supernatants after the MDCK cells are transfected are inoculated to chicken embryos, allantoic fluid of the chicken embryos is collected to obtain rescued H5N1 tiger source influenza viruses, the rescued H5N1 tiger source influenza viruses are subjected to amplification culture in a chicken embryo culture mode, the amplification cultured H5N1 tiger source influenza viruses are inactivated and then purified to obtain purified H5N1 tiger source influenza viruses as antigens.
3. The method for purifying equine anti-influenza A H5N1 immunoglobulin according to claim 1 or 2, wherein the horse is immunized with the antigen to obtain whole blood of the horse, and the serum separated from the whole blood is subjected to ammonium sulfate precipitation, enzyme digestion and proteinA column affinity chromatography to obtain purified IgG immunoglobulin and F (ab') 2 immunoglobulin, specifically:
using the purified influenza virus A H5N1 as an antigen, quantitatively determining the concentration of the purified influenza virus A H5N1 by protein, and adding an adjuvant in a ratio of 1: 1 volume ratio, injecting the mixture into a horse, respectively immunizing the horse at intervals of 7-14 days, collecting whole blood of the horse before each immunization, centrifugally separating the whole blood into serum and precipitate, collecting the serum, collecting a large amount of blood when the HI antibody titer in the serum reaches at least 1:5120, refining the obtained whole blood of the horse, aseptically preparing hyperimmune serum, separating IgG immunoglobulin in the hyperimmune serum by an ammonium sulfate precipitation method, carrying out enzyme digestion and chromatography on the IgG immunoglobulin, and obtaining F (ab') 2 immunoglobulin.
4. The method for purifying equine anti-influenza A H5N1 immunoglobulin according to claim 3, wherein the method for isolating IgG immunoglobulin from hyperimmune serum by ammonium sulfate precipitation comprises the following steps:
step a: mixing 10ml of the obtained hyperimmune serum with physiological saline with one time volume, uniformly mixing, dripping saturated ammonium sulfate solution with the volume 0.5 time that of the hyperimmune serum into the mixture to ensure that the final concentration of the serum volume is 20 percent, uniformly mixing, standing the mixture at room temperature for 30min, centrifuging the mixture at the rotating speed of 5000rpm at the temperature of 4 ℃ for 20min, retaining the supernatant, and discarding the precipitate;
step b: dripping saturated ammonium sulfate solution with volume 1.5 times of the supernatant into the retained supernatant to make the final volume concentration of the supernatant 50%, mixing, standing at room temperature for 30min, centrifuging at 4 deg.C at 5000rpm for 20min, discarding the supernatant, and retaining the precipitate;
step c: adding 1 time of physiological saline into the reserved precipitate to fully dissolve the precipitate, then dripping 0.5 time of saturated ammonium sulfate solution into the reserved precipitate to ensure that the final concentration of the reserved precipitate is 33 percent, uniformly mixing, standing at room temperature for 30min, centrifuging at the rotating speed of 5000rpm at 4 ℃ for 20min, discarding supernatant, reserving the precipitate, repeating the step c for 3 times, adding 0.5 time of physiological saline into the obtained precipitate to fully dissolve the precipitate to obtain protein liquid, filling the protein liquid into a dialysis bag, dialyzing for 24 hours, changing the liquid once every 8 hours, and collecting the IgG immunoglobulin.
5. The method for purifying equine anti-influenza A H5N1 immunoglobulin according to claim 3, wherein the IgG immunoglobulin is subjected to enzymatic cleavage and chromatography to obtain F (ab') 2 immunoglobulin by: adding 0.0099g of pepsin activated by sodium acetate into each milliliter of IgG immunoglobulin solution, diluting the immunoglobulin solution to double volume by deionized water, adjusting the pH of the diluted immunoglobulin solution to 3.3, carrying out enzyme digestion treatment on the immunoglobulin solution with the pH adjusted under the water bath condition of the temperature of 30 ℃ for 3 hours, and adjusting the pH of the immunoglobulin solution to 7.2 after the enzyme digestion is finished to obtain the immunoglobulin after the enzyme digestion;
separating and purifying the F (ab') 2 immunoglobulin from the enzyme-digested immunoglobulin by using a ProteinA column affinity chromatography, adjusting a constant flow pump and a computer ultraviolet monitor, taking out air in the chromatographic column by using an injector, connecting equipment, connecting the ProteinA column to the constant flow pump, adjusting the T value of the computer ultraviolet monitor to be 100, adjusting the A value to be 0, adding 10ml of enzyme-digested immunoglobulin solution into PB solution with twice volume, diluting and sampling; repeatedly passing through the column for 3 times; continuously washing by using 30mM PB solution, and collecting the effluent until the value on the ultraviolet detector is not changed; cleaning the column bed with 0.1M glycine until the value of the ultraviolet detector is not changed; washing the column bed by using 30mM PB solution, and collecting eluent until the numerical value of the ultraviolet detector is not changed; adjusting the pH value of the collected eluent to 7.2; the eluate is the F (ab') 2 immunoglobulin.
6. A equine anti-A H5N1 tiger derived influenza virus immunoglobulin purified by the purification method according to any one of claims 1 to 5.
7. The equine anti-H5N 1 Tiger derived influenza virus specific immunoglobulin as claimed in claim 6, which is refined from an H5 subtype influenza 2.3.4.4 lineage A/Tiger/Heilongjiang/HDHZ/2016 of an original HA protein bound to an equine anti-H5N 1 Tiger derived influenza virus immunoglobulin.
8. The equine anti-H5N 1 Tiger-derived influenza virus-specific immunoglobulin according to claim 7, wherein the original HA protein of H5 subtype influenza 2.3.4.4 lineage A/Tiger/Heilongjiang/HDHZ/2016 was specifically prepared as follows: HA protein and NA protein exist on the surface of H5 subtype influenza 2.3.4.4 lineage A/Tiger/Heilongjiang/HDHZ/2016 virus, wherein the nucleotide sequence for coding HA protein is shown in SEQ ID NO.1, an insect baculovirus expression system is used for constructing and expressing HA protein with His labels, a P-FsatBac1 vector containing HA protein is constructed, DH10Bac competent cells are transformed, shuttle plasmids are constructed, insect SF-9 cells are transfected and transmitted for 3 generations, the P3 generation of cells containing baculovirus and cell supernatant are obtained, the cells are centrifuged at 3000rpm and 4 ℃, cells are collected, after the cells are lysed by lysate, the cells are centrifuged at 12000rpm and the supernatant is collected and purified by a Ni + column, and the original HA protein of the H5 subtype influenza 2.3.4.4 lineage A/Tiger/Heilongjiang/HDHZ/2016 virus is obtained.
9. The method for refining the equine anti-influenza A H5N1 Tiger source specific immunoglobulin according to claim 7 or 8, wherein the original HA protein of the H5 subtype influenza 2.3.4.4 lineage A/Tiger/Heilongjiang/HDHZ/2016 is passed through an affinity chromatography column together with the equine anti-influenza A H5N1 source immunoglobulin, 10mg to 15mg of the H5N1 original HA protein is combined with 1ml of equine anti-influenza A H5N1 source immunoglobulin, the eluate is collected and concentrated by ultrafiltration, and the equine anti-influenza A H5N1 Tiger source specific immunoglobulin is obtained.
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