CN118667888A - Baculovirus expressed chicken interferon recombinant transfer plasmid and preparation, recombinant bacmid and application thereof - Google Patents

Abstract

The invention relates to the technical field of molecular biology, in particular to a baculovirus expressed chicken interferon recombinant transfer plasmid, and preparation, recombinant bacmid and application thereof. The recombinant transfer plasmid integrates two sections of chicken IFN-alpha genes shown as SEQ ID No.1 and SEQ ID No.2, wherein the SEQ ID No.1 contains a melittin signal peptide gene, a His tag protein gene and a stop codon. According to the invention, the optimized chicken IFN-alpha sequence is obtained by modifying the chicken interferon core region sequence, the high-efficiency expression recombinant is constructed, and the IFN-alpha is successfully and efficiently expressed by selecting the double-promoter recombinant baculovirus, so that the invention is expected to provide a new thought and method for large-scale production of the soluble chicken interferon IFN-alpha.

Description

A baculovirus-expressed chicken interferon recombinant transfer plasmid and its preparation, recombinant bacmid and application

Technical Field

The invention relates to the technical field of molecular biology, and in particular to a baculovirus-expressed chicken interferon recombinant transfer plasmid and its preparation, recombinant bacmid and application.

Background Art

Interferon IFN is one of the important cell regulatory factors in the body. It can improve the body's immune function and induce the body to produce a variety of antiviral proteins, especially playing an important role in the early stages of viral infection. Compared with traditional drugs such as antibiotics, interferon has the advantages of not producing drug resistance, less toxic side effects, quick effect, no residue, and safety, and has broad application prospects.

At present, interferons can be divided into three categories according to their receptor specificity, sequence homology and induced ISG: type I interferons that bind to IFNαR1 and IFNαR2 receptors, type II interferons that bind to IFNγR1 and IFNγR2 receptors, and type III interferons that bind to IL-28Rα and IL-10Rβ heterodimer receptors. Among them, type I and type III interferons have many similarities in function and signal transduction.

As for type I interferon, there are many members of mammalian type I interferon, but only two acid- and heat-stable type I interferons have been found in poultry: IFN-α and IFN-β, which have serological differences. Among them, IFN-α has a relatively low amino acid sequence homology with mammals, about 20%. However, the protein core region structure of chicken IFN-α has 80% similarity with that of mammals, proving that their core functions are similar.

Avian IFN-α plays a very important role in antiviral treatment. Studies have shown that it has a good inhibitory effect on influenza virus (AIV), infectious bursal disease virus (IBDV), infectious bronchitis virus (IBV) and other viruses. The antiviral activity of type I interferon is mainly produced by inducing ISGs by binding to IFNαR1 and IFNαR2 receptors. However, due to the different binding abilities of the two to the receptors, the clinical effect of IFN-α is significantly stronger than that of IFN-β.

Chicken IFN-α can induce the body to produce immune proteins and inhibit the replication and spread of viruses during the immune regulation process. However, the existing traditional chicken interferon production methods have the following disadvantages: poor effect, long production cycle, low yield, low effective purity, unstable activity and other problems, resulting in poor use effect. The present invention transforms and constructs the baculovirus-expressed chicken interferon recombinant transfer plasmid, and successfully obtains soluble chicken interferon IFN-α in vitro using the baculovirus expression system, which can effectively reduce the replication of avian influenza virus after clinical use.

Summary of the invention

The present invention provides a baculovirus-expressed chicken interferon recombinant transfer plasmid and its preparation, recombinant bacmid and application, transforms the chicken interferon core region sequence to obtain an optimized chicken IFN-α sequence, constructs a high-efficiency expression recombinant, selects a double promoter to express the recombinant baculovirus to successfully and efficiently express IFN-α, and is expected to provide a new idea and method for the large-scale production of soluble chicken interferon IFN-α, solving the problems existing in the prior art.

One of the technical solutions adopted by the present invention is:

Provided is a baculovirus-expressed chicken interferon recombinant transfer plasmid, wherein the recombinant transfer plasmid integrates two chicken IFN-α genes as shown in SEQ ID No. 1 and SEQ ID No. 2, wherein SEQ ID No. 1 comprises a bee pollen signal peptide gene, a His tag protein gene and a stop codon.

Furthermore, SEQ ID No.1 and SEQ ID No.2 were obtained by optimizing the original sequence of the target gene, chicken IFN-α. The original sequence of chicken IFN-α is shown in SEQ ID No.3:

SEQ ID No.3:

CTACGCCTGCAACCACCTTCGCCCCAAGGATGCCACCTTCTCTCACGACAGCCTCCAG

CTCCTCCGGGACATGGCTCCCACACTACCCCAGCTGTGCCCACAGCACAACGCGTCTT

GCTCCTTCAACGACACCATCCTGGACACCAGCAACACCCGGCAAGCCGACAAAACCA

CCCACGACATCCTTCAGCACCTCTTCAAAATCCTCAGCAGCCCCAGCACTCCAGCCCA

CTGGAACGGCAGCCAACGCCAAAGCCTCCTCAACCGGATCCACCGCTACACCCAGCA

CCTCGAGCAATGCTTGGACAGCAGCGACACGCGCTCCCGGACGCGGTGGCCTCGCAA

CCTTCACCTCACCATCAAAAAACACTTCAGCTGCCTCCACACCTTCCTCCAAGACAAC

GATTACAGCGCCTGCGCCTGGGAACACGTCCGCCTGCAAGCTCGTGCCTGGTTCCTGCACATCCACAACCTCACAGGCAACACGCGCACT.

Furthermore, SEQ ID No. 1 was obtained by optimizing the above target gene and is:

ATGAAGTTCCTGGTGAACGTGGCCCTGGTGTTCATGGTCGTGTACATCTCCTACATCTACGCT bee pollen signal peptide sequence

TGCAACCACCTGCGTCCCCAGGACGCTACTTTCAGCCACGACTCCCTGCAGCTGCTGC

GCGACATGGCTCCCACTCTGCCCCAGCTGTGCCCTCAGCACAACGCCAGCTGCTCCTT

CAACGACACTATCCTGGACACCTCCAACACTCGTCAGGCCGACAAGACTACTCACGA

CATCCTGCAGCACCTGTTCAAGATCCTGTCCAGCCCCTCCACTCCCGCCCACTGGAAC

GGCTCCCAGCGCCAAAGCCTGCTGAACCGCATCCACCGCTACACCCAGCACCTGGAA

CAGTGCCTGGACTCCTCCGACACTCGCTCCCGCACCCGCTGGCCAAGGAACCTGCAC

CTGACCATCAAGAAGCACTTCTCCTGCCTGCACAACCTTCCTGCAGGACAACGACTACA

GCGCCTGCGCTTGGGAACACGTGCGCCTGCAGGCCCGCGCTTGGTTCCTGCACATCCACAACCTGACCGGCAACACTCGTACC CACCACCACCACCATCAC (6X His tag)TAA

SEQ ID No.2 (the reverse complement of SEQ ID No.1) is:

TTAGTGATGGTGGTGGTGGTGGGTACGAGTGTTGCCGGTCAGGTTGTGGATGTGCAGGAACCAAGCGCGGGCCTGCAGGCGCACGTGTTCCCAAGCGCAGGCTGTAGTCGTTGTCCTGCAGGAAGGTGTGCAGGCAGGAGAAGTGCTTCTTGATGGTCAGGTGCAGGTTCCTTGGCCAGCGGGTGCGGGAGCGAGTGTCGGAGGAGTCCAGGCACTGTTCCAGGTGCTGGGTGTAGCGGTGGA TGCGGTTCAGCAGGCTTTGGCGCTGGGAGCCGTTCCAGTG GGCGGGAGTGGAGGGGCTGGACAGGATCTTGAACAGGTGCTGCAGGATGTCGTGAGTAGTCTTGTCGGCCTGACGAGTGTTGGAGGTGTCCAGGATAGTGTCGTTGAAGGAGCAGCTGGCGTTGTGCTGAGGGCACAGCTGGGGCAGAGTGGGAGCCATGTCGCGCAGCAGCTGCAGGGAGTCGTGGCTGAAAGTAGCGTCCTGGGGACGCAGGTGGTTGCAAGCGTAGATGTAGGAGATGTACA CGACCATGAACACCAGGGCCACGTTCACCAGGAACTTCAT.

Furthermore, the construction of the baculovirus-expressed chicken interferon recombinant transfer plasmid is to simultaneously construct the chicken IFN-α genes shown in SEQ ID No. 1 and SEQ ID No. 2 into the cloning sites of the plasmid pFastBac-Dual vector: between BamHI--HindIII and XhoI--KpnI to obtain a recombinant plasmid.

Furthermore, the recombinant transfer plasmid was named pFastBac Dual-DBL-IFN-α.

Furthermore, the complete sequence of the recombinant transfer plasmid finally constructed is shown in SEQ ID No.4.

SEQ ID No.4:

TTAGTGATGGTGGTGGTGGTGGGTACGAGTGTTGCCGGTCAGGTTGTGGATGTGCAGGAA CCAAGCGCGGGCCTGCAGGCGCACGTGTTCCCAAGCGCAGGCCTGTAGTCGTTGTCCTGCAGGAAGGTGTGCAGG CAGGAGAAGTGCTTCTTGATGGTCAGGTGCAGGTTCCTTGGCCAGCGGGTGCGGGAGCGAGTGTCGGAGGAGTCCA GGCACTGTTCCAGGTGCTGGGTGTAGCGGTGG ATGCGGTTCAGCAGGCTTTGGCTGGGAGCCGTTCCAGTGGGC GGGAGTGGAGGGGCTGGACAGGATCTTGAACAGGTGCTGCAGGATGTCGTGAGTAGTCTTGTCGGCCTGACGAGTG TTGGAGGTGTCCAGGATAGTGTCGTTGAAGGAGCAGCTGGCGTTGTGCTGAGGGCACAGCTGGGGCAGAGTGGGAG CCATGTCGCGCAGCAGCTGCAGGGAGTCGTGGCTGAAAGTAGCGTCCTGGGGACGCAGGTGGTTGCAAGCGTAGAT GTAGGAGATGTACACGACCATGAACACCAGGGCCACGTTCACCAGGAACTTCAT ggatcccgcgcccgatggtgggacggtatgaataatccggaatatttataggtttttttattacaaaactgttacgaaaacagtaaaatacttatttattt gcgagatggttatcattttaattatctccatgatctattaatattccggagtatacggacctttaattcaacccaacaatatattatagttaaataagaattattatcaaatcatttgtatattaattaaaatactatactgtaaattacattttatttacaatcactcgacgaagacttgatcacccgggatctcgag ATGAAGTTCCTGGTGAACGTGGCC CTGGTGTTCATGGTCGTGTACATCTCCTACATCTACGCTTGCAACCACCTGCGTCCCCAGGACGCTACTTTCAGCC ACGACTCCCTGCAGCTGCTGCGCGACATGGCTCCCACTCTGCCCCAGCTGTGCCCTCAGCACAACGCCAGCTGCTC CTTCAACGACACTATCCTGGACACCTCCAACACTCGTCAGGCCGACAAGACTACTCACGACATCCTGCAGCACCTG TTCAAGATCCTGTCCAGCCCTCCACTCCCGCCCACTG GAACGGCTCCCAGCGCCAAAGCCTGCTGAACCGCATCC ACCGCTACACCCAGCACCTGGAACAGTGCCTGGACTCCTCCGACACTCGCTCCCGCACCCGCTGGCCAAGGAACCT GCACCTGACCATCAAGAAGCACTTCTCCTGCCTGCACACCTTCCTGCAGGACAACGACTACAGCGCCTGCGCTTGG GAACACGTGCGCCTGCAGGCCCGCGCTTGGTTCCTGCACATCCACAACCTGACCGGCAACACTCGTACCCACCACCACCACCATCACTAA .

Furthermore, the nucleotide sequence of the bee pollen signal peptide gene is shown in SEQ ID No.5:

ATGAAGTTCCTGGTGAACGTGGCCCTGGTGTTCATGGTCGTGTACATCTCCTACATCTA CGCT; the His tag protein gene sequence is shown in SEQ ID No.6: CACCACCACCACCATCAC.

The second technical solution adopted by the present invention is:

A recombinant bacmid is provided. The baculovirus-expressed chicken interferon recombinant transfer plasmid as described above is transformed into Escherichia coli DH5α competent cells. After the obtained plasmid DNA is correctly identified, it is further transformed into DH10Bac competent cells to obtain the recombinant bacmid rBacmid-double IFN-α.

Furthermore, when transforming DH10Bac competent cells, the ratio was 50 μL competent cells: 1 μL plasmid.

The third technical solution adopted by the present invention is:

Provided is a method for applying the above-mentioned recombinant bacmid, comprising the following steps:

(1) Transfecting sf9 cells with the recombinant bacmid rBacmid-double IFN-α to obtain recombinant baculovirus;

(2) purifying the obtained recombinant baculovirus;

(3) The purified recombinant baculovirus is used to infect a large number of sf9 cells to purify the recombinant protein and obtain recombinant interferon.

Furthermore, the recombinant interferon is used to prepare a preparation for resisting H9N2 AIV virus infection.

Furthermore, the preparation for resisting H9N2 AIV virus infection is a subunit vaccine.

Furthermore, the antiviral activity of the recombinant interferon is 5.0×10 ⁶ U/ml.

Furthermore, the recombinant interferon is used to prepare a product for detecting H9N2 AIV virus infection.

Furthermore, the product for detecting H9N2 AIV virus infection is a fluorescence quantitative kit.

Beneficial effects of the present invention:

1. The present invention utilizes an insect cell-baculovirus expression system to simultaneously connect two segments of chicken IFN-α, and adds a beekeeper signal peptide sequence at the front end of the recombinant sequence to facilitate its secretory expression. Protein expression is regulated by optimizing GC content and codon usage, thereby optimizing expression yield and quality, breaking through the bottleneck of traditional IFN production and use, and obtaining soluble chicken interferon IFN-α with excellent clinical use effect.

2. The two segments of chicken IFN-α sequences optimized by the present invention are doubled expressed using the pFastBac-Dual vector, which has the best match with the vector and the host system. Compared with the recombinant plasmid obtained by using the unoptimized chicken IFN-α original sequence, the protein expression yield can be increased by 15%, and the anti-H9N2 AIV virus activity is also increased by 10%.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a PCR identification result of the recombinant bacmid of the present invention;

FIG2 is a comparison of virus-infected cells rescued by the recombinant baculovirus of the present invention and normal growth cells;

FIG3 is the result of indirect immunofluorescence identification after inoculating the recombinant baculovirus stock solution into adherent sf9 cells;

FIG4 is the result of Western-Blot identification of the expression of recombinant baculovirus IFN.

Among them, in Figure 1, M: Marker DL2000 (2000, 1000, 750, 500, 250, 100 bp from top to bottom); 1-4: recombinant bacmid; 5: empty vector control;

The left picture in Figure 2 shows cells infected with the virus, and the right picture shows normal growing cells;

The left picture in Figure 3 shows that cells inoculated with recombinant baculovirus showed obvious green fluorescence under a fluorescence microscope, and the right picture shows that uninfected empty vector cells showed no fluorescence;

In Figure 4, 1: sample precipitate; 2: sample supernatant; 3: blank control; M: protein marker.

DETAILED DESCRIPTION

In order to clearly illustrate the technical features of this solution, the present invention is described in detail below through specific implementation methods and in combination with the accompanying drawings. In the embodiments of the present invention, unless otherwise specified, all parts and percentages are weight units, and the equipment and raw materials used can be purchased from the market or are commonly used in the art. The experimental methods, detection methods, etc. involved in the following embodiments, unless otherwise specified, are all conventional experimental methods, detection methods, etc. in the prior art.

1. Materials and Methods

1.1 Materials

1.1.1 Cells and vectors

The sf9 cell line was preserved by the Poultry Research Institute of Shandong Academy of Agricultural Sciences; Escherichia coli DH5α competent cells and DH10Bac competent cells were purchased from MyBio Corporation; SPF chickens were from the SPF Chicken Experimental Animal Center of the Poultry Research Institute of Shandong Academy of Agricultural Sciences.

1.1.2 Reagents and kits

Restriction endonucleases, PCR Mix, DL2000/5000 DNA Marker, protein Marker, etc. were purchased from TaKaRa; nucleic acid extraction kit was purchased from Hangzhou Biori Co., Ltd.; transfection reagent cellfectin II was purchased from Invitrogen; culture medium, serum, etc. were purchased from Gibco.

1.1.3 Preparation of main reagents

Ampicillin (100 mg/mL), kanamycin (10 mg/mL), and tetracycline (10 mg/mL): Weigh and dissolve in sufficient sterile water, and finally make up to 10 mL. After sterilization by filtering with a 0.22 μm filter, divide into small portions in light-proof 1.5 mL centrifuge tubes and store at -20°C.

X-gal (20 mg/mL): Add 40 mL of dimethylformamide to a 50 mL centrifuge tube. Use an electronic balance to weigh 1 g of X-gal and mix thoroughly to dissolve it in the centrifuge tube. Make up to 50 mL of the solution. After sterilization by filtering with a 0.22 μm filter, store at -20°C away from light.

IPTG (24 mg/mL): Weigh 1.2 g of IPTG using an electronic balance, place in a 50 mL centrifuge tube, add 40 mL of sterile water and mix thoroughly to dissolve, then make up to 50 mL, filter with a 0.22 μm filter for sterilization, aliquot and store at -20°C.

Triple-antibody LB medium: Sterilize the LB liquid medium by high pressure, add kanamycin (50 μg/mL), tetracycline (10 μg/mL) and gentamicin (7 μg/mL), and store at 4°C.

1.2 Methods

1.2.1 Design and modification of target genes

Chicken IFN-α prosequence:

CTACGCCTGCAACCACCTTCGCCCCAAGGATGCCACCTTCTCTCACGACAGCCTCCAG

CTCCTCCGGGACATGGCTCCCACACTACCCCAGCTGTGCCCACAGCACAACGCGTCTT

GCTCCTTCAACGACACCATCCTGGACACCAGCAACACCCGGCAAGCCGACAAAACCA

CCCACGACATCCTTCAGCACCTCTTCAAAATCCTCAGCAGCCCCAGCACTCCAGCCCA

CTGGAACGGCAGCCAACGCCAAAGCCTCCTCAACCGGATCCACCGCTACACCCAGCA

CCTCGAGCAATGCTTGGACAGCAGCGACACGCGCTCCCGGACGCGGTGGCCTCGCAA

CCTTCACCTCACCATCAAAAAACACTTCAGCTGCCTCCACACCTTCCTCCAAGACAAC

GATTACAGCGCCTGCGCCTGGGAACACGTCCGCCTGCAAGCTCGTGCCTGGTTCCTGCACATCCACAACCTCACAGGCAACACGCGCACT (SEQ ID No. 3).

The sequence was optimized in the insect baculovirus expression system and named pFastBac Dual-DBL-IFN-α, with a C-terminal-His tag, and the optimized sequence was simultaneously constructed into the cloning sites of the pFastBac-Dual vector: between BamHI--HindIII and XhoI--KpnI.

The optimized sequence is shown in SEQ ID No. 1 below:

SEQ ID No.1:

ATGAAGTTCCTGGTGAACGTGGCCCTGGTGTTCATGGTCGTGTACATCTCCTACATCTACGCT bee pollen signal peptide sequence

TGCAACCACCTGCGTCCCCAGGACGCTACTTTCAGCCACGACTCCCTGCAGCTGCTGC

GCGACATGGCTCCCACTCTGCCCCAGCTGTGCCCTCAGCACAACGCCAGCTGCTCCTT

CAACGACACTATCCTGGACACCTCCAACACTCGTCAGGCCGACAAGACTACTCACGA

CATCCTGCAGCACCTGTTCAAGATCCTGTCCAGCCCCTCCACTCCCGCCCACTGGAAC

GGCTCCCAGCGCCAAAGCCTGCTGAACCGCATCCACCGCTACACCCAGCACCTGGAA

CAGTGCCTGGACTCCTCCGACACTCGCTCCCGCACCCGCTGGCCAAGGAACCTGCAC

CTGACCATCAAGAAGCACTTCTCCTGCCTGCACACCTTCCTGCAGGACAACGACTACA

GCGCCTGCGCTTGGGAACACGTGCGCCTGCAGGCCCGCGCTTGGTTCCTGCACATCCACAACCTGACCGGCAACACTCGTACC CACCACCACCACCATCAC (6X His tag)TAA

The above bee pollen signal peptide sequence is: SEQ ID No.5:

ATGAAGTTCCTGGTGAACGTGGCCCTGGTGTTCATGGTCGTGTACATCTCCTACATCTA CGCT; the 6XHis tag sequence is: SEQ ID No. 6: CACCACCACCACCATCAC.

The above SEQ ID No.1 and its reverse complementary sequence SEQ ID No.2 were constructed into two cloning sites of pFastBac-Dual vector: between BamHI--HindIII and XhoI--KpnI. The full sequence of the vector finally constructed is shown in SEQ ID No.4:

TTAGTGATGGTGGTGGTGGTGGGTACGAGTGTTGCCGGTCAGGTTGTGGATGTGCAGGAA CCAAGCGCGGGCCTGCAGGCGCACGTGTTCCCAAGCGCAGGCCTGTAGTCGTTGTCCTGCAGGAAGGTGTGCAGG CAGGAGAAGTGCTTCTTGATGGTCAGGTGCAGGTTCCTTGGCCAGCGGGTGCGGGAGCGAGTGTCGGAGGAGTCCA GGCACTGTTCCAGGTGCTGGGTGTAGCGGTGG ATGCGGTTCAGCAGGCTTTGGCTGGGAGCCGTTCCAGTGGGC GGGAGTGGAGGGGCTGGACAGGATCTTGAACAGGTGCTGCAGGATGTCGTGAGTAGTCTTGTCGGCCTGACGAGTG TTGGAGGTGTCCAGGATAGTGTCGTTGAAGGAGCAGCTGGCGTTGTGCTGAGGGCACAGCTGGGGCAGAGTGGGAG CCATGTCGCGCAGCAGCTGCAGGGAGTCGTGGCTGAAAGTAGCGTCCTGGGGACGCAGGTGGTTGCAAGCGTAGAT GTAGGAGATGTACACGACCATGAACACCAGGGCCACGTTCACCAGGAACTTCAT ggatcccgcgcccgatggtgggacggtatgaataatccggaatatttataggtttttttattacaaaactgttacgaaaacagtaaaatacttatttattt gcgagatggttatcattttaattatctccatgatctattaatattccggagtatacggacctttaattcaacccaacaatatattatagttaaataagaattattatcaaatcatttgtatattaattaaaatactatactgtaaattacattttatttacaatcactcgacgaagacttgatcacccgggatctcgag ATGAAGTTCCTGGTGAACGTGGCC CTGGTGTTCATGGTCGTGTACATCTCCTACATCTACGCTTGCAACCACCTGCGTCCCCAGGACGCTACTTTCAGCC ACGACTCCCTGCAGCTGCTGCGCGACATGGCTCCCACTCTGCCCCAGCTGTGCCCTCAGCACAACGCCAGCTGCTC CTTCAACGACACTATCCTGGACACCTCCAACACTCGTCAGGCCGACAAGACTACTCACGACATCCTGCAGCACCTG TTCAAGATCCTGTCCAGCCCTCCACTCCCGCCCACTG GAACGGCTCCCAGCGCCAAAGCCTGCTGAACCGCATCC ACCGCTACACCCAGCACCTGGAACAGTGCCTGGACTCCTCCGACACTCGCTCCCGCACCCGCTGGCCAAGGAACCT GCACCTGACCATCAAGAAGCACTTCTCCTGCCTGCACACCTTCCTGCAGGACAACGACTACAGCGCCTGCGCTTGG GAACACGTGCGCCTGCAGGCCCGCGCTTGGTTCCTGCACATCCACAACCTGACCGGCAACACTCGTACCCACCACCACCACCATCACTAA .

1.2.2 Construction of recombinant bacmid

Transform the pFastBac Dual-DBL-IFN-α clone constructed above into DH5α competent cells: The specific steps are as follows:

Take the DH5α competent cells out of the -80℃ refrigerator and place them on ice. When the bacteria are in an ice-water mixture, add them quickly, place them on ice for 25 minutes, place them in a 42℃ water bath for 45 seconds, and place them on ice for 2 minutes. Add 700uL of LB medium without antibiotics to the clean bench and mix them well. Resuscitate them at 200rpm at 37℃ for 1 hour. Centrifuge at 5000rpm for 1 minute, discard 600uL of supernatant, blow the bacteria gently and apply them on the ampicillin-resistant LB plate, and incubate them inverted in a 37℃ incubator overnight. Pick a monoclonal colony and inoculate it into 5mL of LB medium containing ampicillin resistance, and shake and culture it at 37℃ for 8-12 hours. Extract the plasmid according to the instructions of the plasmid small-scale extraction kit, use 50uL of elution buffer to obtain plasmid DNA, send part of it for sequencing, and store the rest at -20℃.

The correctly identified recombinant plasmid was transformed into DH10Bac competent cells, with a ratio of 50 μL competent cells: 1 μL plasmid. The steps are the same as those for transforming DH5α. The transformed DH10Bac was added to 500 μL SOC non-antibiotic liquid medium and shaken for 6 hours in the dark. Centrifuge at 5000 rpm for 1 minute, discard 400 μL of supernatant, resuspend the remaining bacteria, dilute with SOC medium in multiple ratios, and spread on several blue-white screening plates, incubate in a 37°C constant temperature incubator for 48 hours, select 3-5 white monoclonal colonies that are the most open and long, and perform another partitioning and streaking separation and purification to obtain pure white colonies. Pick the white plaques and place them in a liquid medium with the same resistance, shake and culture at 37°C for 16 hours, extract the recombinant bacmid in the bacterial solution and name it rBacmid-doubleIFN-α. Extract the recombinant bacmid using the bacmid extraction kit, add 50 μL ddH ₂ O to dissolve, and store at -20°C.

Before use, adjust the concentration of the recombinant bacmid to 10 ³ ng/μL, and use PCR to identify whether the recombinant bacmid is successfully constructed. Detection primers: F-5, AGGATGTCGTGAGTAGTCTT 3,, R-5, GTTCATGGTCGTGTACATCT 3,, fragment length 213bp. Reaction system: 2×Premix Taq buffer (Dye Plus) 25μL, recombinant bacmid 0.5μL, F/R Primer 0.2μM each, and sterile water to 50μL.

The reaction conditions were as follows: 98°C for 3 min, 98°C for 10 sec, 55°C for 30 sec, 72°C for 30 sec, 35 cycles, and 72°C for 2 min. After the reaction, 1% agarose gel electrophoresis was performed and the amplification results were observed under ultraviolet light.

1.2.3 Rescue of recombinant baculovirus

Adherently culture sf9 cells, plate the cells in 6-well plates, ensure that the cells are in good growth state and in the logarithmic growth phase, culture them in 1% double-antibody sf900 II medium at 27°C, and subculture the cells after about 48-72 hours of culture.

Transfection of recombinant bacmid DNA into sf9 cells:

Transfect the Sf9 cells in the logarithmic growth phase with the recombinant bacmid identified correctly in 1.2.2. Plate Sf9 cells in a 6-well plate, 8×10 ⁵ cells/well, 2mL per well, and let it stand for 20 minutes until it adheres to the wall. Transfect with cellfectinⅡ transfection reagent. Steps: Add 100μL Grace medium containing 2μg recombinant bacmid to 100μL Grace medium containing 8μL cellfectinⅡ transfection reagent, mix and incubate. Discard the original SF9 cell culture medium and replace it with serum-free Grace medium containing double antibodies, 2.5mL/well, add the incubated liposome mixture dropwise to the 6-well plate, seal it with a sealing film, and place it in a 27℃ biochemical incubator for transfection for 4h. Replace the culture medium with sf900-Ⅱ serum-free culture medium, observe for 3-5d, and when there is obvious cytopathic effect CPE, collect the culture medium and centrifuge it at 1000rpm for 10min. The supernatant obtained is the P1 recombinant baculovirus stock solution, which is the original virus. According to this method, the recombinant baculovirus was blindly propagated to the P4 generation, and the virus solution was stored at 4°C in the dark, and for long-term storage, it can be placed at -80°C. The P4 generation DNA was extracted according to the method provided by the DNA extraction kit and PCR identification was performed.

1.2.4 Expression and identification of recombinant baculovirus

1.2.4.1 Indirect immunofluorescence identification

After the recombinant baculovirus PCR identification was correct, the P4 generation was inoculated into the adherent sf9 cells and continued to be cultured for 48 hours for indirect immunofluorescence identification: the virus solution was inoculated into a 6-well cell plate, 100 μL per well; after 72 hours, the nutrient solution was discarded and washed 3 times with sterile PBS; 200 μL of pre-cooled 4% paraformaldehyde fixative was added to each well, fixed for 30 minutes, washed 3 times with PBS, and allowed to stand for 2 minutes each time. 200 μL of 0.2% TritonX-100 was added to each well and allowed to stand at room temperature for 15 minutes; 200 μL of the freshly prepared 1% BSA blocking solution was added to each well, blocked at room temperature for 30 minutes, and the blocking solution was discarded and washed 3 times with PBS, each time standing for 2 minutes; monoclonal antibodies diluted 1:200 with PBS, 200 μL per well, incubated at 37°C for 1 hour, washed 3 times with PBS, and allowed to stand for 2 minutes each time; Alexa diluted 1:5 000 was added to each well The cells were incubated with 488-labeled goat anti-mouse IgG secondary antibody at 37°C for 1 h in the dark, washed three times with PBS, and left to stand for 2 min each time; the cells were observed under an inverted fluorescence microscope.

1.2.4.2 SDS-PAGE and Western-Blot Identification

SDS-PAGE: Prepare separation gel and 5% concentrated gel according to the kit instructions. Set normal Sf9 cells as control and add recombinant baculovirus to each well, 20 μL.

Western-Blot: Mix the collected recombinant baculovirus stock solution with 4×SDS loading buffer at a ratio of 4:1, dilute the monoclonal antibody 1000 times with Western primary antibody diluent, and dilute the HRP-labeled goat anti-mouse IgG antibody 5000 times with blocking buffer. Expose using the Chemi Doc MP gel imaging system.

1.2.5 Purification of recombinant baculovirus

Sf9 cells were collected by centrifugation and disrupted by ultrasonication in an ice bath for 3S, 5S intervals, and 10min. The cells were centrifuged at 12000rpm and 4℃ for 20min to collect the supernatant and precipitate. The supernatant was filtered through a 0.22um filter membrane and then purified by Ni column affinity enrichment. The purified product was added to loading buffer and boiled at 100℃ for SDS-PAGE analysis of purity.

1.2.6 Biological activity detection of purified protein

The antiviral activity of recombinant interferon was detected by VSV microlesion inhibition method. Cells were plated in 96-well plates and cultured at 37°C in a 5% CO ₂ incubator for 2 hours. The purified protein was diluted in DMEM medium and incubated for 12-24 hours. The supernatant was discarded, and 100 TCID50 of VSV diluted in 100 μL DMEM medium was added. At the same time, a negative control with only virus added and a blank control without virus were set up. Place in a 37°C, 5% CO ₂ incubator for 48-72 hours, observe the cell lesions, and finally calculate the antiviral activity of IFN according to the Reed-Muench method.

1.2.7 Clinical Application - Effects on Viral Replication

Fifty 10-day-old SPF chickens were randomly divided into 5 groups, 10 in each group. Groups 1, 2, and 3 were the test groups injected with 1000U, 2000U, and 5000U interferon at multiple points in the pectoral muscle, respectively; Group 4 was the challenge group injected with AIV (H9N2) without interferon; and Group 5 was the negative control group injected with sterile saline. Each group was raised separately.

Groups 1-3 were simultaneously nasally and ocularly instilled with 10 ⁴ EID50/0.2ml H9N2 subtype avian influenza virus solution and observed for 15 days under conventional feeding. On the 3rd, 6th, 9th, 12th, and 15th days after the challenge, 5 mice were randomly selected from each group, and laryngeal and cloacal swabs were collected to detect the detoxification status by fluorescence quantitative PCR. The detection kit of GM Biotechnology was used, and the reaction system was 25μL: Mixbuffer 20μL, enzyme Mix 2μL, and template 3μL. The positive control (plasmid) and negative control (ddH ₂ O) were carried out according to the same system. Prepare n portions of reaction solution at low temperature, mix thoroughly, and then divide into reaction tubes. Select the corresponding reaction channel and amplification program: 40℃10min, 95℃95℃3min, followed by 95℃10s, 55℃30s for 40 cycles, and collect fluorescence signals at the same time.

When the positive control Ct value is ≤28 and a typical amplification curve appears, and the negative control has no Ct value and no amplification curve, the test is established.

When the sample shows a typical amplification curve and the Ct value is ≤30.0, it is judged as positive; when the sample has no Ct value or the Ct value is ≥35, it is judged as negative; for samples with a Ct value between 30-35 and a typical amplification curve, if the above results are still obtained after repeated testing, it is judged as positive, otherwise it is judged as negative.

2. Results

2.1 Identification of recombinant bacmid rBacmid-double IFN-α

The recombinant plasmid was transformed into DH10Bac competent cells, and the recombinant bacmid was screened out by triple-antibody blue-white screening plate. The recombinant bacmid rBacmid-double IFN-α was identified by PCR. The results showed that a fragment (213 bp) with the size of the target fragment was successfully amplified. At the same time, sequencing confirmed that the recombinant bacmid was successfully constructed. See Figure 1.

2.2 Rescue of recombinant baculovirus rBacmid-double IFN-α

The recombinant bacmid was transfected into the attached SF9 cells and observed continuously. After about 96 hours, the transfected cells began to dissociate and fall off, and the cell nuclei gradually became filled, etc. See the left picture in Figure 2.

2.3 Identification of recombinant bacmid rBacmid-double IFN-α

The recombinant baculovirus stock solution was inoculated into the adherent sf9 cells, and the culture medium was discarded after continued cultivation at 27°C for 48 hours, and indirect immunofluorescence identification was performed. The results showed that obvious green fluorescence could be observed under a fluorescence microscope after the inoculation of baculovirus, while no fluorescence appeared in the uninfected cells, see Figure 3. This indicates that the recombinant protein can react with serum antibodies in an antigen-antibody specific manner, proving that the recombinant baculovirus was successfully constructed.

The expression of recombinant baculovirus IFN was further identified by Western-Blot, and a band of about 22KD was observed (as shown in Figure 4), which was consistent with the expected result. This result showed that IFN-α was successfully expressed in the baculovirus system, and most of it was present in the supernatant.

2.4 Antiviral activity detection

The antiviral activity of recombinant interferon was detected by VSV microlesion inhibition method. According to the Reed-Muench method, the antiviral activity of IFN was calculated to be 5.0×10 ⁶ U/ml.

2.5 Effect of expressing recombinant interferon on H9N2 AIV virus infection

Groups 1, 2, and 3 were the experimental groups injected with 1000U, 2000U, and 5000U of interferon, respectively. Group 4 was the AIV (H9N2) challenge group. Group 5 was the negative control group injected with sterile saline. Each group was isolated and raised separately. Five mice were randomly selected from each group at 3, 6, 9, 12, and 15 days after the challenge, and larynx and cloaca swabs were collected to detect the detoxification.

After inoculation of H9N2 AIV, the survival of the chickens was recorded every day, and the protection rate of each group of chickens expressed by interferon α was analyzed. The chickens in the negative control group ate normally and no deaths occurred. Group 4 was the challenge group injected with AIV (H9N2) only, with a mortality rate of 30%. No deaths occurred in groups 1 to 3 of the interferon test group.

Table 1 Detoxification at different times after infection

After the virus attack, the changes in virus excretion from laryngeal and cloacal cotton swabs were recorded at different times in each group, as shown in Table 1 above.

At 3dpi-9dpi after infection, AIV was detected in groups 1 to 4; at 12-15dpi, no virus was detected in groups 1-3, but AIV was still detected in chickens in group 4. This shows that different doses of baculovirus-expressed interferon can inhibit the proliferation of AIV, and compared with group 4, groups 1-3 can reduce the excretion rate of chickens. Considering the cost-effectiveness, ¹ ×10 ³ U, 2×10 ³ U, and 5×10 ³ U can be used. However, the attenuation effect of 5×10 ³ U is better. The above results lay the foundation for further research and preparation of poultry interferon preparations.

In summary, the present invention transforms the chicken interferon core region sequence to obtain the optimized chicken IFN-α sequence, constructs a highly efficient expression recombinant, and selects a dual promoter to express the recombinant baculovirus. The genome PCR identification results show that a specific product consistent with the size of the corresponding target fragment was successfully amplified; Western-Blot identification shows a protein band with a molecular weight of about 22kD.

The recombinant baculovirus obtained after the modification of the present invention has high expression amount, good activity, easy purification, high purity, and low protein expression yield compared to the baculovirus obtained by not optimizing the original sequence. The expression amount of the recombinant baculovirus after the modification of the present invention is increased by 15%, and the antiviral activity is also increased by 10%. The clinical use effect is good, the cost is low, and the proliferation of the virus can be effectively reduced. The present invention successfully and efficiently expresses IFN-α using the baculovirus expression system, and is expected to provide new ideas and methods for the large-scale production of soluble chicken interferon IFN-α.

The above specific implementation manner cannot be used as a limitation on the protection scope of the present invention. For those skilled in the art, any substitution, improvement or change made to the implementation manner of the present invention falls within the protection scope of the present invention.

The matters not described in detail in the present invention are all known technologies to those skilled in the art.

Claims (8)

1. A baculovirus-expressed chicken interferon recombinant transfer plasmid, characterized in that the recombinant transfer plasmid integrates two chicken IFN-α genes as shown in SEQ ID No.1 and SEQ ID No.2, and SEQ ID No.1 contains a bee pollen signal peptide gene, a His tag protein gene and a stop codon. 2. The preparation of the baculovirus-expressed chicken interferon recombinant transfer plasmid as claimed in claim 1, characterized in that the chicken IFN-α genes shown in SEQ ID No.1 and SEQ ID No.2 are simultaneously constructed into the cloning sites of the plasmid pFastBac-Dual vector: between BamHI--HindIII and XhoI--KpnI to obtain a recombinant plasmid. 3. The preparation of the baculovirus-expressed chicken interferon recombinant transfer plasmid according to claim 2, characterized in that the full sequence of the recombinant transfer plasmid is shown in SEQ ID No.4. 4. The baculovirus-expressed chicken interferon recombinant transfer plasmid according to claim 1, wherein the nucleotide sequence of the beekeeper signal peptide gene is as shown in SEQ ID No.5, and the His tag protein gene sequence is as shown in SEQ ID No.6. 5. A recombinant bacmid, characterized in that the baculovirus-expressed chicken interferon recombinant transfer plasmid according to claim 1 is transformed into Escherichia coli DH5α competent cells, and after the obtained plasmid DNA is identified as correct, it is further transformed into DH10Bac competent cells to obtain the recombinant bacmid rBacmid-double IFN-α. 6. The use of the recombinant bacmid according to claim 5, characterized in that it comprises the following steps: (1) Transfecting sf9 cells with the recombinant bacmid rBacmid-double IFN-α to obtain recombinant baculovirus; (2) purifying the obtained recombinant baculovirus; (3) The purified recombinant baculovirus is used to infect a large number of sf9 cells to purify the recombinant protein and obtain recombinant interferon. 7. The use according to claim 6, characterized in that the recombinant interferon is used to prepare a preparation for resisting H9N2 AIV virus infection. 8. The use according to claim 6, characterized in that the recombinant interferon is used to prepare a product for detecting H9N2 AIV virus infection.