CN114736878A - Recombinant baculovirus co-expressing porcine interferon L3 and porcine interleukin 22 and application thereof - Google Patents

Abstract

The invention discloses a recombinant baculovirus applying a baculovirus/insect cell expression system to co-express porcine interferon L3 and porcine interleukin 22. The co-expressed rPoIFN-L3 and rPoIL-22 can be secreted and expressed in the cell supernatant through indirect immunofluorescence and Western-blot. The co-expressed rPoIFN-L3 and rPoIL-22 have good biological activity and can respectively and effectively inhibit TGEV/PEDV/PDCoV infection in vitro. In addition, rPoIFN-L3 and rPoIL-22 were found to have synergistic anti-TGEV/PEDV effects in vitro at a concentration of 50 ng/mL. A large amount of rPoIFN-L3 and rPoIL-22 with biological activity can be obtained through the protein expression system, and the invention lays a foundation for further realizing the industrialized preparation for resisting the porcine enteric coronavirus infection.

Description

Recombinant baculovirus co-expressing porcine interferon L3 and porcine interleukin 22 and application thereof

Technical Field

The invention relates to the field of genetic engineering, in particular to a recombinant baculovirus applying a baculovirus/insect cell expression system to co-express porcine interferon L3 and porcine interleukin 22 and application thereof.

Background

Diarrhoea caused by enteroviruses is very common and of great economic importance in the pig industry, with coronaviruses being the most predominant, consisting of 4 genera α, β, γ and δ. Among several porcine enteroviruses that have been found, transmissible gastroenteritis virus (TGEV), Porcine Epidemic Diarrhea Virus (PEDV) and the porcine acute diarrhea syndrome coronavirus (SADS-CoV) newly appeared in 2017 belong to the alpha coronavirus and the porcine delta coronavirus (PDCoV) belongs to the delta coronavirus. Results of a survey of porcine diarrhea-associated viruses in southern areas in 2012-2018 showed that PEDV is the most commonly detected virus with a prevalence in samples of 50.21% to 62.10% followed by PDCoV. Besides individual infections, mixed infections are also very common with several porcine enterocoronaviruses. Several porcine enterocoronaviruses, however, differ in their antigen and do not provide cross protection. A vaccine or therapeutic agent against a single virus is needed, but no reports on PDCoV and SADS-CoV vaccines have been reported so far, which makes how to effectively inhibit several porcine enterocoronavirus infections an urgent problem to solve.

Several porcine enterocoronaviruses have been found to act primarily on intestinal epithelial cells, although their cellular receptors differ. The IL-10 family cytokines IFN-L and IL-22 act selectively on mucosal epithelial cells, such as respiratory tract, intestinal tract and the like. It plays an important role in the mucosal epithelial barrier anti-infection process. Earlier laboratory studies prove that prokaryotic expression IL-22 and IFN-L can obviously inhibit porcine enteric coronavirus infection, and IFN-L3 is superior to IFN-L1 and type I interferon (IFN-alpha) in the aspect of resisting porcine enteric coronavirus. In addition, research indicates that IFN-L and IL-22 can synergistically inhibit rotavirus infection, and since porcine enterocoronavirus and rotavirus mainly act on intestinal epithelial cells, the IFN-L3 and IL-22 are presumed to synergistically inhibit porcine enterocoronavirus infection.

The baculovirus/insect cell expression system has the advantages of high product expression level, post-translational processing of the expressed product, similarity of the biological activities of immunogenicity, antigenicity, function and the like of foreign protein to natural protein, low cost of large-scale production of gene engineering products and the like in the practical application process, and is one of four expression systems of gene engineering. There has been no report of co-expressing porcine interferon L3 and porcine interleukin 22 using baculovirus/insect cell expression system so far.

Disclosure of Invention

In view of the above situation, in order to solve the problems of the above technologies, the present invention proposes to co-express porcine interferon L3 and porcine interleukin 22 recombinant baculovirus by using a baculovirus/insect cell expression system, and proves that co-expressed rpoIFN-L3 and rpoIL-22 can be secreted and expressed in cell supernatant through indirect immunofluorescence and Western-blot. The co-expressed rPoIFN-L3 and rPoIL-22 have good biological activity and can respectively effectively inhibit TGEV/PEDV/PDCoV infection in vitro. In addition, rPoIFN-L3 and rPoIL-22 were found to have synergistic anti-TGEV/PEDV effects in vitro at a concentration of 50 ng/mL. A large amount of rPoIFN-L3 and rPoIL-22 with biological activity can be obtained through the protein expression system, and a foundation is laid for further realizing the industrialized preparation for resisting the porcine intestinal coronavirus infection.

The recombinant baculovirus coexpressing porcine interferon L3 and porcine interleukin 22 comprises the following steps:

(1) construction of baculovirus expression vectors

Amplifying the full length of rPoIFN-L3 and rPoIL-22 genes to obtain PCR products; performing gel cutting purification on the obtained PCR product, performing double enzyme digestion on the pFastBacdual carrier by using EcoRI and Hind III, performing double enzyme digestion on the rPoIFN-L3 subjected to gel cutting purification by using EcoRI and Hind III, and performing PCR purification on the enzyme digestion product; the enzyme-cut pFastBacdual is connected with rPoIFN-L3 by using T4 ligase; carrying out double enzyme digestion on pFastBacdual-rPoIFN-L3 plasmid with correct sequencing by using Xho I and Sph I, and carrying out double enzyme digestion on rPoIL-22 obtained by gel cutting and purification by using Xho I and Sph I; then extracting the plasmid to obtain pFastBacdual-rPoIFN-L3-rPoIL-22 plasmid;

(2) construction of recombinant baculovirus

Converting pFastBacdual-rPoIFN-L3-rPoIL-22 into competence DH10Bac, screening by blue-white spots for 3 times, and then extracting bacmid;

(3) expression of rPoIFN-L3 and rPoIL-22

Firstly, insect cells are transfected, and SF21 cells are cultured by Grace's culture solution containing 10% fetal calf serum; mixing bacmid DNA and Cellffectin reagent in a sterilizing tube, and centrifuging to remove cells and large debris; filtering the supernatant by using a filter, transferring the supernatant into a new centrifugal tube, and keeping away from light to obtain a P1 strain substitute; inoculating the P1 generation virus into cells, collecting supernatant to obtain P2 generation virus; inoculating the P2 generation virus into insect cells to obtain a P3 generation virus; the P3 generation virus was inoculated into cells, the supernatant was collected, centrifuged to remove cells and large debris, and after the supernatant was filtered using a filter, the supernatant was transferred to a new centrifuge tube to obtain a supernatant containing rPoIFN-L3 and rPoIL-22.

According to the coexpression porcine interferon L3 and porcine interleukin 22 recombinant baculovirus, in the step (1), the PCR product is subjected to gel cutting purification, and the specific steps are as follows:

1) cutting the gel sheet containing the DNA fragments by using a clean scalpel, putting the gel into an EP tube weighed in advance, weighing, and recording the weight of the gel;

2) adding 1 volume of Binding Buffer into the gel slice;

3) incubating the gel mixture at 58 deg.C for about 10min until the gel sheet is completely dissolved, and inverting the mixing tube every 2min to promote thawing;

4) transferring the dissolved gel solution into a Gene JET purification column, centrifuging at 12000r/min for 1min, discarding liquid in a collection pipe, and putting the purification column into a recovery collection pipe;

5) adding the Wash Buffer into a purification column, centrifuging for 1min at 12000r/min, discarding liquid in a collecting pipe, and putting the purification column into a recovery collecting pipe;

6) centrifuging an empty Gene JET purification column 12000r/min for 1 min;

7) transferring the purification column to a new EP tube, adding 65 deg.C sterilized water into the column, standing for 1min, centrifuging at 12000r/min for 1min, measuring the concentration with ultraviolet spectrophotometer, and storing at-20 deg.C.

According to the coexpression porcine interferon L3 and porcine interleukin 22 recombinant baculovirus of the application, in the step (1), the PCR purification step of enzyme digestion products is as follows:

1) transferring the enzyme digestion product into a clean EP tube;

2) adding Binding Solution with the volume of 3 times into the enzyme digestion product, and oscillating and mixing uniformly;

3) putting the Spin Column into a collecting pipe, transferring the mixed liquid in the step 2) into the Spin Column, centrifuging for 1min at 12000r/min, discarding the liquid in the collecting pipe, and putting the Spin Column into a recovery collecting pipe again;

4) adding Wash Solution into the Column, standing for 1min, centrifuging at 12000r/min for 1min, discarding the liquid in the collecting pipe, and putting Spin Column into the recovery collecting pipe again; repeating the steps once;

5) centrifuging at 12000r/min for 5min, transferring Spin Column into new EP tube, adding 65 deg.C sterilized water to Spin Column, standing for 1min, and centrifuging at 12000r/min for 1 min; the concentration was measured by an ultraviolet spectrophotometer and stored at-20 ℃.

According to the application of the co-expression of porcine interferon L3 and porcine interleukin 22 recombinant baculovirus, in step (1), the enzyme-digested pFastBacDual and rPoIFN-L3 are connected by using T4 ligase, and the connection procedure is as follows: the ligation products were transformed at 25 ℃ for 2h and 65 ℃ for 15min, the transformation procedure was as follows:

1) adding 10 μ L of the ligation product into 100 μ L of DH5 α, ice-cooling for 30min, heat-shocking at 42 deg.C for 1min, and standing on ice for 3 min;

2) adding 800 μ L of nonreactive LB, and shaking in a shaker at 37 deg.C and 220r/min for 50 min;

3) centrifuging at 800r/min for 5min, removing supernatant, spreading on ampicillin solid LB plate, and standing at 37 deg.C in incubator for 15 h.

According to the coexpression porcine interferon L3 and porcine interleukin 22 recombinant baculovirus of the application, in the step (1), plasmid extraction is carried out, and the steps are as follows:

1) pouring the bacterial liquid into a 2mL EP tube, centrifuging at 12000r/min for 1min, and discarding the supernatant;

2) taking 250 mu L S1 to the sediment, and suspending the sediment again; putting 250 mu L S2 into a tube, turning the tube up and down for 4-6 times, and uniformly mixing for no more than 5 min;

3) putting 350 mu L S3 into a tube, turning the tube up and down for 6-8 times, and centrifuging the tube for 10min at 12000 r/min;

4) taking out the supernatant, placing the supernatant on a column, placing the column in a 2mL collecting pipe, and centrifuging at 12000r/min for 1 min; discarding the liquid in the collection tube and replacing the column in the recovery header;

5) adding 500 μ L of Wash 1 to the column, and centrifuging at 12000r/min for 1 min; discarding the liquid in the collection tube and replacing the column in the recovery header;

6) adding 700 mu L of Wash 2 to the column, and centrifuging at 12000r/min for 1 min; discarding the liquid in the collection tube and replacing the column in the recovery header; repeating the steps once; the collection tube was changed to a 1.5mL EP tube;

7) 50 mu L of 65 ℃ endotoxin-free water is taken to be put into a column, kept stand for 1min and centrifuged for 1min at 12000 r/min; the concentration was measured by an ultraviolet spectrophotometer and stored at-20 ℃.

According to the application of the co-expression porcine interferon L3 and porcine interleukin 22 recombinant baculovirus, in the step (2), pFastBacdual-rPoIFN-L3-rPoIL-22 is transformed into competent DH10Bac, and then is screened by blue-white spot for 3 times, and the steps are as follows:

1) adding 5 μ L plasmid into DH10Bac competence, mixing, ice-cooling for 30min, heat-shocking for 30s at 42 deg.C, and standing on ice for 3 min;

2) adding 800 μ L of nonreactive LB, placing in a shaking table, and shaking at 37 deg.C and 220r/min for 4 h;

3) diluting the conversion product with nonreactive LB according to a ratio of 1:10, 1:100 and 1:1000 respectively, coating 100 mu L of each conversion product on a solid LB plate which is prepared in advance and used for blue-white screening, and keeping out of the sun at 37 ℃ for 24 h;

4) selecting several white colonies from each plate, streaking on a new solid LB plate subjected to blue-white screening, performing 2 nd round blue-white screening, selecting white colonies after 3 rounds of screening, putting the white colonies into liquid LB containing Kan, Gen and Tet, and shaking for 24 hours at the temperature of 37 ℃ and at the speed of 220 r/min.

According to the co-expression porcine interferon L3 and porcine interleukin 22 recombinant baculovirus of the application, in the step (2), the bacmid extraction is carried out, and the steps are as follows:

1) pouring the bacterial liquid into a 2mL EP tube under the aseptic condition, centrifuging at 12000r/min for 1min, and removing the supernatant;

2) taking 250 mu L S1 to the sediment, and suspending the sediment again;

3) putting 250 mu L S2 into a tube, turning the tube up and down for 4-6 times, and uniformly mixing for less than 5 min;

4) putting 350 mu L S3 into a tube, turning over the tube for 6-8 times, and centrifuging the tube for 15min at 13000r/min and 4 ℃;

5) sucking the supernatant to another EP tube, adding isopropanol with the same volume, reversing, mixing, standing at 4 deg.C for 30min, and centrifuging at 12000r/min at 4 deg.C for 15 min;

6) discarding supernatant, adding 500 μ L70% anhydrous ethanol, turning upside down, washing precipitate, and centrifuging at 12000r/min for 5 min;

7) discarding the ethanol, drying for 10min, and dissolving with 80 μ L endotoxin-free water at 65 deg.C; the concentration was measured by an ultraviolet spectrophotometer and stored at-20 ℃.

According to the recombinant baculovirus co-expressing porcine interferon L3 and porcine interleukin 22, in the step (3), insect cells are transfected, and SF21 cells are cultured by Grace's culture solution containing 10% fetal bovine serum; preparing bacmid DNA and Cellffectin reagent to be mixed in a sterilizing tube, and operating steps are as follows:

1) 2ug of purified bacmid DNA was diluted into 100. mu.L of unsupplemented Grace medium;

2) completely mixing the Cellffectin reagent, and turning and mixing for 5-10 times; aspirate 8. mu.L of Cellffectin reagent and dilute into 100. mu.L of unsupplemented Grace medium;

3) diluted bacmid DNA was ligated using diluted Cellfectin reagent (total volume about 210 ul); lightly mixing, and incubating at room temperature for 30 min;

4) during the nurturing of the DNA/Cellfectin mixture, the medium was removed from the cells and washed with 2mL of unsupplemented Grace medium; discarding the cleaning culture medium;

5) adding 0.8mL of non-supplemented Grace culture medium into each tube containing the mixture, uniformly mixing the mixtures, and adding the mixtures into the holes containing the cells;

6) culturing at 27 deg.C for 5 h; discard the DNA/Cellfectin mixture, add 2mL of whole culture medium to the cells;

7) nurturing for 72h under 27 ℃ humid conditions or until the cells are observed to be diseased;

8) virus-containing cells were collected from each well and transferred to a 15mL centrifuge tube.

An application of recombinant baculovirus co-expressing porcine interferon L3 and porcine interleukin 22 in resisting porcine enterocoronavirus infection.

After the technology provided by the invention is adopted, the recombinant baculovirus coexpressing porcine interferon L3 and porcine interleukin 22 and the application thereof have the following beneficial effects: the invention successfully constructs a baculovirus expression system of rPoIFN-L3 and rPoIL-22 coexpression, and can secrete and express rPoIFN-L3 and rPoIL-22 proteins with biological activity; the invention lays a foundation for further realizing the industrialized preparation for resisting the porcine intestinal coronavirus infection.

Drawings

FIG. 1 shows the PCR results of recombinant bacmids;

wherein 1: rBacmid; 2: rBac-rPoIFN-L3-rPoIL-22; 3: blank control

FIG. 2 is an indirect immunofluorescence assay for the expression of rPoIFN-L3 and rPoIL-22 proteins;

FIG. 3 shows Western blot for detecting the expression sites of rPoIFN-L3 or rPoIL-22;

FIG. 4 shows the results of SDS-PAGE (a) and Western blot (b) of the purified products;

wherein 1: rPoIFN-L3 protein purified samples; 2: rPoIL-22 protein purified sample

FIG. 5 is a graph showing the measurement of antiviral activity of rPoIFN-L3 protein;

FIG. 6 is a graph of rPoIFN-L3/rPoIL-22 modulating expression of the relevant genes in IPEC-J2 cells;

FIG. 7 shows that rPoIFN-L3/rPoIL-22 inhibits infection by TGEV, PEDV or PDCoV;

FIG. 8 is a graph of the synergistic inhibition of TGEV, PEDV or PDCoV infection by rPoIFN-L3 and rPoIL-22.

Detailed Description

Various preferred embodiments of the present invention will be described below with reference to the accompanying drawings. The following description with reference to the accompanying drawings is provided to assist in understanding the exemplary embodiments of the invention as defined by the claims and their equivalents. It includes various specific details to assist understanding, but they are to be construed as merely illustrative. Accordingly, those skilled in the art will recognize that various changes and modifications can be made to the embodiments described herein without departing from the scope and spirit of the present invention. Also, in order to make the description clearer and simpler, a detailed description of functions and configurations well known in the art will be omitted.

1 construction of baculovirus expression vector

rPoIFN-L3 and rPoIL-22 were subjected to gene-optimized synthesis, with rPoIFN-L3 containing an FC-tag and rPoIL-22 containing a His-tag. The following primers are designed according to the synthetic gene sequence and the restriction enzyme cutting site of the vector, and the full length of the rPoIFN-L3 and rPoIL-22 genes is amplified:

rPoIFN-L3-EcoRⅠ-F：5'CGGAATTCATGCGTGTGCTGGTC3'

rPoIFN-L3-HindⅢ-R：5'CCCAAGCTTTTACTTGCCTTGGGTCTTG3'

rPoIL-22-XhoⅠ-F：5'CCGCTCGAGATGCGTGTGCTGGTG3'

rPoIL-22-SphⅠ-R：5'ACATGCATGCTTAGTGGTGGTGATGGTGG3'

the PCR reaction system is as follows: sterilized Water 30.5. mu.l, reaction buffer (5X) 10. mu.L, dNTP (2.5 mmol. multidot.L)^-1) mu.L, PrimeStar enzyme 0.5. mu.L, 2. mu.L each of the upstream and downstream primers (10. mu. mol. L-1, heavy and light chains), and 1. mu.L of template. The PCR reaction conditions of rPoIFN-L3 were as follows: the PCR procedure was: 35 cycles of 95 ℃ for 5min, 98 ℃ for 10s, 60 ℃ for 5s, and 72 ℃ for 60 s; carrying out 1% agarose gel electrophoresis on the PCR product obtained by amplification at 72 ℃ for 5min to identify whether the PCR product existsDNA. The PCR conditions for rPoIL-22 were all as follows: the PCR procedure was: 35 cycles of 95 ℃ for 2min, 94 ℃ for 30s and 68 ℃ for 90 s; and (3) carrying out 1% agarose gel electrophoresis on the PCR product obtained by amplification at 72 ℃ for 5min to identify whether DNA exists.

And (3) performing gel cutting purification on the obtained PCR product, and specifically comprising the following steps:

1) the gel pieces containing the DNA fragments were cut with a clean scalpel as close to the DNA as possible to reduce the gel volume. The gel was placed in a pre-weighed 1.5mL EP tube and weighed, and the gel weight was recorded.

2) Add 1 volume of Binding Buffer to the gel slice.

3) The gel mixture was incubated at 58 ℃ for about 10min until the gel pieces were completely dissolved. During which the mixing tube was inverted every 2min to facilitate thawing.

4) Transferring the dissolved gel solution to a Gene JET purification column, centrifuging at 12000r/min for 1min, discarding the liquid in the collection tube, and placing the purification column in the recovery collection tube.

5) Add 700. mu.L of Wash Buffer to the purification column (diluted with ethanol), centrifuge for 1min at 12000r/min, discard the liquid in the collection tube, and place the purification column in the recovery tube.

6) The empty Gene JET purification column was centrifuged at 12000r/min for 1 min.

7) The purification column was transferred to a new 1.5mL EP tube, 20. mu.L of 65 ℃ sterile water was added to the column, left to stand for 1min, and centrifuged at 12000r/min for 1 min. The concentration was measured by an ultraviolet spectrophotometer and stored at-20 ℃.

Firstly, carrying out double enzyme digestion on a pFastBacDual vector by using EcoRI and Hind III, wherein the enzyme digestion system is as follows: reaction buffer (10X) 2. mu.L, EcoRI, HindIII enzymes 1. mu.L each, sterile water 6. mu.L, pFastBacDual vector 10. mu.L. The enzyme digestion program is as follows: the obtained enzyme digestion product is subjected to gel cutting purification at 37 ℃ for 4h and 80 ℃ for 20min, and the steps are the same as the above.

The gel-cut purified rPoIFN-L3 was double digested with EcoRI, HindIII and the cleavage products were PCR purified because the fragments were smaller. The PCR purification steps were as follows:

1) the cleavage products were transferred to a clean EP tube.

2) Adding Binding Solution with the volume of 3 times into the enzyme digestion product, and oscillating and mixing uniformly.

3) Placing the Spin Column in a collecting tube, transferring the mixed liquid in the step (2) to the Spin Column, centrifuging at 12000r/min for 1min, discarding the liquid in the collecting tube, and placing the Spin Column in a recovery collecting tube.

4) Add 700. mu.L of Wash Solution to the Column, let stand for 1min, centrifuge at 12000r/min for 1min, discard the liquid from the collection tube, place Spin Column in the recovery header again. This step was repeated once.

5) Centrifuging at 12000r/min for 5min, transferring Spin Column into new EP tube, adding 30 μ L of 65 deg.C sterilized water to Spin Column, standing for 1min, and centrifuging at 12000r/min for 1 min. The concentration was measured by an ultraviolet spectrophotometer and stored at-20 ℃.

The cleaved pFastBacdual was ligated to rPoIFN-L3 using T4 ligase. The linking system is as follows: reaction buffer (10X) 1. mu.L, T4 ligase 1. mu.L, rPoIFN-L3 fragment 6.5. mu.L, pFastBacdual vector 1.5. mu.L. The connection procedure is as follows: the ligation product was transformed at 25 ℃ for 2h and 65 ℃ for 15 min. The conversion steps are as follows:

1) mu.L of the ligation product was added in its entirety to 100. mu.L of DH 5. alpha. competence, ice-cooled for 30min, heat-shocked at 42 ℃ for 1min, and then kept on ice for 3 min.

2) 800 μ L of nonreactive LB was added to the mixture, and the mixture was shaken in a shaker at 37 ℃ and 220r/min for 50 min.

3) Centrifuging at 800r/min for 5min, discarding the supernatant, and spreading on ampicillin-resistant solid LB plate. The mixture was placed in an incubator at 37 ℃ for 15 hours.

After removing the plate from the 37 ℃ incubator, the larger rounder single colony was picked up in 4mL ampicillin-resistant liquid LB, placed in a shaker, and shaken at 37 ℃ at 220r/min for 13 h. Then, plasmid extraction is carried out, and the steps are as follows:

1) the bacterial solution was poured into a 2mL EP tube, centrifuged at 12000r/min for 1min, and the supernatant was discarded.

2) 250 μ L S1 was taken up in the pellet and the pellet was resuspended. (3) And (3) putting 250 mu L S2 into a tube, turning the tube up and down for 4-6 times, and uniformly mixing for no more than 5 min.

3) Putting 350 mu L S3 into a tube, turning the tube up and down for 6-8 times, and centrifuging the tube at 12000r/min for 10 min.

4) The supernatant was removed and placed on a column, which was placed in a 2mL collection tube and centrifuged at 12000r/min for 1 min. The liquid in the collection tube was discarded and the column was replaced in the recovery header.

5) 500. mu.L of Wash 1 was applied to the column and centrifuged at 12000r/min for 1 min. The liquid in the collection tube was discarded and the column was replaced in the recovery header.

6) 700. mu.L of Wash 2 was applied to the column and centrifuged at 12000r/min for 1 min. The liquid in the collection tube was discarded and the column was replaced in the recovery header. This step was repeated once. The collection tube was changed to a 1.5mL EP tube.

7) 50 μ L of 65 ℃ endotoxin-free water was applied to the column, left to stand for 1min and centrifuged at 12000r/min for 1 min. The concentration was measured by UV spectrophotometry and stored at-20 ℃. The obtained plasmid was verified by double digestion, and the specific operation was the same as above. The correct plasmid pFastBacdual-rPoIFN-L3 is subjected to enzyme digestion verification and sent to Jilin province Cumei biotechnology limited company for sequencing.

The pFastBacdual-rPoIFN-L3 plasmid with the correct sequencing was double digested with Xho I and Sph I. The enzyme digestion system is as follows: reaction buffer (10X) 5u L, Xho I, SphI enzyme each 1.5 u L, sterile water 32 u L, pFastBacdual-rPoIFN-L3 plasmid 10u L. The enzyme digestion procedure is 12h at 37 ℃, 20min at 65 ℃, and the obtained enzyme digestion product is subjected to gel cutting purification, and the steps are the same as the above.

rPoIL-22, purified by gel cleavage, was digested simultaneously with Xho I and Sph I. The enzyme digestion system is as follows: reaction buffer (10X) 5u L, Xho I, SphI enzyme 1.5 u L, rPoIL-22 glue recovery product 42 u L. The digestion procedure was 12h at 37 ℃ and 20min at 65 ℃ and the resulting digestion product was purified by PCR as described above. The cleaved pFastBacdual-rPoIFN-L3 was T4 ligated to rPoIL-22.

The linking system is as follows: reaction buffer (10X) 2. mu.L, T4 ligase 1. mu.L, rPoIL-22 fragment 3. mu.L, pFastBacdual-rPoIFN-L3 vector 6. mu.L. The connection procedure is as follows: the ligation products were transformed at 16 ℃ for 12h and 65 ℃ for 15 min. The transformation procedure was as above.

Then, after the plate was taken out of the 37 ℃ incubator, a single colony of a larger circle was picked up in 4mL of ampicillin-resistant liquid LB, and placed in a shaker, and shaken at 37 ℃ and 220r/min for 13 hours. And then extracting the plasmid, wherein the plasmid extraction step is the same as the plasmid extraction step, and the double enzyme digestion verification is carried out on the obtained plasmid, and the specific operation is the same as the above. Enzyme digestion verification of correct pFastBacdual-rPoIFN-L3-rPoIL-22 plasmid sent to Jilin province U.S. Biotech limited for sequencing.

To this end, the optimally synthesized genes encoding rPoIFN-L3 and rPoIL-22 mature proteins were successfully inserted into the donor plasmid pFastBacDual, under the control of the PH and P10 promoters, respectively.

2 construction and identification of recombinant baculovirus

pFastBacdual-rPoIFN-L3-rPoIL-22 was transformed into competent DH10Bac and subjected to blue-white screening 3 times. The method comprises the following specific steps:

1) 5 mu L of plasmid is taken and put into DH10Bac competence, and evenly mixed, ice-washed for 30min, heat shock is carried out for 30s at 42 ℃, and the mixture is placed on ice for 3 min.

2) 800. mu.L of nonreactive LB was added to the mixture and the mixture was shaken in a shaker at 37 ℃ and 220r/min for 4 hours.

3) The resultant was diluted with nonreactive LB at 1:10, 1:100 and 1:1000, and 100. mu.L of each was applied to a solid LB plate prepared in advance for screening blue and white spots, and was kept at 37 ℃ for 24 hours in the dark.

4) Selecting several white colonies (big and isolated) on each plate, streaking on a solid LB plate of a new blue-white spot screening, carrying out 2 nd round blue-white spot screening, selecting white colonies to liquid LB containing Kan, Gen and Tet after 3 rounds of screening, and shaking for 24h at 37 ℃ and 220 r/min.

Then, extracting the bacmid, which comprises the following steps:

1) under the aseptic condition, the bacterial liquid is poured into a 2mL EP tube, centrifuged at 12000r/min for 1min, and the supernatant is discarded.

2) 250 μ L S1 was taken up in the pellet and the pellet was resuspended.

3) And (3) putting 250 mu L S2 into a tube, turning the tube up and down for 4-6 times, and uniformly mixing the tube for no more than 5 min.

4) Putting 350 mu L S3 into a tube, turning the tube up and down for 6-8 times, and centrifuging the tube for 15min at the temperature of 13000r/min and 4 ℃.

5) Sucking the supernatant to another EP tube, adding equal volume of isopropanol, reversing, mixing, standing at 4 deg.C for 30min, and centrifuging at 12000r/min at 4 deg.C for 15 min.

6) Carefully discard the supernatant, add 500. mu.L 70% absolute ethanol, reverse the top and bottom, wash the precipitate, and centrifuge at 12000r/min for 5 min.

7) Discard the ethanol, dry for 10min, dissolve with 80 μ L endotoxin-free water at 65 ℃. The concentration was measured by an ultraviolet spectrophotometer and stored at-20 ℃.

Preparation of blue-white screened LB solid plate: ampicillin (Ampicillin): 100mg of ampicillin was dissolved in 1mL of ddH2O, filtered for use, stored at-20 ℃ and finally used at 1:1000 (v/v). Kanamycin (Kanamycin): 50mg of kanamycin were dissolved in 1ml of ddH2O and filtered through a 0.22 μm filter at 1:1000(v/v) in-20 ℃. Gentamicin (Gentamicin): 7mg of gentamicin were dissolved in 1ml of ddH2O, filtered through a 0.22 μm filter, used at 1:1000(v/v), and stored at-20 ℃. Tetracycline (tetracyline): 10mg of tetracycline was dissolved in 1mL of absolute ethanol and used at 1:1000(v/v) at-20 ℃. Galactoside (X-gal): 100mg of galactoside was dissolved in 1mL of DMSO, used at 1:1000(v/v), and stored at-20 ℃. Isopropyl thiogalactoside (IPTG): 40mg of IPTG were dissolved in 1mL of ddH2O, filtered through a 0.22 μm filter, and used at 1:1000(v/v) at-20 ℃. After the preparation of the LB solid plate screened by the blue-white spot is finished, the LB solid plate is stored at 4 ℃ in a dark place.

The recombinant bacmid rBac-rPoIFN-L3-rPoIL-22 is identified by a PCR method: recombinant bacmids were PCR-identified using M13 upstream and downstream primers according to Bac-to-Bac baculovirus expression system instructions.

M13 Forward：5′GTTTTCCCAGTCACGAC3′

M13 Reverse：5′CAGGAAACAGCTATGAC3′

And (3) PCR reaction system: ex Taq enzyme 12.5. mu.L, M13 upstream and downstream primers 0.25. mu.L, sterile water 10. mu.L, plasmid diluted and added 1. mu.L (no more than 10 ng). And (3) PCR reaction conditions: at 94 ℃ for 4min, at 94 ℃ for 30s, at 55 ℃ for 30s, at 72 ℃ for 5min, for 35 cycles; and (3) carrying out 1% agarose gel electrophoresis on the PCR product obtained by amplification at 72 ℃ for 7min to determine whether the size of the DNA is in accordance with the expectation.

Expression of 3 rPoIFN-L3 and rPoIL-22

Insect cells were first transfected and SF21 cells were cultured in Grace's medium containing 10% fetal bovine serum. Inoculation of 9X 10 wells with 6-well plates per well⁵Sf21 cells were exposed for 2h at 27 ℃. For each transfected sample, bacmid DNA, Cellfectin reagent, was prepared to be mixed in a 1.5mL sterile tube. The specific operation is as follows:

1) 2ug of purified bacmid DNA was diluted into 100. mu.L of unsupplemented Grace medium.

2) And completely mixing the Cellffectin reagent, and turning and mixing for 5-10 times. Aspirate 8. mu.L of Cellfectin reagent diluted into 100. mu.L of unsupplemented Grace medium.

3) Diluted bacmid DNA (total volume approximately 210ul) was ligated using diluted Cellfectin reagent. Gently mixing, and incubating at room temperature for 30 min.

4) During the nurturing of the DNA/Cellfectin mixture, the media was removed from the cells and washed with 2mL of unsupplemented Grace media. The wash medium was discarded.

5) To each tube containing the mixture was added 0.8mL of unsupplemented Grace medium, the mixture was mixed and added to the wells containing the cells.

6) Culturing at 27 deg.C for 5 h. The DNA/Cellfectin mixture was discarded and 2mL of whole medium was added to the cells.

7) Incubate at 27 ℃ for 72h in moist condition or until the cells are observed to develop lesions.

8) Virus-containing cells were collected from each well and transferred to a 15mL centrifuge tube.

Cells and large debris were removed by centrifugation at 500r/min for 5 min. After the supernatant was filtered through a 0.45um filter, the supernatant was transferred to a new 15mL centrifuge tube and stored at 4 ℃ in the dark. This is the P1 strain. Inoculating the P1 toxin substitute to 2 × 10 density at MOI of 0.05⁶In the cell of (a). And collecting the supernatant after 72h, and specifically performing the operation (9) obtaining the virus of P1 generation. Thus, the P2 generation virus was obtained. The P2 generation virus was inoculated to insect cells as above to obtain the P3 generation virus. Seed virus titers were determined by plaque assay.

P3 generation virus was inoculated at a cell density of 2 × 10 at MOI of 5⁶The supernatant was collected for 96 hours, centrifuged at 4 ℃ at 1000r/min for 10 minutes, and the cells and large debris were removed. After filtering the supernatant using a 0.45um filter, the supernatant was transferred to a new centrifuge tube. Thus, a supernatant containing rPoIFN-L3 and rPoIL-22 was obtained.

4 detection of rPoIFN-L3 and rPoIL-22 expression by indirect immunofluorescence and Western blot

The indirect immunofluorescence was performed as follows: identifying correct recombinant bacmid transfected SF21 insect cells in logarithmic growth phase, culturing at 27 ℃ for 72h, fixing with 4% paraformaldehyde for 30min when the morphology appears pathological changes, penetrating with 0.05% Triton X-100 for 15min, blocking with 5% skim milk at 37 ℃ for 2h, and taking a mouse anti-His tag antibody diluted by 1:500 as a primary antibody, 1: alexa Fluor 546 gat anti-mouse IgG antibody diluted 500 times and goat anti-pig IgG (abcom) diluted 1:800 times are used as secondary antibodies, and fluorescence is observed by an inverted fluorescence microscope after DAPI staining for 10 min.

The Western blot is specifically operated as follows: collecting 96h virus infection supernatant, centrifuging at 1000r/min for 10min, adding 5 xSDS Buffer, boiling, performing SDS-PAGE electrophoresis, and wet transferring to nitrocellulose membrane. And carrying out Western blot detection by using goat anti-pig IgG diluted by 1:5000 times and mouse anti-His label diluted by 1:2000 times as primary antibodies and using IRDye 800-labeled anti-goat IgG antibody and anti-mouse IgG antibody diluted by 1:10000 times as secondary antibodies, and carrying out imaging by using a near-infrared fluorescence scanning imaging system.

Purification of 5 rPoIFN-L3/rPoIL-22

Collecting supernatant of the infectious virus for 96h, centrifuging for 10min at 1000r/min, filtering with a 0.45 μm filter membrane to remove impurities, and purifying with Protein A affinity chromatography column and cobalt affinity chromatography column in sequence. The purified protein samples were then subjected to SDS-PAGE and wet-transferred to nitrocellulose membranes. And carrying out Western blot detection by using goat anti-pig IgG diluted by 1:5000 times and mouse anti-His label diluted by 1:2000 times as primary antibodies and using IRDye 800-labeled anti-goat IgG antibody and anti-mouse IgG antibody diluted by 1:10000 times as secondary antibodies, and carrying out imaging by using a near-infrared fluorescence scanning imaging system.

6 rPoIFN-L3 antiviral activity assay

1) Spreading appropriate amount of MDBK cells to 96-well cell culture plate;

2) diluting a sample to be detected by multiple times, wherein the dilution is 10 < -1 > to 10 < -8 >;

3) adding 100 mu L of diluted sample into a 96-well plate, setting 3 repeated wells for each dilution, and setting an untreated group as a control;

4) culturing at 37 deg.C for 24 hr, removing supernatant, inoculating VSV GFP virus, and inoculating virus to untreated group as virus control group;

5) when the cytopathic effect (CPE) of the virus control group was about 100%, the expression of GFP fluorescent protein was observed by an inverted fluorescence microscope.

7 rPoIFN-L3/rPoIL-22 activity and swine enterocoronavirus resistant activity detection

IPEC-J2 cells were seeded into 24-well plates and grown for 1 day to generate a confluent monolayer of cells. IPEC-J2 cells were treated with rPoIFN-L3 or rPoIL-22 at final concentrations of 10ng/mL, 100ng/mL, 1000ng/mL, and F12 DMEM as a negative control. After 24h of culture, collecting cells to detect related gene expression.

After 24h of treatment with rPoIFN-L3 or rPoIL-22 with final concentration of 10ng/mL, 100ng/mL or 1000ng/mL, the virus is inoculated with TGEV, PEDV or PDCoV with MOI of 1, absorbed at 37 ℃ for 2h, virus solution is removed, the virus solution is washed for 3 times, F12 DMEM is added to be cultured at 37 ℃ for 48h, and cells and supernatant are collected to detect the virus infection.

Detection of 8 rPoIFN-L3 and rPoIL-22 in vitro synergistic antiviral action

IPEC-J2 cells were seeded into 24-well plates and grown to a confluent monolayer. Treating cells with rPoIFN-L3, rPoIL-22 and mixed rPoIFN-L3 and rPoIL-22 for 24h, inoculating TGEV, PEDV or PDCoV with MOI of 1, adsorbing at 37 ℃ for 2h, removing virus liquid, washing for 3 times, adding F12 DMEM, culturing at 37 ℃ for 48h, and collecting cells to detect virus infection. Wherein the concentrations of rPoIFN-L3 and rPoIL-22 used alone were 100ng/mL, and the concentrations of rPoIFN-L3 and rPoIL-22 mixed were 50ng/mL, respectively.

In the embodiment of the invention, the baculovirus transfer vector is presented in the laboratory of the teacher of Liuchang Ming; insect cells SF21 were stored by the inventors laboratory; coli (e.coli) DH10Bac competent cells, Grace's insect cell culture medium, Fetal Bovine Serum (FBS) were purchased from zemer feishel technologies ltd; coli (e.coli) DH5 α competent cells were purchased from TaKaRa;

sf9 insect cell serum-freeMedium II was purchased from Shanghai culture Biotech GmbH. Gene-optimized synthesis of rPoIFN-L3 and rPoIL-22 was performed by Jinzhi Biotech, Inc., Suzhou.

The results of the above construction method are analyzed below.

1 PCR analysis of recombinant bacmids

To determine whether the transposition of pFastBacdual-rPoIFN-L3-rPoIL-22 was successful. In the research, PCR amplification of recombinant bacmid DNA is carried out by adopting M13 upstream and downstream primers, and whether the PCR amplification is successful or not can be judged according to the size of a product band. The pIFN-L3 gene sequence size is 1311bp, and the pIL-22 gene sequence size is 549 bp. The results showed that the band size of the transposable product of pFastBacdual-rPoIFN-L3-rPoIL-22, rBac-rPoIFN-L3-rPoIL-22PCR, was around 5000bp, while the band size of the transposable product of pFastBacdual, rBacmid PCR, was around 2500bp, as shown in FIG. 1. That is, it was shown that the transposition of pFastBacdual-rPoIFN-L3-rPoIL-22 was successful.

Expression of 2 rPoIFN-L3/rPoIL-22 protein

After rBac-rPoIFN-L3-rPoIL-22 bacmid was transfected into SF21 insect cells, to verify whether rPoIFN-L3 or rPoIL-22 protein was expressed, the study used antibodies of different fluorescent signals and detected by indirect immunofluorescence. The results show that after 72H of transfection, rPoIFN-L3 showed green fluorescence signals detected by goat anti-pig IgG H & L (FITC) antibody, while rPoIL-22 showed red fluorescence signals detected by mouse anti-His tag antibody-fluorescent gene 546goat anti-mouse IgG antibody, and the 2 fluorescence signals almost completely coincided, as shown in FIG. 2. To further explore the expression sites of rPoIFN-L3 and rPoIL-22 proteins, equal amounts of cell supernatants, cell lysates, and cell pellets were detected by Western blot method, wherein rPoIFN-L3 was detected by goat anti-porcine IgG-DyLight 700-labeled anti-goat IgG antibody and rPoIL-22 was detected by mouse anti-His monoclonal antibody-DyLight 700-labeled anti-mouse IgG antibody. The results show that the protein is mainly present in the supernatant and the content in the cell lysate or cell pellet is small, as shown in FIG. 3. The above results indicate that rBac-pIFN-L3-pIL-22 bacmid was successfully expressed after transfection into insect cells, and the expressed protein was mainly present in the supernatant.

Purification and identification of 3 rPoIFN-L3/rPoIL-22 protein

rPoIFN-L3 or rPoIL-22 Protein is centrifugally concentrated by an ultrafiltration tube, and then purified by a Protein A affinity chromatographic column and a cobalt affinity chromatographic column in sequence. To identify the effect of protein purification, this study was examined using SDS-PAGE. The results are shown in FIG. 4a, which shows that neither rPoIFN-L3(48Ku) nor rPoIL-22(20Ku) protein bands are unique. The rPoIFN-L3 protein purified sample infers that a band with the size of 50Ku is rPoIFN-L3 protein according to the size of the protein, and the lower band can be an shed Fc label or a partial protein of rPoIFN-L3 formed due to degradation and the like. The several bands of the rPoIL-22 protein purified sample are assumed to be rPoIL-22 protein, and the reason for the formation of the bands is probably that the sizes of the proteins are slightly different due to different glycosylation of the proteins in the protein expression process. Therefore, the present study was further verified by the Western blot method using an antibody specific to Fc tag or His tag. The results are shown in FIG. 4b, which shows that 2 bands of the rPoIFN-L3 protein purified sample can be recognized by the Fc tag specific antibody, and several bands of the rPoIL-22 protein purified sample can be recognized by the His tag specific antibody, which is expected. The results prove that the rPoIFN-L3 or rPoIL-22 protein has better purification effect and no impurities.

Analysis of antiviral Activity of 4 rPoIFN-L3

To investigate whether baculovirus-expressed rPoIFN-L3 has antiviral activity and units of antiviral activity, this study used the VSV GFP virus to perform assays for rPoIFN-L3 antiviral activity. The results are shown in FIG. 5, indicating that rPoIFN-L3 is antiviral active, and the antiviral activity of calculated purified rPoIFN-L3 is in units of 5X 105 AU/mg. The antiviral activity of the supernatant containing rPoIFN-L3 was 1X 103 AU/mL. For uniform quantification in subsequent studies, the antiviral activity units were not used in this study, but protein concentration units were used.

Assay for 5 rPoIFN-L3 or rPoIL-22 protein Activity

In order to investigate whether the baculovirus-expressed rPoIFN-L3 or rPoIL-22 has biological activity, the expression of related genes in cells is detected after IPEC-J2 cells are treated for 24 hours by rPoIFN-L3 or rPoIL-22 with certain concentration. The results show that rpoen-L3 can dose-dependently induce the expression of interferon stimulated gene 15(ISG15) (fig. 6a), mucosal virus resistance gene a (mxa) (fig. 6b), interferon induced transmembrane protein 1(IFITM1) (fig. 6c) or 3(IFITM3) (fig. 6d), 2'-5' -oligoadenylate synthetase-like protein (OASL) (fig. 6 e). rPoIFN-L3 at a concentration of 1000ng/mL was able to upregulate approximately 150-fold, 1500-fold, 160-fold, 40-fold, or 2000-fold expression of ISG15, MXA, IFITM1, IFITM3, or OASL, respectively, after treatment of the cells (FIGS. 6 a-e). That is, rPoIFN-L3 successfully stimulates the transcription of interferon-stimulated gene (ISG) in IPEC-J2 cells to promote the cells to have antiviral state. Similarly, rPoIL-22 was dose-dependent to up-regulate the expression of the antimicrobial protein beta defensin 2(BD-2) in IPEC-J2 cells, 1000ng/mL of rPoIL-22 up-regulated BD-2 expression by about 8-fold, 100ng/mL of rPoIL-22 up-regulated BD-2 expression by about 5-fold, and 10ng/mL of rPoIL-22 up-regulated BD-2 expression by about 4-fold (FIG. 6 f). The above results demonstrate that rPoIFN-L3 and rPoIL-22 have good biological activity, respectively.

6 rPoIFN-L3 or rPoIL-22 inhibiting porcine enterocoronavirus infection

Previous research work in this laboratory has shown that prokaryotic expression of IFN-L or IL-22 can inhibit the infection of enterocoronavirus. In order to detect whether the rPoIFN-L3 or rPoIL-22 expressed by baculovirus can inhibit virus infection, IPEC-J2 cells are pretreated for 24h by using rPoIFN-L3 or rPoIL-22 with a certain concentration, then TGEV, PEDV or PDCoV are respectively inoculated according to MOI (maximum average molecular weight) of 1, and the cells are collected after being cultured for 48h and then detected by a relative fluorescence quantitative PCR method. The results are shown in FIG. 7c and FIG. 7a, showing that rPoIFN-L3 and rPoIL-22 can inhibit TGEV, PEDV or PDCoV infection in a dose-dependent manner, respectively. To ensure the reliability of the results, the study also examined the supernatant for TCID50, which is shown in fig. 7d and 7b, and which is consistent with the results of the relative fluorescent quantitative PCR method. rPoIFN-L3 was shown to reduce TGEV infection by more than 70%, PEDV by more than 80%, and PDCoV by more than 90% at concentrations of 1000ng/mL and 100ng/mL (FIGS. 3-3 d). rPoIL-22 reduced TGEV or PEDV infection by about 80% and PDCoV infection by about 85% at concentrations of 1000ng/mL and 100ng/mL (FIG. 7 b). The above results indicate that baculovirus-expressed rPoIFN-L3 or rPoIL-22 was effective in inhibiting TGEV/PEDV/PDCoV infection in IPEC-J2 cells.

7 rPoIFN-L3 and rPoIL-22 synergistically inhibit infection by porcine enterocoronavirus

To investigate whether the baculovirus-expressed rPoIFN-L3 and rPoIL-22 can synergistically inhibit porcine enteric coronavirus infection. After the cells of IPEC-J2 were co-treated with rPoIFN-L3 and rPoIL-22 for 24h in advance, TGEV, PEDV or PDCoV were inoculated at MOI ═ 1 for 48h, and the cells were collected and detected by the relative fluorescent quantitative PCR method. The results show that rPoIFN-L3 and rPoIL-22 have a good synergistic effect at a concentration of 50 ng/mL. Compared with a virus control group, cells treated by rPoIFN-L3 or rPoIL-22 with the concentration of 100ng/mL alone can inhibit TGEV infection by 2.2 times or 1.4 times, PEDV infection by 178 times or 12 times and PDCoV infection by 20.5 times or 4.8 times. And after the cells are co-treated by 50ng/mL rPoIFN-L3 and 50ng/mL rPoIL-22, TGEV infection can be inhibited by 4.5 times, PEDV infection can be inhibited by 334 times, and PDCoV infection can be inhibited by 24.3 times, namely TGEV, PEDV or PDCoV infection is inhibited by 75%, 99.6% or 95% (figure 8). The above results indicate that the anti-TGEV/PEDV effect of co-treated cells with rPoIFN-L3 and rPoIL-22 at a concentration of 50ng/mL is superior to that of rPoIFN-L3 or rPoIL-22 alone at a concentration of 100 ng/mL. That is, 50ng/mL of rPoIFN-L3 and rPoIL-22 had a synergistic effect in vitro against TGEV/PEDV infection, while the concentrations of rPoIFN-L3 and rPoIL-22 showed an additive effect in vitro against PDCoV infection.

The present invention has been described in detail, and the principle and embodiments of the present invention are explained herein by using specific examples, which are only used to help understand the method and the core idea of the present invention; meanwhile, for a person skilled in the art, according to the idea of the present invention, there may be variations in the specific embodiments and the application scope, and in summary, the content of the present specification should not be construed as a limitation to the present invention.

From the above description of the embodiments, it will be apparent to those skilled in the art that the present invention may be practiced. Of course, the above listed cases are only examples, and the present invention is not limited thereto. It should be understood by those skilled in the art that other modifications or simplifications according to the technical solution of the present invention may be appropriately applied to the present invention and should be included in the scope of the present invention.

Claims (9)

1. A recombinant baculovirus co-expressing porcine interferon L3 and porcine interleukin 22 is characterized by comprising the following steps:

(1) construction of baculovirus expression vectors

Amplifying the full length of rPoIFN-L3 and rPoIL-22 genes to obtain a PCR product; performing gel cutting purification on the obtained PCR product, performing double enzyme digestion on the pFastBacdual carrier by using EcoRI and Hind III, performing double enzyme digestion on the rPoIFN-L3 subjected to gel cutting purification by using EcoRI and Hind III, and performing PCR purification on the enzyme digestion product; the enzyme-cut pFastBacDual and rPoIFN-L3 are connected by using T4 ligase; carrying out double enzyme digestion on pFastBacdual-rPoIFN-L3 plasmid with correct sequencing by using Xho I and Sph I, and carrying out double enzyme digestion on rPoIL-22 obtained by gel cutting and purification by using Xho I and Sph I; then extracting the plasmid to obtain pFastBacdual-rPoIFN-L3-rPoIL-22 plasmid;

(2) construction of recombinant baculovirus

Converting pFastBacdual-rPoIFN-L3-rPoIL-22 into competence DH10Bac, screening by blue-white spots for 3 times, and then extracting bacmid;

(3) expression of rPoIFN-L3 and rPoIL-22

Firstly, insect cells are transfected, and SF21 cells are cultured by Grace's culture solution containing 10% fetal calf serum; mixing bacmid DNA and Cellffectin reagent in a sterilizing tube, and centrifuging to remove cells and large debris; filtering the supernatant by using a filter, transferring the supernatant into a new centrifuge tube, and keeping away from light to obtain a P1 strain substitute; inoculating the P1 generation virus into cells, collecting supernatant, and obtaining P2 generation virus; inoculating the P2 generation virus into insect cells to obtain a P3 generation virus; the P3 generation virus was inoculated into cells, the supernatant was collected, centrifuged to remove cells and large debris, and after the supernatant was filtered using a filter, the supernatant was transferred to a new centrifuge tube to obtain a supernatant containing rPoIFN-L3 and rPoIL-22.

2. The recombinant baculovirus co-expressing porcine interferon L3 and porcine interleukin 22 according to claim 1, wherein,

in the step (1), the PCR product is subjected to gel cutting and purification, and the specific steps are as follows:

1) cutting the gel sheet containing the DNA fragments by using a clean scalpel, putting the gel into an EP tube weighed in advance, weighing, and recording the weight of the gel;

2) add 1 volume of Binding Buffer to the gel slice;

3) incubating the gel mixture at 58 deg.C for about 10min until the gel sheet is completely dissolved, and inverting the mixing tube every 2min to promote thawing;

4) transferring the dissolved gel solution into a Gene JET purification column, centrifuging at 12000r/min for 1min, discarding the liquid in the collection pipe, and putting the purification column into the recovery collection pipe;

5) adding the Wash Buffer into a purification column, centrifuging for 1min at 12000r/min, discarding liquid in a collecting pipe, and putting the purification column into a recovery collecting pipe;

6) centrifuging an empty Gene JET purification column 12000r/min for 1 min;

7) transferring the purification column to a new EP tube, adding 65 deg.C sterilized water into the column, standing for 1min, centrifuging at 12000r/min for 1min, measuring the concentration with ultraviolet spectrophotometer, and storing at-20 deg.C.

3. The recombinant baculovirus co-expressing porcine interferon L3 and porcine interleukin 22 according to claim 1, wherein,

in the step (1), the PCR purification of the enzyme digestion product comprises the following steps:

1) transferring the enzyme digestion product into a clean EP tube;

2) adding 3 times of volume of Binding Solution into the enzyme digestion product, and oscillating and mixing uniformly;

3) putting the Spin Column into a collecting pipe, transferring the mixed liquid in the step 2) into the Spin Column, centrifuging for 1min at 12000r/min, discarding the liquid in the collecting pipe, and putting the Spin Column into a recovery collecting pipe again;

4) adding Wash Solution into the Column, standing for 1min, centrifuging at 12000r/min for 1min, discarding the liquid in the collecting tube, and putting the Spin Column into the recovery collecting tube again; repeating the steps once;

5) centrifuging at 12000r/min for 5min, transferring Spin Column into new EP tube, adding 65 deg.C sterilized water to Spin Column, standing for 1min, and centrifuging at 12000r/min for 1 min; the concentration was measured by an ultraviolet spectrophotometer and stored at-20 ℃.

4. The recombinant baculovirus co-expressing porcine interferon L3 and porcine interleukin 22 according to claim 1, wherein,

in the step (1), the enzyme-cut pFastBacdual and rPoIFN-L3 are connected by using T4 ligase, and the connection procedure is as follows: the ligation products were transformed at 25 ℃ for 2h and 65 ℃ for 15min, the transformation procedure was as follows:

1) adding 10 μ L of the ligation product into 100 μ L of DH5 α, ice-cooling for 30min, heat-shocking at 42 deg.C for 1min, and standing on ice for 3 min;

2) adding 800 μ L of nonreactive LB, and shaking in a shaker at 37 deg.C and 220r/min for 50 min;

3) centrifuging at 800r/min for 5min, removing supernatant, spreading on ampicillin solid LB plate, and standing at 37 deg.C in incubator for 15 h.

5. The recombinant baculovirus co-expressing porcine interferon L3 and porcine interleukin 22 according to claim 1, wherein,

in the step (1), plasmid extraction is performed, and the steps are as follows:

1) pouring the bacterial liquid into a 2mL EP tube, centrifuging at 12000r/min for 1min, and discarding the supernatant;

2) taking 250 mu L S1 to the sediment, and suspending the sediment again; putting 250 mu L S2 into a tube, turning the tube up and down for 4-6 times, and uniformly mixing for no more than 5 min;

3) putting 350 mu L S3 into a tube, turning the tube up and down for 6-8 times, and centrifuging the tube at 12000r/min for 10 min;

4) taking out the supernatant, placing the supernatant on a column, placing the column in a 2mL collecting pipe, and centrifuging at 12000r/min for 1 min; discarding the liquid in the collection tube and replacing the column in the recovery header;

5) adding 500 μ L of Wash 1 to the column, and centrifuging at 12000r/min for 1 min; discarding the liquid in the collection tube and replacing the column in the recovery header;

6) adding 700 μ L of Wash 2 to the column, and centrifuging at 12000r/min for 1 min; discarding the liquid in the collection tube and replacing the column in the recovery header; repeating the steps once; the collection tube was changed to a 1.5mL EP tube;

7) 50 mu L of 65 ℃ endotoxin-free water is taken to be put into a column, kept stand for 1min and centrifuged for 1min at 12000 r/min; the concentration was measured by an ultraviolet spectrophotometer and stored at-20 ℃.

6. The recombinant baculovirus co-expressing porcine interferon L3 and porcine interleukin 22 according to claim 1, wherein,

in the step (2), after pFastBacdual-rPoIFN-L3-rPoIL-22 is transformed into competent DH10Bac, blue-white screening is carried out for 3 times, and the steps are as follows:

1) adding 5 μ L plasmid into DH10Bac competence, mixing, ice-cooling for 30min, heat-shocking for 30s at 42 deg.C, and standing on ice for 3 min;

2) adding 800 μ L of nonreactive LB, and shaking in a shaker at 37 deg.C and 220r/min for 4 h;

3) diluting the conversion product with nonreactive LB according to a ratio of 1:10, 1:100 and 1:1000 respectively, coating 100 mu L of each conversion product on a solid LB plate which is prepared in advance and used for blue-white screening, and keeping out of the sun at 37 ℃ for 24 h;

4) selecting several white colonies from each plate, streaking on a new solid LB plate subjected to blue-white screening, performing 2 nd round blue-white screening, selecting white colonies after 3 rounds of screening, putting the white colonies into liquid LB containing Kan, Gen and Tet, and shaking for 24 hours at the temperature of 37 ℃ and at the speed of 220 r/min.

7. The recombinant baculovirus co-expressing porcine interferon L3 and porcine interleukin 22 according to claim 1, wherein,

in the step (2), the bacmid is extracted, and the steps are as follows:

1) pouring the bacterial liquid into a 2mL EP tube under the aseptic condition, centrifuging at 12000r/min for 1min, and removing the supernatant;

2) 250 mu L S1 of the suspension was added to the pellet and the pellet was resuspended;

3) putting 250 mu L S2 into a tube, turning the tube up and down for 4-6 times, and uniformly mixing for less than 5 min;

4) putting 350 mu L S3 into a tube, turning over the tube for 6-8 times, and centrifuging the tube for 15min at 13000r/min and 4 ℃;

5) sucking the supernatant to another EP tube, adding isopropanol with the same volume, reversing, mixing, standing at 4 deg.C for 30min, and centrifuging at 12000r/min at 4 deg.C for 15 min;

6) discarding the supernatant, adding 500 μ L70% anhydrous ethanol, inverting, washing the precipitate, and centrifuging at 12000r/min for 5 min;

7) discarding the ethanol, drying for 10min, and dissolving with 80 μ L endotoxin-free water at 65 deg.C; the concentration was measured by UV spectrophotometry and stored at-20 ℃.

8. The recombinant baculovirus co-expressing porcine interferon L3 and porcine interleukin 22 according to claim 1, wherein,

in the step (3), insect cells are transfected, and SF21 cells are cultured by Grace's culture solution containing 10% fetal calf serum; preparing bacmid DNA and Cellffectin reagent to be mixed in a sterilizing tube, and operating steps are as follows:

1) 2ug of purified bacmid DNA was diluted into 100. mu.L of unsupplemented Grace medium;

2) completely mixing the Cellffectin reagent, and turning and mixing for 5-10 times; aspirate 8. mu.L of Cellfectin reagent diluted into 100. mu.L of unsupplemented Grace medium;

3) diluted bacmid DNA was ligated using diluted Cellfectin reagent (total volume about 210 ul); lightly mixing, and incubating at room temperature for 30 min;

4) during nurturing of the DNA/Cellfectin mixture, the media was removed from the cells and washed with 2mL of unsupplemented Grace media; discarding the cleaning culture medium;

5) adding 0.8mL of unsupplemented Grace culture medium into each tube containing the mixture, uniformly mixing the clear solution, and adding the mixture into the holes containing the cells;

6) culturing at 27 deg.C for 5 h; discard the DNA/Cellfectin mixture, add 2mL of whole culture medium to the cells;

7) nurturing for 72h under 27 ℃ humid conditions or until the cells are observed to be diseased;

8) virus-containing cells were collected from each well and transferred to a 15mL centrifuge tube.

9. An application of a recombinant baculovirus co-expressing porcine interferon L3 and porcine interleukin 22 is characterized in that the recombinant baculovirus is applied to resisting porcine enteric coronavirus infection.

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