CN107460173B

CN107460173B - Method for purifying porcine epidemic diarrhea virus, porcine reproductive and respiratory syndrome virus or avian influenza virus

Info

Publication number: CN107460173B
Application number: CN201710807210.5A
Authority: CN
Inventors: 李兰; 郑其升; 乔绪稳; 陈瑾; 杨丽; 张元鹏; 侯立婷; 侯继波
Original assignee: Jiangsu Academy of Agricultural Sciences
Current assignee: Jiangsu Academy of Agricultural Sciences
Priority date: 2017-09-08
Filing date: 2017-09-08
Publication date: 2020-09-22
Anticipated expiration: 2037-09-08
Also published as: CN107460173A

Abstract

The invention provides a method for purifying porcine epidemic diarrhea virus, porcine reproductive and respiratory syndrome virus or avian influenza virus, and relates to the field of biology. The purification method comprises the step of combining a compound formed by fusion protein PGFSN and GEM particles with porcine epidemic diarrhea virus, porcine reproductive and respiratory syndrome virus or avian influenza virus, wherein the amino acid sequence of the fusion protein PGFSN is shown as SEQ ID NO: 1. The method for purifying the porcine epidemic diarrhea virus, the porcine reproductive and respiratory syndrome virus or the avian influenza virus has the advantages of simple operation, high virus recovery rate, high removal rate of foreign proteins and low cost.

Description

Method for purifying porcine epidemic diarrhea virus, porcine reproductive and respiratory syndrome virus or avian influenza virus

Technical Field

The invention relates to the field of biology, and in particular relates to a method for purifying porcine epidemic diarrhea virus, porcine reproductive and respiratory syndrome virus or avian influenza virus.

Background

With the rapid development of the livestock breeding industry and deepening of knowledge of raisers on the safety and side effects of animal vaccines, the market demand of high-quality vaccines is increasing. The antigen is used as the first link of vaccine manufacture, and directly influences the quality of the vaccine. Throughout the development of human vaccines, the purification technology is approximately through three stages of simple separation, primary separation and refined purification.

At present, most of animal vaccines in China are not purified, the vaccines contain a large amount of impurities such as calf serum, cell debris and the like, and researches show that a large amount of foreign proteins in the vaccines are main factors causing animal adverse reactions, and simultaneously, the immune response of target antigens is also seriously interfered, and the generation of neutralizing antibodies is greatly reduced, so that the immune paralysis or the immune failure of organisms is caused, which undoubtedly puts higher requirements on antigen purification technology, and means that the time of purifying animal vaccines is coming.

Disclosure of Invention

The invention aims to provide a method for purifying porcine epidemic diarrhea virus, porcine reproductive and respiratory syndrome virus or avian influenza virus, which has the advantages of simple operation, high virus recovery rate, high removal rate of foreign protein and low cost.

The purpose of the invention is realized by adopting the following technical scheme.

A method for purifying porcine epidemic diarrhea virus, porcine reproductive and respiratory syndrome virus or avian influenza virus comprises the step of combining the porcine epidemic diarrhea virus, the porcine reproductive and respiratory syndrome virus or the avian influenza virus by adopting a compound formed by fusion protein PGFSN and GEM particles, wherein the amino acid sequence of the fusion protein PGFSN is shown as SEQ ID NO: 1.

In the present invention, the GEM particles are obtained by treating lactococcus lactis with an acid and boiling the acid.

In the invention, the fusion protein PGFSN is obtained by inducing and expressing recombinant bacteria carrying the fusion protein PGFSN coding gene.

In a preferred technical scheme, the sequence of the fusion protein PGFSN coding gene is shown as SEQ ID NO. 2.

In the invention, the recombinant bacteria after induced expression are cracked, centrifuged and then the supernatant of the lysate is taken as fusion protein PGFSN.

In the present invention, 2.5 × 10⁹Mixing the GEM particles with 95-105 mu g of fusion protein PGFSN, and incubating to form fusion protein PGFSN and GEM particlesThe complex of (1).

In the preferred technical scheme, the molecular weight is 2.5 × 10⁹Adding 10 into a compound formed by GEM particles and 95-105 mu g of fusion protein PGFSN^8.55TCID₅₀PEDV virus solution of (1), 10^8.09TCID₅₀The PRRSV virus solution or 50mL of H9N2 virus solution with the blood coagulation price of 1: 256.

In the present invention, the complex of the fusion protein PGFSN and GEM particles is bound to the virus, centrifuged, and the pellet is taken.

Compared with the prior art, the invention has the beneficial effects that: in the traditional purification methods such as centrifugation, membrane filtration, chromatography and the like, the centrifugation and the membrane filtration have very limited ability to remove foreign proteins due to low resolution, while the chromatography has high removal rate of foreign proteins, and the use of the purification materials and equipment used in the operation is hindered due to high cost. The applicant skillfully designs a fusion protein PGFSN, displays the fusion protein on the surface of GEM particles through the specific binding of an anchoring domain of the fusion protein PGFSN and GEM particles, and then realizes the purification of the viruses by utilizing the specific binding of a lectin domain of the fusion protein PGFSN to porcine epidemic diarrhea viruses, porcine reproductive and respiratory syndrome viruses and avian influenza viruses. The high specificity of the anchoring structure domain of the fusion protein PGFSN to GEM particles and the high specificity of the agglutinin structure domain to porcine epidemic diarrhea virus, porcine reproductive and respiratory syndrome virus and avian influenza virus in the invention determine that the method has extremely high resolution (namely high removal rate of foreign protein), in addition, the method is simple and convenient to operate, can be realized by two-step low-speed centrifugation, has low requirements on equipment, low cost, virus recovery rate higher than 98 percent and foreign protein removal rate more than 95 percent, and has prominent large-scale application prospect.

Drawings

FIG. 1 shows the result of enzyme digestion and identification of recombinant expression plasmid pET-PGFSN, where M is DNA marker; 1-3 are Nde I and Xho I double digestion products of recombinant plasmid pET-PGFSN.

FIG. 2 shows the identification result of the fusion protein, and FIGS. 2(A) and 2(B) show the identification results of SDS-PAGE and Western-blot, respectively, wherein lane 1 in the two figures is the supernatant of the lysate of the recombinant expression bacteria after induced expression; 2, inducing expression, recombining and expressing the lysate sediment, and M is a protein Marker; 3 is the supernatant of control bacteria lysate; 4, control bacteria lysate precipitate.

FIG. 3 shows the result of the identification of the specific binding between the PGFSN anchoring domain of the fusion protein and the GEM particles, wherein lane 1 is the supernatant separated after the GEM particles are added to the crude extract of the fusion protein PGFSN; m is a protein Marker; lane 2 is the pellet isolated after the GEM particles were added to the crude fusion protein PGFSN extract.

FIG. 4 shows the results of the activity assay of the lectin domain of the fusion protein, with the abscissa as the sample dilution, where PGFSN means the fusion protein PGFSN added to the coated ELISA plate for detection, PGFSN + RNase B means the incubation mixture of PGFSN and RNase added to the coated ELISA plate for detection, and control means the control lysate supernatant added to the coated ELISA plate for detection.

FIG. 5 is Ni²⁺-Sepharose HisTrap HP^TMThe SDS-PAGE identification result of the nickel column purified fusion protein, wherein M is a protein Marker; lane 1 is eluent 100mM imidazole; lane 2 is 200mM imidazole eluate; lanes 3-5 are 300mM imidazole eluates; lane 6 is an eluate of 500mM imidazole.

FIG. 6 is a fusion protein standard curve in which the OD is the abscissa and the fusion protein concentration is the ordinate.

FIG. 7 shows the results of identification of the purification of PEDV cell culture medium, and FIGS. 7(A), (B) and (C) show the results of identification of ELISA, SDS-PAGE and Western-blot, respectively; FIG. 7(A) shows the results of ELISA detection of each sample to be detected, with the abscissa being the number of the sample to be detected; in FIG. 7(B), lane 1 shows a culture solution of PEDV cells; 2 is a precipitate B; 3 is supernatant A; and M is a protein Marker. In FIG. 7(C), 1 is a supernatant A obtained by purifying a PEDV cell culture solution by the virus purification method of the present invention; m is a protein Marker; 2 is a sediment B obtained after the PEDV cell culture solution is purified by the virus purification method; PEDV cell culture medium is shown as 3.

FIG. 8 shows the result of identification of the purified PRRSV cell culture solution, FIGS. 8(A), (B) and (C) show the results of identification by ELISA, SDS-PAGE and Western-blot, respectively, FIG. 8(A) shows the result of ELISA detection of each sample to be tested, the abscissa indicates the number of the sample to be tested, sample 1 to be tested is the supernatant A obtained by purification of the PRRSV cell culture solution by the method of the present invention, sample 2 to be tested is the supernatant obtained by centrifugation after adding fusion protein PGFSN to Marc-145 cell lysate, sample 3 to be tested is the supernatant obtained by centrifugation after adding GEM particles to the PRRSV cell culture solution, sample 4 to be tested is the supernatant obtained by centrifugation after adding control bacteria lysate to the PRRSV cell culture solution, sample 5 to be tested is the unpurified PRRSV cell culture solution, and sample 6 to be tested is the fusion protein PGFSN. In FIG. 8(B), 1 is the supernatant A obtained after the PRRSV cell culture fluid is purified by the method of the present invention; 2 is a precipitate B obtained after the PRRSV cell culture solution is purified by the method of the invention; m is a protein Marker; and 3 is PRRSV cell culture solution. In FIG. 8(C), 1 is PRRSV cell culture; 2 is a precipitate B obtained after PRRSV cell culture fluid is purified by the virus purification method; and 3 is supernatant A obtained after PRRSV cell culture solution is purified by the virus purification method.

Detailed Description

The invention is further illustrated by the following examples.

Example preparation of a fusion protein having anchoring Effect

Design of fusion protein PGFSN: the design of the fusion protein PGFSN with anchoring function is the key for realizing virus purification in the invention, the amino acid sequence of the fusion protein is shown in SEQ ID NO:1, the total length is 258 amino acids, wherein the 1 st to 121 th amino acids are virus binding functional regions (lectin structural domains), the 138 th and 258 th amino acids are GEM particle binding functional regions (anchoring structural domains), and the 122 th and 137 th amino acids are flexible peptides connecting the two functional regions. The sequence of the encoding gene of the fusion protein PGFSN is shown as SEQ ID NO. 2. For protein identification, His-tag protein (HHHHHHHH) was introduced into the C-terminus of PGFSN as a fusion protein, and the His-tag protein-encoding gene was introduced before the stop codon of the sequence shown in SEQ ID NO:2 (CATCATCACCATCACCAT).

The fusion protein PGFSN was synthesized by Kinsery and inserted into the vector PUC57 to obtain recombinant plasmid PUC 57-PG.

(1) Construction of recombinant expression plasmids

Carrying out double enzyme digestion on the recombinant plasmid PUC57-PG and a prokaryotic expression vector pET32a by using restriction enzymes Nde I and Xho I, recovering a target gene and an expression vector fragment, and connecting by using T4DNA ligase to obtain the recombinant expression plasmid.

Wherein, the enzyme digestion reaction system is shown in Table 1.

TABLE 1 digestion reaction System

Components of the System	Dosage of
		10×QuickCut buffer	3μL
QuickCut NdeI	1μL
		QuickCut XhoI	1μL
Plasmid DNA	8μL
		dH₂O	Make up volume to 30. mu.L

The enzyme digestion reaction is carried out in an Eppendorf tube of 0.5mL, the mixture is gently mixed by a pipette and placed in a metal bath at 37 ℃ for reaction for 30min, and all the enzyme digestion products are taken out for electrophoresis.

Wherein, the connection reaction system is shown in Table 2.

TABLE 2 ligation reaction System

Components of the System	Dosage of
		The recovered pET32a enzyme fragment	2μL
Recovered target gene fragment	5μL
		T4DNA ligase	1μL
10×T4DNA lignase Buffer	1μL
		dH₂O	Make up volume to 10. mu.L

The components in the ligation reaction system were mixed well, centrifuged instantaneously and ligated overnight at 16 ℃.

After the ligation reaction is finished, the ligation product is transferred to DH5 alpha competent cells, the transformed recombinant plasmid is coated on an LB plate containing ampicillin and cultured overnight at 37 ℃, a single clone is selected and inoculated in 3mL of LB liquid medium containing ampicillin and cultured overnight at 37 ℃ and 220rpm by a shaking table, plasmid is extracted by a plasmid DNA small quantity extraction kit, Nde I and Xho I double enzyme digestion identification is carried out on the obtained plasmid, fragments of about 5389bp and 792bp are obtained from the positive recombinant plasmid (figure 1), the fusion protein coding gene is correctly inserted into an expression vector pET32a, and the recombinant plasmid with correct enzyme digestion verification is named as pET-PGFSN.

(2) Construction of recombinant expression bacteria

And (3) transforming BL21(DE3) competent cells by the recombinant expression plasmid pET-PGFSN to obtain the recombinant expression strain. Meanwhile, BL21(DE3) was transformed with the plasmid pET32a as a control to obtain a control bacterium.

(3) Inducible expression of fusion proteins

The recombinant expression strain monoclonal is selected and placed in 5mL LB liquid culture medium containing ampicillin, and cultured overnight at 37 ℃ and 220rmp to obtain mother liquor. Inoculating the mother liquor into a fresh LB liquid culture medium containing ampicillin at a volume ratio of 1:100, culturing at 37 deg.C and 220rmp for 4h, adding IPTG with a final concentration of 1mmol/L for induction, and performing induction expression at 15 deg.C and 180rmp for 24 h.

The control bacteria were induced and expressed in the same way.

(4) Extraction and identification of fusion proteins

And (3) centrifuging the recombinant expression bacteria liquid subjected to induction expression at 4 ℃ and 8000rpm for 10min, harvesting bacteria, washing the bacteria with PBS, and then resuspending. And (3) carrying out high-pressure cracking on the thallus suspension under the pressure of 800MPa, centrifuging the obtained thallus lysate for 15min at the temperature of 4 ℃ and the rpm of 12000, collecting the supernatant (namely the fusion protein PGFSN crude extract) and the precipitate of the thallus lysate, and carrying out SDS-PAGE electrophoresis and Western-blot identification. Meanwhile, the same method is adopted to obtain the supernatant and the precipitate of the control bacterium lysate.

FIG. 2A is SDS-PAGE electrophoresis, which shows that clear target protein bands appear in the supernatant and precipitate of lysate of recombinant expression bacteria with expected size of 27kDa after induced expression, and 50-70% of target proteins realize soluble expression, compared with the control bacteria. In Western-blot identification, a mouse anti-His tag monoclonal antibody (purchased from Nanjing Shengxing Biotech Co., Ltd.) is used as a primary antibody, a horseradish peroxidase-labeled goat anti-mouse IgG antibody (purchased from Wuhan doctor Drehd bioengineering Co., Ltd.) is used as a secondary antibody, a DAB color development kit is used for developing color, a specific band appears on a PVDF membrane, and the band at 27KDa is proved to be fusion protein PGFSN (figure 2B).

EXAMPLE Activity characterization of the PGFSN anchoring Domain of the Secondary fusion protein

1. Method for preparing GEM particles

A method of making GEM particles comprising the steps of:

(1) culturing lactococcus lactis MG1363 in GM17 culture medium on a shaking table at 30 deg.C overnight;

(2) centrifuging lactococcus lactis MG1363 fermentation liquor at room temperature and 6000rpm for 5min, and collecting thalli;

(3) fully resuspending the thallus with PBS, centrifuging at room temperature and 6000rpm for 5min, and washing for 1 time;

(4) adding 0.1M hydrochloric acid into the washed thallus, and boiling in water bath for 30 min;

(5) centrifuging the boiled thallus at room temperature and 6000rpm for 5min, and washing the precipitate with PBS for 3 times;

(6) and (4) resuspending the precipitate washed in the step (5) by using PBS to obtain the lactococcus lactis skeleton (namely the GEM particles). 1 GEM particle is the backbone of 1 lactococcus lactis MG 1363.

2.5 × 10⁹Individual GEM particles are defined as 1 unit GEM particle. The GEM granules were stored at-70 ℃ until use.

2. Binding of fusion protein PGFSN anchoring structure domain and GEM particle and specificity identification

A crude extract of fusion protein PGFSN was prepared as in example one, 1 unit of GEM particles was added to the crude extract, incubated at room temperature for 10min, then centrifuged at 9000rpm for 3min, and the supernatant and pellet were collected and subjected to SDS-PAGE for identification. As can be seen in FIG. 3, a specific band of interest of 27kDa appears in the lane of the pellet, indicating that the fusion protein PGFSN anchor domain binds specifically to the GEM particle.

EXAMPLES Activity identification of PGFSN lectin Domain of triple fusion proteins

The binding properties of the lectin domain of the fusion protein PGFSN to ribonuclease B (RNase B) were identified by ELISA.

The ELISA method for detecting the binding capacity of the fusion protein PGFSN and the RNase B comprises the following specific steps:

(1) coating: the ELISA plate was coated with RNase B to examine the binding ability of the fusion protein PGFSN to RNase B. The coating amount of the RNase B is 100 ng/hole, and the diluent of the RNase B is a carbonate buffer solution with the pH value of 9.6; coating in a refrigerator at 4 ℃ overnight, and washing with PBST (1% Tween-20 added to PBS buffer solution (pH7.2)) for 3 times;

(2) and (3) sealing: adding 1% BSA solution (adding 1% BSA at final concentration to PBST) into a 96-well plate, incubating at 37 ℃ for 3h at 200. mu.L/well, and washing PBST; BSA isBovine serum albumin。

(3) Adding 800 mu g/mL RNase B into 200 mu g/mL fusion protein PGFSN for co-incubation to obtain an incubation mixture of the fusion protein PGFSN and the RNase B. Adding fusion protein PGFSN crude extract (initial concentration is 200 mug/mL) with different dilutions or fusion protein PGFSN and RNase B incubation mixture into each hole of the ELISA plate, taking contrast bacteria lysate supernatant as contrast, adding 100 mug L/hole of each solution, incubating for 1h at 37 ℃, and washing by PBST;

(4) incubating the primary antibody: adding a mouse anti-His tag monoclonal antibody, incubating for 1h at 37 ℃, and washing by PBST;

(5) incubation of secondary antibody: adding a goat anti-mouse IgG antibody marked by HRP, incubating at 37 ℃ for 1h, and washing by PBST;

(6) developing with TMB developing solution;

(7) terminating the reaction by concentrated sulfuric acid;

(8) OD values were read at a wavelength of 450 nm.

As can be seen in FIG. 4, the fusion protein PGFSN lectin domain binds RNase B, whereas control lysate supernatant does not. In addition, the fusion protein PGFSN is incubated with RNase B solution in advance, and then an ELISA plate coated with RNaseB is added for ELISA reaction, so that the fusion protein PGFSN can inhibit or block the combination of the fusion protein PGFSN lectin domain and the RNase B on the ELISA plate, which shows that the combination of the fusion protein PGFSN and the RNase B is strong and has high specificity.

Example quantitative analysis of the four fusion proteins PGFSN

The fusion protein ELISA detection method comprises the following steps: the invention discovers that the fusion protein PGFSN and the RNase B have stronger combination for the first time, the combination has high specificity, and the ELISA method for detecting the fusion protein is established by utilizing the combination of the high specificity, and the specific method is shown in the third embodiment.

Preparation of fusion protein standard: the fusion protein PGFSN crude extract was prepared by the method of example one using Ni manufactured by GE corporation²⁺-Sepharose HisTrap HP^TMAnd (3) purifying the protein by using a nickel column, washing the protein by using a washing solution containing 20mM imidazole, eluting the protein by using 100mM imidazole, 200mM imidazole, 300mM imidazole and 500mM imidazole in sequence, collecting an eluent containing the fusion protein PGFSN, and carrying out SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) identification. FIG. 5 shows that a single protein band appears at the expected size, resulting in a fusion protein with nearly 100% purity. The fusion protein purified by the nickel column was subjected to imidazole removal using a GE Hitrap 5mL desaling Desalting column using an eluent of 300mM imidazole, and then the concentration thereof was quantified using a BCA protein quantification kit, thereby obtaining a fusion protein PGFSN standard product of a known concentration.

And (3) preparing a standard curve: the fusion protein standards with known concentrations were diluted in a gradient manner, and the OD values corresponding to the fusion protein PGFSN standards at each concentration were measured by the ELISA method described in example three, title 1, and a standard curve was prepared with the OD values as abscissa and the concentration of the fusion protein PGFSN as ordinate (fig. 6), and expressed by a formula. The crude extract of fusion protein prepared in the first example was assayed by the ELISA method, and the obtained OD value was substituted with the X value to determine the Y value, i.e., the concentration of the crude extract of fusion protein. Through calculation, 30mg of fusion protein PGFSN can be obtained in 400mL of fermentation liquor.

Example five quantitative analysis of GEM particle binding fusion protein PGFSN

The concentration of the fusion protein PGFSN crude extract prepared in the first example is determined by the method in the third example, 0.3, 0.5 and 1mL of the fusion protein crude extract are respectively added with 1 unit of GEM particles, the mixture is incubated for 10min at room temperature (20-30 ℃), and centrifuged at 9000rpm for 3min to obtain supernatant and precipitate, the supernatant is determined by the ELISA method in the third example, the maximum volume of the fusion protein not detected in the supernatant is the upper binding limit of 1 unit of GEM particles, and the binding of 95-105 μ g of fusion protein in 1 unit of GEM particles is determined.

EXAMPLE sixthly the method for purifying virus and the identification of the purified product of the present invention

1. Preparation of virus liquid

(1) Preparation of PEDV cell culture solution

PEDV CV777 strain (for one gem, Huang Chuan Juan, in dawn, Houting, Qiao stable, Zheng Sheng, Hou wave, screening of Pedv subunit vaccine immunopotentiator, North China agronomy report 2016,31(2):205-^6.9TCID₅₀/mL。

(2) Preparation of PRRSV cell culture solution

The PRRSV NJ strain (Genebank accession number: GQ375425.1) adopts Marc-145 cells to proliferate, the culture is harvested, repeated freeze thawing is carried out for 3 times, the supernatant is obtained by centrifugation, the titer is determined to be 10^6.5TCID₅₀/mL。

(2) Preparation of avian influenza virus chick embryo allantoic fluid

Avian influenza virus H9N2NJ02 strain and HZ strain (developed from four-linkage vaccine of ripple, newcastle disease, respiratory infectious bronchitis, egg drop syndrome and avian influenza (subtype H9): Nanjing agricultural university.2013) are propagated by adopting chick embryos, chick embryo allantoic fluid is obtained, the virus titer is determined by using hemagglutination test (HA), the hemagglutination price of NJ02 strain is 1:256, and the hemagglutination price of HZ strain is 1: 256.

2. the virus purification method disclosed by the invention is adopted to purify the PEDV CV777 strain, and the specific method is as follows:

(1) 1 unit of GEM particles and 105 mu g of fusion protein PGFSN crude extract are mixed, and shake incubation is carried out for 10min at the temperature of 20-30 ℃.

(2) Centrifuging at 9000rpm for 5min, collecting precipitate, and washing with PBS buffer solution for 3 times to obtain GEM-PGFSN compound;

(3) adding 45mL of PEDV CV777 strain cell culture solution into the GEM-PGFSN compound, placing the mixture in a shaking table at the temperature of 37 ℃ and the rotation speed of 150rpm, and performing shaking incubation for 1 h;

(4) centrifuging at 9000rpm for 5min, respectively collecting supernatant A and precipitate B, washing precipitate B with PBS buffer solution for 3 times to obtain virus purification compound GEM-PGFSN-virus, and here specifically obtaining GEM-PGFSN-PEDV.

In addition, PRRSV NJ strain, avian influenza virus NJ02 strain, and HZ strain were purified by the same method as described above. The difference is that the PRRSV NJ strain cell culture solution added in the step (3) is 39mL, the avian influenza virus NJ02 strain chick embryo allantoic fluid is 50mL, and the avian influenza virus HZ strain chick embryo allantoic fluid is 50 mL.

3. Identification of purified PEDV:

(1) detecting the content of PEDV in the supernatant after the PEDV CV777 strain cell culture solution is purified

The content of PEDV in the supernatant obtained by purifying the PEDV CV777 cell culture solution (i.e., the supernatant a obtained by purifying the PEDV 777 cell culture solution by the method of the title 2 in this example) was determined by sandwich ELISA, which was as follows: wrapping anti-PEDV monoclonal antibody (purchased from Shandong Green city Biotech Co., Ltd.) in ELISA plate, adding sample to be tested, incubating at 37 deg.C for 1h, washing PBST, adding HRP-labeled anti-PEDV monoclonal antibody, incubating at 37 deg.C for 1h, washing PBST, adding TMB color developing solution, acting for 15min, adding concentrated sulfuric acid to terminate reaction, and detecting OD on enzyme-linked immunosorbent assay₄₅₀. The sample to be tested includes supernatant a (marked as sample to be tested 1) obtained by purifying PEDV 777 cell culture solution by the method in title 2 of this embodiment, supernatant (marked as sample to be tested 2) obtained by adding fusion protein PGFSN to Vero cell lysate and then centrifuging, supernatant (marked as sample to be tested 3) obtained by adding GEM particles to PEDV cell culture solution and then centrifuging, supernatant (marked as sample to be tested 4) obtained by adding control bacteria lysate to PEDV cell culture solution and then centrifuging, unpurified PEDV cell culture solution (marked as sample to be tested 5) and fusion protein control (marked as sample to be tested 6), OD obtained by detecting sample to be tested 1-6₄₅₀The values are 0.127, 0.117, 1.356, 1.132, 1.43 and 0.077 in sequence, and the OD of the sample 1 to be detected can be determined from the above₄₅₀The results are equivalent to those of

samples

2 and 6 to be detected, which show that the PEDV residual amount in the supernatant A is almost zero after the PEDV cell culture solution is purified by the virus purification method, and the virus purification method has high purification efficiency, and is shown in FIG. 7 (A).

(2) Purity of virus purification complex GEM-PGFSN-PEDV was checked by SDS-PAGE

The supernatant A and the precipitate B obtained by purifying the PEDV cell culture solution and the PEDV cell culture solution were subjected to SDS-PAGE and identified, whereby FIG. 7(B) was obtained. As can be seen from the figure, compared with PEDV cell culture solution before purification, the impurity protein band in the precipitate B (i.e. GEM-PGFSN-PEDV) obtained after purification is obviously reduced, which shows that the purification method of the invention can remove a large amount of impurity protein, and the fusion protein band (about 27KDa) and a purer PEDV protein band (the M protein of PEDV is between 25 KDa and 35KDa, and the N protein of PEDV is between 75 KDa and 70 KDa) can be seen in the precipitate B after purification.

(3) Detection of virus specificity in virus purification complex GEM-PGFSN-PEDV by using Western-blot

And (3) purifying the PEDV cell culture solution and the PEDV cell culture solution to obtain a supernatant A and a precipitate B, and identifying the specificity of the purification method on the PEDV by using Western-blot. As can be seen from fig. 7(C), clear specific bands consistent with the sizes of PEDV M protein and N protein appear in both the PEDV cell culture solution and the precipitate B lanes, and only weak corresponding bands appear in the supernatant a, which again proves that PEDV can be enriched on the surface of GEM particles through the fusion protein PGFSN to achieve the purpose of purification; and the purification efficiency is higher.

All three methods described above demonstrate that the virus purification method of the present invention is capable of purifying PEDV.

4. Identification of PRRSV purified by the Virus purification method of the invention

The PRRSV NJ strain was purified by the purification method of the present invention, and then the purified products were identified by sandwich ELISA, SDS-PAGE and Western-blot with reference to title 3 of this example, to obtain FIG. 8(A), FIG. 8(B) and FIG. 8 (C). As can be seen from FIG. 8(A), sample 1 to be tested was tested for OD detection₄₅₀The method is equivalent to the

samples

2 and 6 to be detected, and shows that the PRRSV residual quantity in the supernatant A is almost zero after the PRRSV cell culture solution is purified by the virus purification method, thus the virus purification method has higher purification efficiency. As can be seen from FIG. 8(B), compared with the PRRSV cell culture solution before purification, the impurity protein band in the purified precipitate B is obviously reduced, which shows that the purification method of the invention can remove a large amount of impurity protein, and the Western-blot detection is further carried out because the virus concentration multiple is small and the virus protein band is not seen in the SDS-PAGE detection, and the monoclonal antibody of anti-PRRSV GP5 protein is used as a primary antibody (purchased from Nanjing Shiji animal health management Co., Ltd.), and the PRR is seen from FIG. 8(C)Clear specific bands which are consistent with the PRRSV GP5 protein in size are shown in an SV cell culture solution lane and a sediment B lane, and no corresponding band is shown in a supernatant A, so that the PRRSV can be enriched on the surface of a GEM particle through a fusion protein PGFSN to achieve the purpose of purification; and the purification efficiency is higher.

All three methods described above demonstrate that the virus purification method of the present invention is capable of purifying PRRSV.

5. The identification of the purified avian influenza virus by the virus purification method of the invention

(1) The content of the influenza virus in the supernatant A obtained after the purification by the virus purification method is detected by adopting Hemagglutination Assay (HA)

The samples to be tested were as follows: supernatant A (marked as a sample A1 to be detected) obtained by purifying avian influenza virus H9N2NJ02 strain chick embryo allantoic fluid by adopting the virus purification method of the invention, supernatant (marked as a sample A2 to be detected) obtained by adding contrast bacteria cracking fluid into avian influenza virus H9N2NJ02 strain chick embryo allantoic fluid, supernatant (marked as a sample A3 to be detected) obtained by adding GEM particles into avian influenza virus H9N2NJ02 strain chick embryo allantoic fluid, and avian influenza virus H9N2HZ strain chick embryo allantoic fluid (marked as a sample A4 to be detected); and similarly, the avian influenza virus H9N2HZ strain chick embryo allantoic fluid is purified by the virus purification method to obtain a sample A1 to be detected, a sample A2 to be detected, a sample A3 to be detected and a sample A4 to be detected, which correspond to the avian influenza virus H9N2HZ chick embryo allantoic fluid.

The method for detecting the content of the avian influenza virus in the sample to be detected by utilizing the hemagglutination test comprises the following steps: adding PBS buffer solution into a 96-hole U-shaped blood coagulation plate, adding 25 mu L/hole, adding a sample to be detected into the 1 st hole, adding 25 mu L/hole, diluting the sample to be detected by two times, adding the sample to be detected into the 2 nd to 11 th holes, discarding the 11 th hole by 25 mu L, finally adding 1% of chicken red blood cells, standing at room temperature for about 30min, and observing the result, wherein the maximum dilution degree of 100% agglutination of the red blood cells is the blood coagulation price of the influenza virus. Table 3 shows that the hemagglutination values of the samples A1-A4 to be tested of NJ02 strain are 1:2 respectively²、1:2⁸、1:2⁸、1:2⁸Table 4 shows that the blood coagulation prices of the samples A1-A4 to be tested of the HZ strain are 1:2¹、1:2⁸、1:2^8.5、1:2⁸The results show that the avian influenza virus chick embryo allantoisThe residual amount of the avian influenza virus in the supernatant A purified by the virus purification method is almost zero, which indicates that the virus purification method has higher purification efficiency.

TABLE 3 Hemagglutination Assay (HA) test results of test samples from NJ02 strain

Group of	HA blood coagulation valence
		Sample to be tested A1	1:2²
Sample to be tested A2	1:2⁸
		Sample to be tested A3	1:2⁸
Sample to be tested A4	1:2⁸

TABLE 4 hemagglutination test (HA) test results of HZ strain to-be-tested samples

EXAMPLE seventhly recovery determination of the Virus purification method of the present invention

In order to examine the recovery rate of the purified virus by the virus purification method of the present invention in example six, the virus content in the virus solution before purification and in the supernatant a obtained after purification of the virus solution in example six was examined by ELISA method (PEDV and PRRSV) and hemagglutination test (influenza virus).

For PEDV CV777 strain: when the test sample is virus solution before purification and 1/10, 1/20, 1/50, 1/100, 1/120 dilution and Vero cell lysate thereof, the obtained OD values are 1.903, 1.052, 0.873, 0.553, 0.342, 0.299 and 0.072 in sequence, while the OD value obtained by detecting supernatant a obtained after virus solution purification is 0.196 which is lower than the OD value obtained by detecting virus solution with 120 times dilution is lower than the OD value obtained by detecting virus solution after virus solution purification, and the residue of PEDV virus after purification is lower than (1/120) 100 percent, namely the residue of PEDV virus in supernatant a obtained after virus solution purification is lower than 1 percent, so the recovery rate of the purification method of the invention to the PEDV CV777 strain is higher than 99 percent.

For PRRSV NJ strains: when the test sample is virus culture solution before purification and 1/10, 1/20, 1/40, 1/80, 1/100 dilution and Marc-145 cell lysate, the obtained OD values are 2.257, 1.328, 0.964, 0.735, 0.582, 0.509 and 0.402 in sequence, while the OD value obtained by detecting supernatant A obtained after virus solution purification is 0.532, is lower than the OD value obtained by detecting 80 times of dilution virus solution, and the PRRSV residual amount after purification is lower than (1/80) 100%, namely the PRRSV residual amount in the supernatant A obtained after virus solution purification is lower than 2%, so the recovery rate of the PRRSV NJ strain by the purification method of the invention is higher than 99%.

For influenza virus, the data from example six were analyzed and the calculated recoveries were as follows: residual virus content of NJ02 strain 2²/2⁸100%, namely the PRRSV residual amount in the supernatant A obtained after the NJ02 strain virus liquid is purified is lower than 2%, so the recovery rate of the NJ02 strain by the purification method is higher than 98%; residual amount of HZ strain virus 2¹/2⁸100%, namely the PRRSV residual content in the supernatant A obtained after the HZ strain virus liquid is purified is lower than 1%, so that the recovery rate of the HZ strain by the purification method is higher than 99%.

EXAMPLE eighthly, determination of removal rate of foreign protein by Virus purification method of the present invention

To examine the removal rate of the purification method in example six for the impure proteins in the virus solution, the virus purification complex GEM-PGFSN-virus (pellet B, virus PEDV CV777 strain, prrsv j strain, avian influenza virus NJ02 strain, or avian influenza virus HZ strain) obtained in example six was resuspended in 1/10 volume of PBS buffer before purification, then 1% SDS (sodium dodecyl sulfate) was added to the suspension, boiled at 100 ℃ for 10min to dissociate the fusion protein from the GEM particles, centrifuged at 12000rpm for 10min, and the supernatant C was harvested. And (3) determining the total protein content in the supernatant C obtained after dissociation of the virus solution before purification and the virus purification compound GEM-PGFSN-virus by using a BCA protein quantitative kit. The total protein content of PEDV cell culture solution before purification was 3.2mg/mL, and the total protein content of supernatant C obtained after dissociation of virus purification complex GEM-PGFSN-PEDV was 876 μ g/mL, calculated as the antigen recovery efficiency of about 100%, and the removal rate of hetero-proteins was 100% (1- (0.1 × 876/3200) ], which was higher than 95%; the total protein content in the PRRSV cell culture solution before purification was 2.8mg/mL, the total protein content in the supernatant C obtained after dissociation of the virus purification complex GEM-PGFSN-PRRSV was 487 μ g/mL, calculated as the antigen recovery efficiency of about 100%, the removal rate of the foreign proteins was ═ 1- (0.1 × 487/2800) ] 100% and higher than 95%; similarly, the removal rate of the foreign proteins of influenza virus NJ02 strain and HZ strain was 95% or more by the method for purifying the six viruses of examples.

Sequence listing

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atgaccatcc gtagcggcga ctatattgat aaccaagctt tcgaaaccaa catgggtcgt 240

cgttttggtc cgtatggtgg cagcggtggc agcgcgaaca ccctgagcaa cgtgaaggtt 300

attcagatct cgtccgctgc gggcagcgga tccgataaag tccgcattta ttatgaacaa 360

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accggctgtg cgacatggac accgacggcg gtggctggac tgtgttccaa cgtcgtagcg 480

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Claims

1. A method for purifying porcine epidemic diarrhea virus, porcine reproductive and respiratory syndrome virus or avian influenza virus is characterized by comprising the steps of combining the porcine epidemic diarrhea virus, the porcine reproductive and respiratory syndrome virus or the avian influenza virus with a compound formed by fusion protein PGFSN and GEM particles, wherein the amino acid sequence of the fusion protein PGFSN is shown as SEQ ID NO:1, the GEM particles are obtained by treating lactic acid galactococcus with acid and boiling, the fusion protein PGFSN is obtained by inducing and expressing recombinant bacteria carrying fusion protein PGFSN coding genes, the recombinant bacteria after induced and expressed are cracked, lysate supernatant is taken as the fusion protein PGFSN after centrifugation, and 2.5 × 10⁹And mixing the GEM particles with 95-105 mu g of fusion protein PGFSN, and incubating to form a compound of the fusion protein PGFSN and the GEM particles.

2. The method for purifying the porcine epidemic diarrhea virus, the porcine reproductive and respiratory syndrome virus, or the avian influenza virus of claim 1, wherein the sequence of the fusion protein PGFSN encoding gene is shown in SEQ ID NO. 2.

3. The method of purifying porcine epidemic diarrhea virus, porcine reproductive and respiratory syndrome virus, or avian influenza virus of claim 2, wherein the concentration of the purified porcine epidemic diarrhea virus, porcine reproductive and respiratory syndrome virus, or avian influenza virus is 2.5 × 10⁹GEM particles and 95-105 mu g of fusion protein PGFSNAdding 10 into the formed composite^8.55TCID₅₀PEDV virus solution of (1), 10^8.09TCID₅₀The PRRSV virus solution or 50mL of H9N2 virus solution with a hemagglutination valence of 1:256, and is incubated for 1H at 37 ℃.

4. The method for purifying porcine epidemic diarrhea virus, porcine reproductive and respiratory syndrome virus, or avian influenza virus of claim 3, wherein said complex of fusion protein PGFSN and GEM particles binds to said virus, followed by centrifugation and removal of the pellet.