Raccoon dog alpha interferon and beta defensin fusion protein as well as coding gene and application thereof

Abstract

The invention relates to the technical field of genetic engineering, and particularly discloses a racoon dog alpha interferon and beta defensin fusion protein as well as a coding gene and application thereof. The racoon dog alpha interferon and beta defensin fusion protein has an amino acid sequence as shown in SEQ ID NO: 1. Meanwhile, the invention also provides a coding gene, a recombinant expression vector and a host cell of the fusion protein. The fusion protein provided by the invention has dual biological activities of alpha interferon and beta defensin of the raccoon dog, and has certain antiviral activity and antibacterial activity.

Description

Technical Field

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

[0002] Raccoon dogs were domesticated, raised and bred by the Specialty Products Research Institute of the Chinese Academy of Agricultural Sciences in 1957 from wild Ussuri raccoon dogs produced in Heilongjiang. It has a history of more than 60 years. Raccoon dog skin is an important raw material for fur in the world. It is known for its lightness, softness, beauty and warmth. In particular, the popularity of parkas with raccoon dog skin as the lining has led to a sharp increase in the market demand for raccoon dog skin in recent years. my country is the world's largest raccoon dog breeding country, raccoon dog skin processing country, raccoon dog skin product consumer and exporter. With the continuous expansion of raccoon dog breeding scale and the continuous increase in breeding density, pathogens are complex and diverse, and mixed infections of multiple pathogens often occur, causing huge economic losses to the raccoon dog breeding industry. Drug control is one of the effective measures to prevent and control raccoon dog infectious diseases, but with the advent of the era of "banning, reducing and limiting antibiotics" on the feed and breeding ends, green and efficient biological veterinary drugs will become the best choice for the prevention and control of raccoon dog infectious diseases.

[0003] Interferon and defensin are both green and efficient new biological veterinary drugs that can replace antibiotics. Interferon has antiviral and immune-regulating functions; defensin has antibacterial and immune-regulating functions. The combination of the two can resist mixed viral and bacterial infections and enhance immunity. There are no reports on the tandem expression of raccoon interferon and defensin at home and abroad. Summary of the invention

[0004] In view of the above problems, the present invention provides a fusion protein of raccoon dog interferon alpha and defensin beta, its encoding gene and application, and the fusion protein has high antiviral and antibacterial activity.

[0005] In order to achieve the above-mentioned object of the invention, the embodiment of the present invention adopts the following technical solution:

[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 alpha interferon and beta defensin fusion protein as an antibacterial drug.

[0012] The present invention also provides a gene encoding the fusion protein of the raccoon dog interferon-alpha and defensin-beta, and the base sequence of the gene encoding 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 fusion protein of raccoon dog interferon-α and defensin-β.

[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. Add restriction 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 cutting 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 fusion protein of raccoon dog interferon α and defensin β in the present invention is connected with the restriction 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 by Pichia pastoris is more conducive to the formation of the correct spatial conformation of the fusion protein of α-interferon and β-defensin of Raccoon Dog, thereby improving its activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is a structural diagram of a 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 digestion in the embodiment of the present invention, wherein lane M: DL5000 DNA, lane 1: before digestion; lane 2: after digestion;

[0031] Figure 3 It 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 detection diagram for detecting the secretion 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 It is a Western Blot diagram for detecting 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 activity 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 solution 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 used to limit the present invention.

[0037] Example

[0038] Obtaining fusion protein

[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 donated 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 gene sequence of raccoon dog interferon α (IFNα) (accession number: EF543192.1) and raccoon dog defensin β (BDβ) (accession number: XM-055335531.1) registered in GenBank, the signal peptide was removed and the gene sequence was optimized, and 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 overnight and then recovered. The linearized and non-linearized plasmids were detected by electrophoresis. Figure 2 Prepare X33 yeast competent cells, electrotransform the linearized plasmid into X33 yeast competent cells after 2000V voltage stimulation, spread on YPD solid plates containing bleomycin antibiotics, and culture for 2-3 days; select several single colonies and culture them overnight in YPD liquid culture medium, take the bacterial liquid and use the universal primer AOX-1 of yeast for PCR verification, and use the PCR result of the linearized recombinant plasmid as the positive control. The theoretical value is about 1253bp (target gene 707bp + vector fragment 546bp), and the identification result is as follows Figure 3 shown.

[0052] 3.2. Secretory expression test

[0053] Inoculate the strains in the YPD medium in the previous step (including X33 / pPICZαA empty vector) into BMGY medium. Centrifuge the BMGY culture in the previous step, discard the supernatant, replace it with BMMY medium, add 0.5% methanol every 12 hours to induce expression, and collect the bacteria after 72 hours; centrifuge the cultured bacterial solution at 12000rpm for 10 minutes, discard the precipitate, collect the supernatant, add 100% trichloroacetic acid (TCA) to the supernatant, mix well and store at -20℃ overnight, take out the frozen supernatant the next day, centrifuge at 12000rpm for 20 minutes, collect the protein precipitate, obtain the protein concentrate, perform SDS-PAGE detection, and obtain the following Figure 4 The electrophoresis diagram is shown in the figure. Anti-His Mouse MAb was used as the primary antibody and diluted at 1:5000. After being fully mixed, it was 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 and diluted at 1:10000. It was incubated at room temperature for 1 hour. ECL reagent was used for color development. The obtained Western Blot diagram is shown in the figure below. Figure 5 As shown, it is a single band and the protein size is correct, 35KDa. After detection by specific antibodies, it is confirmed that the obtained protein is the target fusion protein.

[0054] 3.3 Expression and purification of fusion protein

[0055] The positive recombinant yeast liquid was inoculated into YPD liquid medium, and cultured at 30℃ and 220rpm for 24h to activate the strain; then inoculated into 25mL BMGY medium at 1% inoculum, cultured at 28℃ and 220rpm until the bacterial OD600 = 4, centrifuged at 2500rpm for 5min, discarded the culture liquid, resuspended the bacterial cell in 100mL BMMY medium, and then cultured at 30℃ and 220rpm, supplemented with 1mL pure methanol every 24h, and cultured continuously for 4d. The cultured bacterial liquid was centrifuged at 12000rpm for 10min, the precipitate was discarded, the supernatant was collected, 20mL of 100% TCA was added to the supernatant, mixed and stored at -20℃ overnight, the frozen supernatant was taken out the next day, centrifuged at 12000rpm for 20min, the protein precipitate was collected, and the protein concentrate was obtained. The obtained protein concentrate was placed in a dialysis bag, and the dialysis bag was placed in a urea-free Binding Buffer and dialyzed 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 subjected to SDS-PAGE detection. The protein concentration was determined using NanoDrop2000, and the average protein concentration determined by Nano Drop2000 was 0.15 mg / mL.

[0056] 4. Antiviral activity of fusion protein

[0057] After vero cells grow to a monolayer in a 96-well cell culture plate, the fusion protein is diluted 4 times, with a total of 8 dilution gradients, 8 replicate wells for each gradient, 100 μL of each gradient is added to the 96-well plate, incubated in a cell culture incubator at 37°C and 5% CO2 for 18-24 hours, and the culture medium is discarded. The VSV solution is diluted to 100TCID50 with DMEM nutrient solution containing 2% FBS, and 100uL is added to each well of the 96-well plate. At the same time, a positive control (only treated with virus) and a negative control (2% FBS DMEM nutrient solution) are designed. When the observed positive control group cells show more than 75% lesions, the result is determined, and the protection rate (antiviral activity) of the recombinant protein to the cells is calculated according to the Reed-Muench method. The higher the protection rate, the higher the corresponding antiviral activity.

[0058] The anti-VSV activity of the fusion protein was determined by the microcytopathic inhibition method 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 detected by agar diffusion method, with Staphylococcus aureus (ATCC 6538) and Escherichia coli (ATCC 25922) as indicator strains. The indicator strain was cultured in LB medium until the logarithmic growth phase, 20 μL of bacterial solution was mixed with 20 mL of LB solid medium to be solidified, and then poured into a sterilized plate with a diameter of 90 mm. After cooling, a hole was punched on the plate with a diameter of 6 mm. 50 μL of 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, and the plate was cultured upright at 37°C for 2 h, then inverted for 10 h, and cultured at 37°C for 16 h to observe whether there was an inhibition zone.

[0063] like Figure 6 As shown, the fusion protein has an inhibitory effect on Staphylococcus aureus, and the diameter of the inhibition zone is 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 modification, equivalent substitution or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A fusion protein of raccoon dog interferon-α and defensin-β, characterized in that: It has the amino acid sequence shown in SEQ ID NO:

1.

2. Use of the fusion protein of raccoon dog interferon-α and defensin-β as claimed in claim 1 as an antiviral drug.

3. Use of the fusion protein of raccoon dog interferon-α and defensin-β as claimed in claim 1 as an antibacterial drug.

4. A gene encoding the fusion protein of raccoon dog interferon-α and defensin-β according to claim 1, characterized in that: The base sequence of the coding gene is as shown in SEQ ID NO:

2.

5. A recombinant expression vector, characterized in that: Contains the coding gene and its expression regulatory element as described in claim 4.

6. The method for preparing a recombinant expression vector according to claim 5, characterized in that: The following steps are involved: a. Add restriction 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 cutting and ligation to obtain the recombinant expression vector.

7. The method for preparing a recombinant expression vector according to claim 6, characterized in that: EcoR I restriction site and Not I restriction site are added to both ends of the coding gene, and the modified gene sequence is as shown in SEQ ID NO: 3; The target plasmid is pPICZαA.

8. A recombinant strain, characterized in that: Comprising the recombinant expression vector according to claim 5.

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

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

Images