A fusion protein of raccoon dog alpha interferon and beta defensin, its encoding gene and application

Abstract

The present invention relates to the field of genetic engineering technology, specifically disclosing a raccoon dog interferon-α and β-defensin fusion protein, its encoding gene, and its application. The raccoon dog interferon-α and β-defensin fusion protein has the amino acid sequence shown in SEQ ID NO:1. The present invention also provides a gene encoding the fusion protein, a recombinant expression vector, and a host cell. The fusion protein provided by the present invention possesses the dual biological activities of raccoon dog interferon-α and β-defensin, and has certain antiviral and antibacterial activities.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, in particular to a raccoon dog alpha interferon and beta defensin fusion protein, a coding gene thereof and an application thereof. Background Art

[0002] The raccoon dog (Ussuri raccoon dog) originated in Heilongjiang Province in 1957 and has a history of over 60 years. Raccoon dog fur is a key source of fine hair in the fur industry worldwide, renowned for its lightness, softness, aesthetic appeal, and warmth. The popularity of parkas lined with raccoon dog fur has led to a surge in demand for raccoon dog fur in recent years. my country is the world's largest producer, processor, consumer, and exporter of raccoon dog fur products. With the continuous expansion of raccoon dog farming and increasing stocking density, the pathogens are complex and diverse, often leading to mixed infections, resulting in significant economic losses for the raccoon dog industry. Drug control is an effective measure for preventing and controlling infectious diseases in raccoon dogs. However, with the advent of the era of "antibiotic bans, antibiotic reductions, and antibiotic restrictions" in both feed and animal husbandry, green and effective biological veterinary drugs are becoming the optimal choice for the prevention and control of infectious diseases in raccoon dogs.

[0003] Interferon and defensins are both green, efficient, and novel biological veterinary drugs that can replace antibiotics. Interferon has antiviral and immune-regulating properties, while defensins have antibacterial and immune-regulating properties. Their combined use can combat mixed viral and bacterial infections and enhance immunity. Currently, there are no reports of tandem expression of raccoon dog interferon and defensins, either domestically or internationally. Summary of the Invention

[0004] In response to the above problems, the present invention provides a fusion protein of raccoon dog α-interferon and β-defensin, its encoding gene and application. The fusion protein has high antiviral and antibacterial activities.

[0005] To achieve the above-mentioned purpose, the embodiment of the present invention adopts the following technical solutions:

[0006] A fusion protein of raccoon dog interferon-alpha and defensin-beta has an amino acid sequence as shown in SEQ ID NO: 1.

[0007] Compared with the prior art, the fusion protein provided by the present invention is obtained by connecting and optimizing two proteins with different functions by repeating GGGGSGGGGSGGGGS as a connecting sequence. The fusion protein has the dual biological activities of raccoon dog α-interferon and β-defensin, and the antiviral activity and antibacterial activity of the fusion protein are high.

[0008] The amino acid sequence shown in SEQ ID NO: 1 is as follows:

[0009] CDLPDTHGLRNWRVLTLLGQMRRLSAGSCDHYTNDFAFPKELFDGQRLQEAQALSVVHVMTQKVFHLFCTDTSSAPWNMTLLEELCSGLSEQLDDLEACPLQEVGLAETPLTHDD STLRTYFQRISLYLQDKNHSPCAWEMVRAEIGRSFFSSTILQERIRRRKGGGGSGGGGSGGGGSGIINTLQRYYCRIRSGRCALLSCLPKEEQIGRCSSTGRKCCRRKKHHHHHH.

[0010] The present invention also provides the use of the raccoon dog alpha interferon and beta defensin fusion protein as an antiviral drug.

[0011] The present invention also provides the use of the raccoon dog α-interferon and β-defensin fusion protein as an antibacterial drug.

[0012] The present invention also provides a gene encoding the raccoon dog interferon-α and defensin-β fusion protein, and the base sequence of the encoding gene is as shown in SEQ ID NO: 2.

[0013] The base sequence of the gene shown in SEQ ID NO: 2 is as follows:

[0014] .

[0015] The coding gene provided by the present invention can accurately encode the raccoon dog α-interferon and β-defensin fusion protein.

[0016] The present invention also provides a recombinant expression vector, which contains the gene and its expression regulatory element.

[0017] The present invention also provides a method for preparing the recombinant expression vector, comprising the following steps:

[0018] a. Adding restriction enzyme sites and protective bases at both ends of the gene to obtain a modified gene sequence;

[0019] b. Introducing the modified gene sequence into the target plasmid by enzyme digestion and ligation to obtain the recombinant expression vector.

[0020] Preferably, an EcoR I restriction site and a Not I restriction site are added to both ends of the gene, and the modified gene sequence is as shown in SEQ ID NO: 3; and the target plasmid is pPICZαA.

[0021] The modified gene base sequence shown in SEQ ID NO: 3 is as follows:

[0022] .

[0023] After the gene encoding the raccoon dog α-interferon and β-defensin fusion protein in the present invention is connected with the enzyme cleavage site and the protective base, it is connected to pPICZαA, which can achieve accurate and efficient expression in red wall yeast and increase the expression amount of the fusion protein.

[0024] The present invention also provides a recombinant strain comprising the above recombinant expression vector.

[0025] Preferably, the recombinant strain is Pichia pastoris.

[0026] The fusion protein of raccoon dog interferon-α and defensin-β obtained through the Pichia pastoris expression system causes minimal damage to cells.

[0027] More preferably, the Pichia pastoris is X33.

[0028] The selection of X33 in Pichia pastoris is more conducive to forming the correct spatial conformation of the fusion protein of raccoon dog α-interferon and β-defensin, thereby improving its activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is a structural diagram of the recombinant expression vector plasmid in an embodiment of the present invention;

[0030] Figure 2 This is a gel electrophoresis diagram of the linearization of the vector digested by enzyme in the embodiment of the present invention, wherein lane M: DL5000 DNA, lane 1: before digestion; lane 2: after digestion;

[0031] Figure 3 This is the PCR verification of the target gene in the embodiment of the present invention, wherein, lane M: DL5000 DNA, lanes 1-2: different positive transformants; lane 3: positive control;

[0032] Figure 4 This is an SDS-PAGE test chart for detecting the secretory expression of the fusion protein in the embodiment of the present invention, wherein lane M: 180KDa marker, lanes 1-7: different positive transformants; lane 8: negative control;

[0033] Figure 5 This is a Western Blot diagram for detecting the fusion protein in the embodiment of the present invention, wherein lane M: 180KDa marker, lanes 1-7: different positive transformants; lane 8: positive control;

[0034] Figure 6 This is a graph showing the antibacterial test of the fusion protein against Staphylococcus aureus in an embodiment of the present invention, wherein 1: streptomycin; 2: gentamicin; 3: fusion protein; 4: uninduced expression yeast supernatant; 5: PBS;

[0035] Figure 7This is a graph showing the antibacterial test of the fusion protein against Escherichia coli in an embodiment of the present invention, wherein 1: streptomycin; 2: gentamicin; 3: fusion protein; 4: uninduced expression yeast supernatant; 5: PBS. DETAILED DESCRIPTION

[0036] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0037] Example

[0038] Obtaining fusion proteins

[0039] Materials, methods, and results

[0040] 1. Materials

[0041] DMEM medium and fetal bovine serum (FBS) were purchased from Gibco, USA; YPD medium, BMGY medium, and BMMY medium were purchased from Beijing Solebow Technology Co., Ltd.

[0042] X33 yeast strain was purchased from Sangon Biotechnology;

[0043] Vesicular stomatitis virus (VSV) and African green monkey kidney cells (Vero cells) were kindly provided by the Veterinary Biological Products Laboratory, College of Veterinary Medicine, Hebei Agricultural University;

[0044] Staphylococcus aureus (ATCC 6538) and Escherichia coli (ATCC 25922) were purchased from Beijing Baozang Biotechnology Co., Ltd.;

[0045] Anti-His Mouse MAb and Goat Anti-Mouse IgG were purchased from Sangon Biotech (Shanghai) Co., Ltd.; genes and primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0046] 2. Design and synthesis of raccoon dog interferon-α and defensin-β genes

[0047] Referring to the GenBank accession number: EF543192.1, the raccoon dog interferon α (IFNα) gene sequence (accession number: XM-055335531.1) and the raccoon dog defensin β (BDβ) gene sequence, the signal peptide was removed and the gene sequence was optimized. The two genes were connected with a connecting peptide (GGGGSGGGGSGGGGS) to obtain the coding gene (SEQ ID NO: 2). EcoR I and Not I restriction sites and protective bases were added to both ends of the coding gene and sent to Sangon Biotech (Shanghai) Co., Ltd. for synthesis to obtain the modified gene sequence (SEQ ID NO: 3);

[0048] The target gene fragment PCR product was amplified by PCR and recombined into the pPICZaA plasmid by double enzyme digestion to obtain a recombinant expression vector named pPICZα-IFNα-BDβ. The map of the recombinant expression vector is shown in Figure 1 shown.

[0049] 3. Transformation of pPICZα-IFNα-BDβ and Expression and Purification of Fusion Protein

[0050] 3.1. Electroporation and PCR Verification

[0051] The recombinant expression vector pPICZα-IFNα-BDβ was digested with Sac1 enzyme overnight and then recovered. The linearized and non-linearized plasmids were detected by electrophoresis. Figure 2 Prepare X33 yeast competent cells, stimulate the linearized plasmid with 2000V voltage and then electrotransform it into X33 yeast competent cells, spread it on YPD solid plates containing bleomycin antibiotics, and culture it for 2-3 days; select several single colonies and culture them overnight in YPD liquid medium, take the bacterial liquid and use the universal yeast primer AOX-1 for PCR verification, and use the PCR result of the linearized recombinant plasmid as the positive control. The theoretical value is about 1253bp (707bp of target gene + 546bp of vector fragment). The identification result is as shown below. Figure 3 shown.

[0052] 3.2. Secretory expression test

[0053] The strains in the YPD medium in the previous step (including the X33 / pPICZαA empty vector) were inoculated into BMGY medium. The BMGY culture in the previous step was centrifuged, the supernatant was discarded, and the medium was replaced with BMMY medium. 0.5% methanol was added every 12 hours to induce expression, and the bacteria were harvested after 72 hours. The cultured bacterial solution was centrifuged at 12000rpm for 10 minutes, the precipitate was discarded, the supernatant was collected, 100% trichloroacetic acid (TCA) was added to the supernatant, mixed and stored at -20℃ overnight. The next day, the frozen supernatant was taken out and centrifuged at 12000rpm for 20 minutes. The protein precipitate was collected to obtain a protein concentrate, which was tested by SDS-PAGE. Figure 4 The electrophoresis diagram is shown in the figure. Anti-His Mouse MAb was used as the primary antibody, diluted at 1:5000, mixed thoroughly, and then covered on a polyvinylidene fluoride membrane (PVDF membrane) and incubated at room temperature for 1 hour. Goat Anti-Mouse IgG was used as the secondary antibody, diluted at 1:10000, and incubated at room temperature for 1 hour. ECL reagent was used for color development. The obtained Western Blot diagram is shown in the figure. Figure 5 As shown, it is a single band and the protein size is correct, 35KDa. After detection with specific antibodies, it was confirmed that the obtained protein is the target fusion protein.

[0054] 3.3 Expression and purification of fusion proteins

[0055] Positive recombinant yeast cultures were inoculated into YPD liquid medium and incubated at 30°C and 220 rpm for 24 hours to activate the strain. A 1% inoculum was then inoculated into 25 mL of BMGY medium and incubated at 28°C and 220 rpm until the cells reached an OD600 of 4. The culture was then centrifuged at 2500 rpm for 5 minutes, discarded, and resuspended in 100 mL of BMMY medium. The culture was then incubated at 30°C and 220 rpm, supplemented with 1 mL of pure methanol every 24 hours for 4 days. The resulting culture was centrifuged at 12,000 rpm for 10 minutes, the precipitate discarded, and the supernatant collected. 20 mL of 100% TCA was added to the supernatant, mixed, and incubated at -20°C overnight. The next day, the supernatant was frozen and centrifuged at 12,000 rpm for 20 minutes to collect the protein precipitate, resulting in a protein concentrate. The resulting protein concentrate was placed in a dialysis bag and dialyzed against urea-free Binding Buffer at 4°C for 12 hours to remove residual TCA. The protein was purified using a His-tag protein purification kit, and the eluted protein solution was collected and analyzed by SDS-PAGE. The protein concentration was determined using a NanoDrop 2000, with an average protein concentration of 0.15 mg / mL.

[0056] 4. Antiviral activity of fusion protein

[0057] After Vero cells were grown to a monolayer in a 96-well cell culture plate, the fusion protein was diluted 4-fold in eight dilution gradients, with eight replicate wells per gradient. 100 μL of each gradient was added to the 96-well plate and incubated in a cell culture incubator at 37°C and 5% CO2 for 18-24 hours. The culture medium was discarded. The VSV solution was diluted to 100 TCID50 with DMEM containing 2% FBS, and 100 μL was added to each well of the 96-well plate. A positive control (treated with virus alone) and a negative control (treated with 2% FBS in DMEM) were also designed. The results were determined when more than 75% of the cells in the positive control group showed lesions. The protection rate (antiviral activity) of the recombinant protein against cells was calculated according to the Reed-Muench method. A higher protection rate corresponds to a higher antiviral activity.

[0058] The anti-VSV activity of the fusion protein was determined by microcytopathic inhibition assay and calculated according to the Reed-Muench method, as shown in Table 1. The antiviral activity of the recombinant protein was calculated to be 4 4+0.9 U / 0.1mL=7.9×10 5 U / mL, the recombinant protein concentration was 0.15 mg / mL, and the specific activity was 5.27×10 6 U / mg.

[0059] Table 1 Antiviral activity of fusion proteins

[0060]

[0061] 5. Antibacterial activity of fusion protein

[0062] The antibacterial activity of the drug was tested using the agar diffusion method, with Staphylococcus aureus (ATCC 6538) and Escherichia coli (ATCC 25922) as indicator strains. The indicator strains were cultured in LB medium until the logarithmic growth phase. 20 μL of the bacterial solution was mixed with 20 mL of solid LB medium to be solidified and poured into a 90 mm diameter sterile plate. After cooling, the plate was punched with a 6 mm diameter hole punch. 50 μL each of the recombinant protein (concentration of 1.5 mg / mL), ampicillin (100 μg / mL), gentamicin (100 μg / mL), PBS, and uninduced supernatant were added to each well. The plates were incubated upright at 37°C for 2 h, then inverted for 10 h and 37°C for 16 h. The presence of inhibition zones was observed.

[0063] like Figure 6 As shown, the fusion protein has an inhibitory effect on Staphylococcus aureus, with an inhibition zone diameter of 5.8 mm; see Figure 7 As shown, the fusion protein has an inhibitory effect on Escherichia coli, and the diameter of the inhibition zone is 4.3 mm.

[0064] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A fusion protein of raccoon dog interferon-α and defensin-β, characterized in that: The amino acid sequence of the raccoon dog interferon-α and β defensin fusion protein is shown in SEQ ID NO: 1; the base sequence of the gene encoding the raccoon dog interferon-α and β defensin fusion protein is shown in SEQ ID NO:

2.

2. Use of the raccoon dog interferon-α and defensin-β fusion protein according to claim 1 in the preparation of anti-VSV virus drugs.

3. Use of the fusion protein of raccoon dog interferon-alpha and defensin-beta according to claim 1 in the preparation of drugs for inhibiting Staphylococcus aureus and Escherichia coli.

4. A recombinant expression vector, characterized in that: Comprising the coding gene and expression regulatory element thereof as described in claim 1.

5. The method for preparing a recombinant expression vector according to claim 4, wherein: The following steps are involved: a. Adding restriction enzyme sites and protective bases at both ends of the coding gene to obtain a modified gene sequence; b. Introducing the modified gene sequence into the target plasmid by enzyme digestion and ligation to obtain the recombinant expression vector.

6. The method for preparing a recombinant expression vector according to claim 5, wherein: The two ends of the coding gene are respectively added EcoR I restriction enzyme site and Not I restriction enzyme cutting site, the modified gene sequence is as SEQ ID NO: 3; The target plasmid is pPICZαA.

7. A recombinant strain, characterized in that: Comprising the recombinant expression vector according to claim 4.

8. The recombinant strain according to claim 7, wherein: The engineered bacteria of the recombinant strain is Pichia pastoris.

9. The recombinant strain according to claim 8, characterized in that: The Pichia pastoris is X33.

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