Long-acting forest musk deer interferon fusion protein as well as preparation method and application thereof

Abstract

The invention discloses a long-acting forest musk deer interferon fusion protein as well as a preparation method and application thereof, and relates to the technical field of genetic engineering, and the technical key points are as follows: the amino acid sequence of the fusion protein is as shown in SEQ ID NO.2, and the fusion protein is formed by connecting an IFN alpha protein with an amino acid sequence as shown in SEQ ID NO.3 and an FSA protein with an amino acid sequence as shown in SEQ ID NO.4 through a flexible linker. The invention determines that the fusion protein IFN alpha-FSA has a certain inhibition effect on MDBK cell proliferation, and the inhibition effect is reduced along with the reduction of dosage. The IFN alpha-FSA is safe when being used for mice in vivo, both the IFN alpha-FSA and the IFN alpha have antiviral activity, and the half-life period of the IFN alpha-FSA is longer than that of the IFN alpha.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to a long-acting forest musk interferon fusion protein, its preparation method, and its application. Background Technology

[0002] The forest musk deer (Moschus berezovskii), a Class I protected wild animal in China, produces musk, a substance with high economic and medicinal value. Currently, artificial musk deer farming techniques are advancing resource conservation and sustainable utilization, and the conditions for large-scale and industrialized development are largely in place. Although my country has relatively rich experience in artificial musk deer breeding, many obstacles remain due to environmental factors and technical limitations. Current research reports on forest musk deer diseases include numerous cases of bacterial infections leading to illness, as well as reports of viral infections causing death.

[0003] Interferons are an important family of cytokines with functions including resisting viral infections, inhibiting tumor growth, and regulating the body's immune function. They are frequently used to treat viral or bacterial diseases such as hepatitis, multiple sclerosis, tuberculosis, and brucellosis. The specificity and short half-life of interferons limit their clinical use. While human serum albumin-interferon fusion protein biopharmaceuticals have been used in human clinical practice to prolong the drug's half-life, research in this area in veterinary medicine is still insufficient. Interferons exert their antiviral effects by stimulating the body to produce antiviral-related immune responses upon exposure to external stimuli. Therefore, in-depth research on interferons is beneficial for the prevention and treatment of viral diseases.

[0004] With the increasing effectiveness of interferon in treating viral and neoplastic diseases in livestock and poultry in recent years, veterinary researchers are paying closer attention to the immune properties of interferon in animals and the research on related clinical products. The genes of interferon in more and more animals, such as giant pandas, foxes, minks, and raccoon dogs, are being studied and reported. The application of interferon gene-engineered products in the veterinary field will become increasingly widespread. However, research on musk deer serum albumin is still relatively limited. Research on biopharmaceuticals fused with musk deer interferon α and serum albumin is beneficial for the prevention and treatment of viral diseases associated with musk deer.

[0005] Therefore, this invention integrates musk deer interferon α and musk deer serum albumin, and uses the Pichia pastoris expression system to exogenously express the fusion protein, providing a feasible measure to extend the half-life of musk deer interferon α and providing ideas for the prevention and control of viral diseases of musk deer. Summary of the Invention

[0006] The purpose of this invention is to provide a long-acting lin-musk interferon fusion protein, its preparation method, and its application. By evaluating the anti-cell proliferation ability, antiviral activity, and biosafety of this fusion protein, a reference is provided for the clinical application of lin-musk interferon α.

[0007] To achieve the above objectives, the technical solution of the present invention is as follows:

[0008] This invention provides a long-acting forest musk interferon fusion protein, the amino acid sequence of which is shown in SEQ ID NO.2, and is formed by linking IFNα protein (amino acid sequence shown in SEQ ID NO.3) and FSA protein (amino acid sequence shown in SEQ ID NO.4) via a flexible linker.

[0009] SEQ ID NO.2:MCHLPHTHSLANRRVLMLLRQLRRVSPSSCLQDRNDFAFPQEALGGS QLQKAQAISVLHEVTQHTFQLFSTEGSAAAWDESLLDKLRTALDQQLTDLQACLRQEEGLQGAPLLKEDSSLAVRKYFHRITLYLQEKGLSPCAWEVVRAEVMRAFSSSTNLQERFRRKDGGGGSKWVTFISLLLLFSSAYSRGVFRRDTHKSEIAHRFKDLGEDHFQGLVLIAFSQYLQQCPFDEHVKLVKELTDFAKTCVADESHAGCEKSLHTLFGDELCKVASLRETYGDMADCCEKQEPERNECFLKHKDDSPDLPKLKPDPDTLCAEFKADEKKFWGKYLYEVSRRHPYFYAPELLYYANKYSGVFQECCQAEDKGACLLPKIETMKEKVLASSATQRFRCASIQKFGERALKAWAVARLSQRFPKADFTEITKIVTDVTKVHKECCHGDVLECADDRADLIKYICDNQDAISSKLKECCDKPLLEKSHCIAEIEKDAVPENLPPLTADFAEDKEVCKNYQEAKDAFLGTFLYEYARRHPEYSVSLLLRLAKGYETTLEECCAKDDPPACYATVFDKLKHLVDEPQNLVKQNCELFEKTGEYGFQNVLIVRYTRKVPQVSTPTLVEVSRKLGKVGSKCCKKPESERLSCAEDYLGLILNRLCVLHEKTPVSEKVTKCCTESLVNRRPCFSALTPDETYVPKPFDEKLFTFHADICTLSDTEKQIKKQTALVELLKHKPKATEEQLKAVMGNFVAFVDKCCAADDKEACFAVEGPKLVASTQAALA;

[0010] SEQ ID NO.3: MAPAWSLLLALLLLSCNAICSLGCHLPHTHSLANRRVLMLLRQLRRVS PSSCLQDRNDFAFPQEALGGSQLQKAQAISVLHEVTQHTFQLFSTEGSAAAWDESLLDKLRT ALDQQLTDLQACLRQEEGLQGAPLLKEDSSLAVRKYFHRITLYLQEKGLSPCAWEVVRAEV MRAFSSSTNLQERFRRKD;

[0011] SEQ ID NO.4: MKWVTFISLLLLFSSAYSRGVFRRDTHKSEIAHRFKDLGEDHFQGLV LIAFSQYLQQCPFDEHVKLVKELTDFAKTCVADESHAGCEKSLHTLFGDELCKVASLRETYGDMADCCEKQEPERNECFLKHKDDSPDLPKLKPDPDTLCAEFKADEKKFWGKYLYEVSRRHPYFYAPELLYYANKYSGVFQECCQAEDKGACLLPKIETMKEKVLASSATQRFRCASIQKFGERALKAWAVARLSQRFPKADFTEITKIVTDVTKVHKECCHGDVLECADDRADLIKYICDNQDAISSKLKECCDKPLLEKSHCIAEIEKDAVPENLPPLTADFAEDKEVCKNYQEAKDAFLGTFLYEYARRHPEYSVSLLLRLAKGYETTLEECCAKDDPPACYATVFDKLKHLVDEPQNLVKQNCELFEKTGEYGFQNVLIVRYTRKVPQVSTPTLVEVSRKLGKVGSKCCKKPESERLSCAEDYLGLILNRLCVLHEKTPVSEKVTKCCTESLVNRRPCFSALTPDETYVPKPFDEKLFTFHADICTLSDTEKQIKKQTALVELLKHKPKATEEQLKAVMGNFVAFVDKCCAADDKEACFAVEGPKLVASTQAALA.

[0012] The present invention provides a gene encoding the amino acid sequence of the above fusion protein, and the nucleotide sequence of the gene is as shown in SEQ ID NO.1.

[0013]

[0014] The present invention provides a recombinant vector containing the gene sequence of SEQ ID NO.1 described above.

[0015] The present invention provides an engineered bacterium containing the above-mentioned recombinant vector.

[0016] This invention provides a method for preparing the above-mentioned fusion protein, comprising the following steps:

[0017] S1, Construction of recombinant plasmid pMD19-T-IFNα-FSA;

[0018] S2. Construction of recombinant Pichia pastoris strain with exogenous expression of fusion protein IFNα-FSA;

[0019] S3, induced expression of the fusion protein IFNα-FSA;

[0020] S4. Purification and refolding.

[0021] Preferably, step S1 includes the following steps:

[0022] S11. Design overlapping PCR primers to amplify the FSA gene and IFNα gene;

[0023] The amplification primers for the FSA gene include pf1 with the sequence shown in SEQ ID NO.5 and pf2 with the sequence shown in SEQ ID NO.6; the amplification primers for the IFNα gene include pf3 with the sequence shown in SEQ ID NO.7 and pf4 with the sequence shown in SEQ ID NO.8.

[0024] S12. The FSA gene and IFNα gene obtained in step S11 are amplified using overlap PCR technology to obtain the gene of the fusion protein.

[0025] S13. Ligate the recombinant plasmid with the pMD19-T vector to construct a cloning plasmid. Digest the recombinant plasmid with two enzymes to obtain the target fragment. Ligate the recombinant plasmid with a eukaryotic expression vector to construct the recombinant plasmid pPICZαA-IFNα-FSA.

[0026] Preferably, step S2 includes: digesting the recombinant plasmid pPICZαA-IFNα-FSA with a single enzyme of SacⅠ restriction endonuclease, and then electroporating the linearized plasmid into Pichia pastoris to construct the recombinant strain X33-pPICZαA-IFNα-FSA.

[0027] Preferably, step S3 includes: inducing the expression of the fusion protein IFNα-FSA under the following conditions: induction at a methanol induction concentration of 0.5% for 96 h.

[0028] This invention provides the application of the above-mentioned long-acting musk deer interferon fusion protein in the preparation of antiviral drugs for musk deer.

[0029] This invention provides the application of the above-mentioned long-acting musk deer interferon fusion protein in the preparation of antiviral biological agents for musk deer.

[0030] Compared with existing technologies, the beneficial effects of this solution are:

[0031] 1. This invention successfully cloned the nucleic acid sequence of musk deer serum albumin using homologous sequence cloning, with a full length of 1824 bp encoding 608 amino acids, the first 18 of which are signal peptides. The recombinant plasmid pPICZα-IFNα-FSA was successfully constructed. Secondary and tertiary structure predictions of the fusion gene IFNα-FSA showed that the basic structure of IFNα, the important binding sites IFNAR-1 and IFNAR-2, and the interferon α / β domain were not altered, and the biological activity of IFNα was not affected.

[0032] 2. In this invention, the recombinant plasmid pPICZαA-IFNα-FSA was linearized and then electrotransformed into Pichia pastoris. The fusion protein IFNα-FSA was successfully expressed under methanol induction, and the induction conditions for IFNα-FSA were determined. It was also determined that IFNα-FSA has a certain inhibitory effect on the proliferation of MDBK cells, and the inhibitory effect decreases with decreasing dosage. IFNα-FSA is safe for use in mice in vivo. Both IFNα-FSA and IFNα have antiviral activity, but the half-life of IFNα-FSA is longer than that of IFNα. Attached Figure Description

[0033] Figure 1 The results are the cloning results of the serum albumin gene (A is the amplification of FSA; M: DL2000 Marker, 1: negative, 2: target gene; B is the identification of pMD19 T-FSA, M: Marker IV; 2-5: amplification results of M13 primer, 7-10: amplification results of FSA-specific primer, 1: negative control of M13 primer, 6: negative control of FSA primer).

[0034] Figure 2 It is the amplification of IFNα and FSA target fragments (M: DL2000 Marker, 1: pf3 / pf4 negative, 2: pf3 / pf4 PCR product, 3: pf1 / pf2 negative, 4: pf1 / pf2 PCR product);

[0035] Figure 3The identification of recombinant plasmid pMD19T-IFNα-FSA (M: Marker IV, 1-4: Amplification results of M13 primers, 6-9: Amplification results of pf1 / pf4 primers, 5: Negative control of M13 primers, 10: Negative control of pf1 / pf4 primers);

[0036] Figure 4 The recombinant plasmid pPICZαA-IFNα-FSA was identified (M: Marker Ⅳ, 1-4: pf1 / pf4 amplification results, 7-10: AOX1 amplification results, 5: pf1 / pf4 primer negative control, 6: AOX1 primer negative control);

[0037] Figure 5 The expression vector was verified by enzyme digestion (M: Marker Ⅳ, 1: plasmid pPICZαA-IFNα-FSA, 2: plasmid digested with EcoR I, 3: plasmid digested with NotI, 4: plasmid digested with both enzymes).

[0038] Figure 6 This is a sequence alignment of serum albumin (blue background indicates conserved amino acid regions, and red triangles indicate the amino acid sequence of protein FSA);

[0039] Figure 7 It is a phylogenetic tree constructed based on the protein amino acid sequence;

[0040] Figure 8 This is the signal peptide prediction result from FSA;

[0041] Figure 9 This is a prediction of the secondary structure of the mature FMD-FSA protein;

[0042] Figure 10 These are the tertiary structures of proteins FMD-FSA and IFNα-FSA (A is the tertiary structure of FMD-FSA; B is the tertiary structure of IFNα-FSA);

[0043] Figure 11 These are predictions of the domains of mature proteins (A represents the conserved domain predictions of FMD-FSA; B represents the conserved domain predictions of IFNα-FSA).

[0044] Figure 12 It is a linearized plasmid pPICZαA-IFNα-FSA (M: Marker Ⅳ, 1: circular plasmid, 2: linearized plasmid digested with Sac I).

[0045] Figure 13 It is a positive transformant identification (M: Marker IV, 2-4: AOX1 amplification results, 6-8: pf1 / pf4 amplification results, 1: AOX1 negative, 5: pf1 / pf4 negative);

[0046] Figure 14 These are the results of IFNα-FSA induction (A is SDS-PAGE electrophoresis; B is Western blotting results; M: protein molecular weight standard (15-120kDa); 1: IFNα-FSA).

[0047] Figure 15 This is the result of methanol concentration optimization (M: protein molecular weight standard, 1-5: methanol concentration 0.5%-2.5%);

[0048] Figure 16 This is the result of induction time optimization (M: protein molecular weight standard, 1-6: induction time 0-120h);

[0049] Figure 17 This refers to changes in mouse body weight;

[0050] Figure 18 The effect of IFNα-FSA on cell proliferation (* indicates significant difference (P<0.05));

[0051] Figure 19 These are the results of IFNα-FSA half-life assays (A represents plasma residual activity assay; B represents in vivo half-life assay). Detailed Implementation

[0052] To enable those skilled in the art to better understand the present invention, the technical solution of the present invention will be described in further detail below with reference to the embodiments and accompanying drawings. Obviously, the described embodiments are merely some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort should fall within the scope of protection of the present invention.

[0053] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0054] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0055] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other. The present invention will now be described in detail with reference to the embodiments.

[0056] Example 1: Amplification of the serum albumin gene of the musk deer

[0057] Primers FSA were designed based on the nucleotide sequences of serum albumin from closely related species such as bovine and ovine, and according to the homology and similarity comparison of conserved serum albumin sequences. These primers were synthesized by Hangzhou Youkang Biotechnology Co., Ltd. Universal primers M13-F / R for the pMD19-T vector and AOX1-F / R for pPICZαA were also synthesized. Primer information is shown in Table 1.

[0058] Table 1 Primer Information

[0059]

[0060] Total RNA was extracted from the liver tissue of the musk deer (collected from the Sichuan Musk Deer Research Institute) according to the instructions of the RNA extraction kit. The extracted total RNA from the musk deer lymphocytes was reverse transcribed into cDNA using a reverse transcription premix kit. The reverse transcription system was: 2 μL of 5×Evo M-MLV RT Master, 8 μL of RNA, and a total volume of 10 μL. The reverse transcription reaction conditions were: 37℃ for 15 min, 85℃ for 2 min. Using the cDNA obtained from reverse transcription as a template, and the FSA primer pair as upstream and downstream primers, PCR amplification was performed. The PCR reaction program was set as follows: pre-denaturation—95℃ for 5 min; denaturation—95℃ for 30 s, annealing—58℃ for 30 s, extension—72℃ for 1 min, cycle number 30; total extension—72℃ for 10 min. The PCR reaction system (total 50 μL) included: 2 μL each of FSA-F and FSA-R, 1 μL of template, 25 μL of 2×Pfu PCRMix, and 20 μL of ddH2O.

[0061] Add 5 μL of 2×Taq PCR MasterMix to the PCR product, incubate at 72℃ for 10 min, add polyA, and analyze by 1% agarose gel electrophoresis to obtain the target fragment of approximately 1824 bp (see [link to product]). Figure 1 The target fragment was purified by gel extraction and ligated into the pMD19-T vector. The ligation system (total 10 μL) included: 1 μL pMD19-T, 1 μL T4 DNALigase, 1 μL 10X T4 DNALigase Buffer, 4 μL purified fragment, and 3 μL ddH2O.

[0062] After thorough mixing, centrifuge and ligate overnight at 16°C. DH5α competent cells were prepared using the calcium chloride method. Under aseptic conditions, 100 μL of spare DH5α competent cells stored at -70°C were thawed on ice and gently mixed. 10 μL of ligation product was added, and the mixture was shaken well and incubated on ice for 30 min; then incubated in a 42°C water bath with a heat shock for 90 s; incubated on ice for 3-5 min; 1 mL of antibiotic-free LB liquid medium was added; and the mixture was incubated in a 37°C water bath with gentle shaking at 150 rpm for 1 h; the bacterial culture was centrifuged at room temperature for 3 min at 8000 rpm, retaining only the bacterial pellet. The pellet was resuspended in 100 μL of antibiotic-free LB liquid medium and plated onto LB agar plates (Amp+), and incubated at 37°C for 12 h.

[0063] Positive transformants from ampicillin-resistant plates were inoculated into LB ampicillin-resistant culture medium and cultured in a water bath for 12 h. Colony PCR was performed using specific and universal primers for identification. PCR conditions were as follows: 95℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 58℃ annealing for 30 s (annealing temperature of pMD19-T vector universal primer M13 was set to 55℃), 72℃ extension for 30 s, for a total of 30 cycles; 72℃ extension for 5 min; storage at 4℃. The PCR reaction system (total 20 μL) included: 1 μL each of FSA-F / M13F and FSA-R / M13R, 1 μL template, 1 μL 2×Taq PCR MasterMix, and 1 μL ddH2O. Positive transformants were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing identification. The PCR results were as expected (see [link to PCR results]). Figure 1 ).

[0064] Positive transformants with correct sequencing results were aliquoted into sterile EP tubes at a ratio of 7:3 (bacterial culture: glycerol) and stored at -70°C. Recombinant plasmids were extracted using a plasmid miniprep kit, and their concentration was determined. Plasmid pMD19-T-FSA was stored at -20°C.

[0065] Example 2: Construction of the pPICZαA-IFNα-FSA recombinant plasmid

[0066] 1. Design of overlapping PCR primers

[0067] The mature protein genes of IFN-α and FSA were amplified using primer pairs pf1 / pf2 and pf3 / pf4, respectively. During the PCR process, upstream and downstream restriction sites EcoRI, Not I, and the flexible linker GGGGS were introduced. The two gene segments were fused using overlap PCR technology. The primer sequences are shown in Table 2.

[0068] Table 2 Primers used for overlap extension PCR amplification

[0069] Note: Underlined base sequences represent the cleavage sites and protective bases of restriction endonucleases EcoRI and NotI, respectively; bolded sequences represent linker sequences.

[0070] 2. Amplification of FSA and IFNα genes

[0071] PCR amplification program for the IFNα mature protein gene and the complete FSA gene: pre-denaturation 95℃ for 5 min; denaturation 95℃ for 30 s, annealing 65℃ for 30 s, extension 72℃ for 20 s. The reaction mixture (total 50 μL) included: 25 μL 2×Pfu PCR Mix, 2 μL each of forward and reverse primers, 1 μL plasmid, and 20 μL ddH2O. The primers for amplifying the IFNα mature protein gene were pf1 and pf2, and the template was plasmid pMD19-T-IFNα. The primers for amplifying the complete FSA gene were pf3 and pf4, and the template was plasmid pMD19-T-FSA (see [link to relevant documentation]). Figure 2 ).

[0072] 3. PCR amplification of fusion genes

[0073] The purified products of the target fragments from the two reaction systems were mixed in equimolar amounts and then subjected to overlap PCR under the following conditions: 94℃ for 3 min (pre-denaturation); 94℃ for 30 s (denaturation), 60℃ for 30 s (annealing), and 72℃ for 2.5 min (extension), for 10 cycles. After removal, primers were added to amplify the fusion protein gene, and the reaction conditions were the same as above, for 20 cycles, with a total extension program of 10 min at 72℃; stored at 4℃. The overlap PCR reaction system (50 μL) included: 25 μL of 2×Pfu PCR Mix, 1 μL each of pf1 and pf4, 1 μL of fusion product, and 22 μL of ddH2O. 5 μL of 2×Taq PCR MasterMix was added to the PCR product, and the reaction was carried out at 72℃ for 10 min. After adding the A tail, the target fragment of approximately 2408 bp was purified and recovered by 1% agarose gel electrophoresis (see...). Figure 3 The reaction solution is used directly for the connection reaction.

[0074] 4. Construction of recombinant plasmid pMD19-T-IFNα-FSA

[0075] (1) Preparation of the target fragment: The pMD19-T-IFNα-FSA recombinant plasmid and the pPICZαA vector plasmid were digested with EcoRI and NotI. The digestion system (20 μL) included 2 μL of 10×Buffer, 1 μL each of EcoRI and NotI, 10 μL of vector / plasmid, and 6 μL of ddH2O. After electrophoresis analysis, the fragments of the vector and the target gene were purified by gel purification and fused with T4 DNA ligase. The ligation system (10 μL) included 1 μL of 10×Buffer, 1 μL of T4 DNA Ligase, 5 μL of FSA-IFNα target fragment, and 3 μL of pPICZαA vector.

[0076] (2) Transformation, screening and identification of positive strains: The product of the above ligation was transformed into DH5α competent cells. The bacterial pellet was resuspended in 100 μL of LB medium and plated onto LB bleomycin-resistant agar plates and incubated at 37°C for 12 h. The positive transformants were inoculated into LB bleomycin-resistant medium and incubated in a water bath for 12 h. The bacterial culture was identified by PCR using specific primers and universal primers (see [link to relevant documentation]). Figure 4 PCR reaction conditions: 95℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 58℃ annealing for 30 s (annealing temperature of pPICZαA vector universal primer AOX1-F / R primer set to 53℃), 72℃ extension for 30 s, for a total of 30 cycles; 72℃ extension for 5 min; storage at 4℃. The PCR reaction system (20 μL) included: 1 μL each of pf1 / AOX1-F and pf2 / AOX1-R, 1 μL template, 10 μL 2×TaqPCRMasterMix, and 7 μL ddH2O.

[0077] The recombinant plasmid pPICZαA-IFNα-FSA was extracted and verified by enzyme digestion with EcoRI and NotI. Positive transformants identified by PCR and enzyme digestion were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The results showed that after single enzyme digestion, a clear band was observed at 5833 bp, consistent with the band size of the linearized recombinant plasmid. After double enzyme digestion, bands were observed at approximately 2297 bp, 3536 bp, and 5833 bp, representing the digested target fragment, the vector pPICZαA, and the linearized recombinant plasmid, respectively. The sequencing results of the positive transformants were correct, and the expression vector was successfully constructed (see...). Figure 5 ).

[0078] Positive transformants with correct sequencing results were aliquoted into sterile EP tubes at a ratio of 7:3 (bacterial culture: glycerol) and stored at -70°C for later use.

[0079] Example 3: Bioinformatics Analysis of IFNα-FSA Fusion Gene

[0080] The sequence of serum albumin FSA from the forest musk deer has been uploaded to NCBI, accession number PV278754.

[0081] Homology analysis of the amino acid sequences of serum albumin from musk deer, cattle, sheep, and goats was performed using DNAMAN software (see [link to DNAMAN software]). Figure 6 Phylogenetic trees of these serum albumin amino acid sequences were constructed using MEGA 7.0 software with the nearest neighbor method and 1000 replicates. The phylogenetic results showed that the musk deer is relatively distantly related to human and equine serum albumin, but its amino acid sequences are distributed in the same major branch with those of cattle, sika deer, goats, and sheep (100% confidence level). Within this branch, cattle and sheep form separate branches. Row comparisons of these sequences showed that although the serum albumin from these animals has different origins, its amino acid sequences are highly conserved (see [link to study]). Figure 7 ).

[0082] The signal peptide of FMD-FSA was predicted using SignalP 5.0Server software, and the results are as follows: Figure 8 The results showed that the first 18 amino acids of the sequence constituted the signal peptide, and the following 589 amino acids constituted the mature protein. Analysis of the physicochemical properties of proteins FSA and IFNα-FSA revealed that, after removing the signal peptide, the mature FSA protein had a molecular weight of 66.85 kDa and an isoelectric point (pI) of 5.98, while the mature IFNα-FSA protein had a molecular weight of 88.17 kDa and a pI of 6.38. Secondary predictions for the mature FSA protein are shown below. Figure 9 It can be seen that the secondary structure of the FSA protein is mainly composed of 23 major α-helices and random coils.

[0083] Tertiary structures of FMD-FSA and IFNα-FSA were predicted using SWISS-MODEL. The IFNα and FSA proteins are linked by a flexible linker, allowing them to maintain their conformation while possessing a high degree of freedom, which is beneficial for each protein to perform its respective function (see...). Figure 10 ).

[0084] Conserved domains and functional sites of mature FMD-FSA and IFNα-FSA proteins were analyzed and predicted using NCBI CD-search online software. The results showed that FMD-FSA possesses multiple domains with multiple albumin-binding sites. IFNα-FSA, on the other hand, has two additional IFNAR-1 binding sites, two IFNAR-2 binding sites, and one N-glycosylation site. These sites are functional sites on conserved domains of IFNα itself, preserving the important functions of both IFNα and FSA proteins (see [link to relevant documentation]). Figure 11 ).

[0085] Example 4: Secretory expression of the fusion protein IFNα-FSA in Pichia pastoris

[0086] 1. Construction of recombinant Pichia pastoris expressing exogenous fusion protein IFNα-FSA

[0087] (1) Preparation of Pichia pastoris competent cells: Pichia pastoris X33 strain was streaked onto YPD plates. After colonies grew, a single colony was selected and inoculated into 25 mL of YPD medium. The culture was incubated at 30℃ and 220 r / min for 20 h. After microscopic examination showed no contamination, 0.2 mL of the bacterial culture was inoculated into 50 mL of fresh YPD medium and incubated at 30℃ and 220 r / min until the OD600 reached 1.3-1.5. The culture was then centrifuged at 1500×g and 4℃ for 5 min and then chilled in 500 mL of ice water. Resuspend the bacterial precipitate in cold sterile water; centrifuge the resuspended solution at 1500×g, 4℃ for 5 min, and resuspend the bacterial precipitate in 250 mL of ice-cold 1 mol / L sorbitol solution; centrifuge again at 1500×g, 4℃ for 5 min, and resuspend the bacterial precipitate in 20 mL of ice-cold 1 mol / L sorbitol solution; centrifuge again at 1500×g, 4℃ for 5 min, and resuspend the bacterial precipitate in 1.0 mL of ice-cold 1 mol / L sorbitol solution. The final bacterial volume is approximately 1.5 mL. Aliquot into 80 μL tubes and store at -80℃.

[0088] (2) Transformation and screening of Pichia pastoris

[0089] 1) Take the prepared Pichia pastoris X33 competent cells, thaw them on ice, and add 2-5 μL of recombinant plasmid pPICZαA-IFNα-FSA (approximately 5-10 μg) linearized with Sac I to each tube. The linearization enzyme digestion system (20 μL) includes 2 μL of 10×Buffer, 2 μL of Sac I, 10 μL of plasmid, and 6 μL of ddH2O. After mixing, transfer the mixture to a pre-chilled electroporation cuvette and incubate on ice for 5 min. Perform electroporation according to the transformation conditions: 400 Ω, 25 μF, 1500 V. After transformation, aspirate the bacterial culture from the electroporation cuvette and mix it with 1 mL of pre-chilled 1 mol / L sorbitol solution. Incubate at 30℃ for 2 h, then centrifuge briefly, discard the supernatant, mix the lower bacterial layer, and spread it onto YDP antibiotic plates (Zeocin content 100 μg / mL). Incubate at 30℃ for 2-3 days. Agarose gel electrophoresis analysis showed that the total band size of the recombinant plasmid pPICZαA-IFNα-FSA was 5833 bp, which was in line with expectations (see [link]). Figure 12 ).

[0090] 2) PCR identification of recombinant Pichia pastoris: Yeast transformant plasmids were extracted according to the yeast plasmid miniprep kit instructions. Using the extracted plasmids as templates, PCR identification was performed using pf1 and pf4 specific primers and AOX1-F / R primers, respectively. The PCR product band sizes were all as expected (see...). Figure 13 PCR-positive recombinants are used to induce expression.

[0091] 2. Induction and Condition Optimization of the Fusion Protein IFNα-FSA

[0092] (1) Inducible expression of recombinant pPICZαA-IFNα-FSA

[0093] The selected positive recombinants were inoculated into 500 mL Erlenmeyer flasks containing 100 mL of BMGY medium and cultured at 30 °C and 250 rpm until the OD600 reached 2.0. The precipitate was collected by centrifugation and resuspended in 100 mL of BMGY medium. The precipitate was cultured at 30 °C and 250 rpm for 72 h. Methanol was added every 24 h to a final concentration of 1%. After induction, the supernatant was collected by centrifugation and then concentrated by ultrafiltration to collect the protein.

[0094] (2) SDS-PAGE / Western blot analysis of the target protein

[0095] 1) Mix the concentrated supernatant with 5×SDS loading buffer and prepare the sample. After boiling in a water bath for 10 min, perform SDS-PAGE. Prepare the gels according to the SDS-PAGE gel preparation kit instructions: a 5% stacking gel and a 10% separating gel. The electrophoresis procedure is as follows: electrophoresis at 80V until the indicator reaches the separating gel, then switch to 120V until the indicator is close to the bottom of the separating gel. After electrophoresis, retain only the separating gel and immerse it in Coomassie Brilliant Blue staining solution for 1 h. After the procedure, destain the separating gel in a boiling water bath, changing the water several times during the process, until the background of the separating gel is clear, then photograph and record the results.

[0096] 2) After SDS-PAGE of the protein, remove the stacking gel, cut out the target protein band, and cut the PVDF membrane to the same size as the PAGE gel. Transfer conditions: voltage 150V, current 300mA, transfer time 60min. After transfer, wash the PVDF membrane with TBST buffer, 5min / wash, 3 times. Block in protein-free blocking buffer for 10min, then wash again using the same method as the previous step. Add the primary antibody, mouse anti-6×His Tag monoclonal antibody (1:4000 dilution), and incubate overnight at 4℃. After washing with the primary antibody, use the secondary antibody HRP-labeled goat anti-mouse IgG (1:5000 dilution) and incubate at 37℃ for 1-2h. After washing, develop the membrane using ultrasensitive ECL chemiluminescence solution according to the manufacturer's instructions.

[0097] SDS-PAGE results showed that the pPICZαA-IFNα-FSA recombinant yeast expressed a protein of approximately 80 kDa after induction, and Western blotting also showed a specific 80 kDa band, consistent with experimental expectations. Figure 14 ).

[0098] (3) Optimization of induction expression conditions

[0099] 1) Methanol concentration optimization: Expression was induced by methanol concentrations of 0.5%, 1.0%, 1.5%, 2.0% and 2.5%, respectively. After 72 h of induction, the supernatant of the fermentation broth was collected for SDS-PAGE detection.

[0100] The results showed that the induced expression of the target protein was most effective when the methanol concentration was 0.5% (see [link to study]). Figure 15 ).

[0101] 2) Expression time optimization: After methanol concentration optimization, expression was induced for 120 h at the optimal methanol induction concentration. Starting from 0 h, the supernatant of fermentation broth was taken every 24 h for SDS-PAGE detection.

[0102] The results showed that the protein concentration reached its maximum after 96 hours of expression (see...). Figure 16 ).

[0103] Therefore, the optimal protein was obtained by induction at a methanol concentration of 0.5% for 96 hours. Finally, the fusion protein IFNα-FSA was purified using the Ni-NTA protein purification kit, concentrated and desalted by ultrafiltration centrifuge tubes, mixed with glycerol at a 3:1 ratio, and stored at -20℃ for later use.

[0104] Example 5: Activity analysis of the fusion protein IFNα-FSA

[0105] 1. Test materials

[0106] 1.1 Bacterial strains and plasmids

[0107] Recombinant yeast X33-pPICZαA–IFNα and pseudorabies virus were preserved in the Animal Quarantine Laboratory of Sichuan Agricultural University. Bovine kidney cells MDBK(NBL-1) were purchased from Wuhan Shangen Biotechnology Co., Ltd.

[0108] 1.2 Experimental Animals

[0109] SPF-grade Kunming mice were purchased from Chengdu Dashuo Experimental Animal Co., Ltd.

[0110] 1.3 Experimental Materials and Reagents

[0111] Wokawi (Beijing) Biotechnology Co., Ltd.: DMEM culture medium; Beyotime Biotechnology Co., Ltd.: trypsin cell digestion solution; Wuhan Boster Biological Engineering Co., Ltd.: Cell Counting Kit–8; Shenzhen Shangwei Biotechnology Co., Ltd.: fetal bovine serum (New Zealand).

[0112] 2. Test methods

[0113] 1.1 Safety test in mice

[0114] Mice were pre-fed for one week and then randomly divided into three groups of 10 mice each. Each group received an intraperitoneal injection of an equal volume of the IFNα fusion protein IFNα-FSA. A negative control group was also included, receiving an equal volume of PBS. The observation and detection indicators are as follows:

[0115] (1) Observation of general condition and toxic reaction: After administration, observe the mental state, respiration, skin, feces and general activity of mice within 14 days, and record the presence of poisoning symptoms and death in a timely manner;

[0116] (2) Body weight determination: The body weight of mice was measured and recorded 1 day after administration;

[0117] (3) Organ index determination: On day 14 after drug administration, mice were euthanized by cervical dislocation and dissected. The heart, lungs, liver, and kidneys of each group were removed. The color and shape of each organ were visually inspected for any abnormalities and the results were recorded. After observation, the organs were weighed and the organ index of each organ was calculated.

[0118] Organ Index (%) = Organ wet weight (g) / Body weight (g) × 100%

[0119] 1.2 Cytotoxicity assay

[0120] MDBK cells (NBL-1, Madin-Darby Bovine Kidney cells) in the logarithmic growth phase were collected, digested with trypsin, and then subjected to a 1×10⁻⁶ solution. 5 Cells were seeded at a density of 100 μL / mL in 96-well plates and incubated at 37°C with 5% CO2 for 24 h. FMD-IFNα and IFNα-FSA were added to final concentrations of 400 ng / mL, 200 ng / mL, 100 ng / mL, and 50 ng / mL, respectively. Wells without added protein served as controls. Each group was set up in 5 replicates. After incubation for 24 h, 10 μL of CCK-8 was added to each well and incubated for 1.5 h. The absorbance at 450 nm was measured in each well using a microplate reader, and the cell growth inhibition rate was calculated.

[0121] Cell growth inhibition rate = (1 - OD value of experimental group / OD value of control group) × 100%.

[0122] 1.3 Antiviral activity assay

[0123] (1)TCID 50 Measurement

[0124] PRV (Porcine Pseudorabies Virus) was diluted 10-fold with serum-free DMEM medium. A dilution of 10 was selected. -3 Up to 10 -7 Viral solutions were used for experiments, with 10 wells per gradient. MDBK cells in logarithmic growth phase were trypsinized with approximately 1 × 10⁻⁶ cells / well. 5 Cells were seeded at a density of 100 μL / mL in 96-well plates and incubated at 37°C with 5% CO2 until a monolayer was formed. The culture medium was then aspirated, and diluted virus solution was added. The plates were then incubated at 37°C with 5% CO2. The cells in the diseased wells were observed and recorded daily. TCID was calculated using the Reed-Muench method. 50 .

[0125] Distance ratio = (Percentage above 50% - 50%) / (Percentage above 50% - Percentage below 50%)

[0126] TCID 50 = Negative logarithm of viral dilution above 50% + distance ratio

[0127] (2) Antiviral activity assay

[0128] The expression method of *Corydalis musk deer* IFNα in *Pichia pastoris* was the same as that of the *Corydalis musk deer* IFNα-FSA fusion protein in *Pichia pastoris*. FMD-FSA and IFNα-FSA were serially diluted 4-fold with 10% FBSDMEM, resulting in 6 serial dilutions, which served as the test samples.

[0129] MDBK cells were cultured routinely at 37°C and 5% CO2. After digestion, they were inoculated at 1×10⁻⁶ cells / cells. 5 Seedlings were placed at a density of 100 μL / mL in 96-well cells culture plates. Cells were incubated at 37°C and 5% CO2 until well adherent and well-grown. The culture medium was aspirated, and the sample solution was transferred to each well of a 96-well plate containing MDBK cells (100 μL). Control wells (no sample, no virus) and control wells (no sample, no virus) were also included. Cells were incubated at 37°C and 5% CO2 for 18–24 h, and the supernatant was discarded. Cells were then cultured with 100 TCID50. 50 Add PRV at a concentration of 100 μL / mL to each well, incubate at 37°C with 5% CO2 for approximately 2 hours, discard the virus-containing DMEM culture medium, add 100 μL / well of 2% FBSDMEM, and incubate at 37°C with 5% CO2. Observe and record the cytopathic effect in the wells daily until the virus control wells without interferon show complete cytopathic effect. Repeat the experiment three times, and calculate the cytopathic effect inhibition rate according to the Reed-Muench method. The highest interferon dilution that protects half of the cells from viral damage is defined as 1 unit of interferon activity. The calculation formula is as follows:

[0130] Distance ratio = (Percentage of lesions with a rate higher than 50% - 50%) / (Percentage of lesions with a rate higher than 50% - Percentage of lesions with a rate lower than 50%)

[0131] lg4 (highest dilution of interferon with 50% cytopathic effect inhibition rate) = distance ratio + lg4 (dilution with higher than 50% cytopathic effect inhibition rate)

[0132] 1.4 Half-life determination

[0133] (1) In vitro half-life determination

[0134] Anticoagulated blood was collected from SPF-grade KM mice. Plasma was collected by centrifugation at 3000 r / min for 5 min and mixed with an equal volume of PBS solution to prepare 50% plasma. Protein IFNα and IFNα-FSA were added separately, with a final protein concentration of 10 μg / mL in the plasma. Plasma without protein was set up as a negative control group. Samples were taken after incubation at 37℃ for 0, 0.25, 0.5, 1, 3, 6, 12 and 24 h to detect antiviral activity and record the cytopathic effect inhibition rate.

[0135] (2) In vivo half-life determination

[0136] Four SPF-grade KM mice were subcutaneously injected with IFNα 10 μg / kg, IFNα 100 μg / kg, IFNα-FSA 10 μg / kg, and IFNα-FSA 100 μg / kg, respectively, with a negative control set up. Anticoagulated blood was collected by tail disconnection at 0 h, 24 h, 48 h, 72 h, and 96 h after injection. Plasma was collected by centrifugation at 3000 r / min for 5 min, and the antiviral activity of interferon in plasma was detected. The cytopathic effect inhibition rate was also recorded.

[0137] 2. Test Results

[0138] 2.1 Safety Test

[0139] Changes in body weight of mice in each group after intraperitoneal injection of protein are as follows: Figure 17 As shown in Table 3, the growth status of the mice was as follows. One day after injection, the weight of all mice decreased, but recovered and increased after one day. No obvious symptoms or abnormal behavior were observed during the observation period, and the overall mental state was good. The weight changes in all groups showed an overall upward trend, and there was no significant difference compared to the negative control group. On day 14, the mice were euthanized by cervical dislocation. Dissection revealed no obvious lesions in the heart, liver, lungs, spleen, and both kidneys. The organ indices for each group are recorded in Table 4, and there was no significant difference compared to the negative control group.

[0140] Table 3. Mouse growth status

[0141]

[0142] Table 4 Organ Index of Mice

[0143]

[0144] 2.2 Cytotoxicity assay

[0145] The inhibitory effect of protein IFNα-FSA on the proliferation of MDBK cells was detected according to the method specified in the CCK-8 kit instructions. Figure 18 As shown, 400 ng / mL IFNα-FSA significantly inhibited the proliferation of MDBK cells more than 200 ng / mL IFNα-FSA (P<0.05); 100 ng / mL IFNα-FSA significantly inhibited the proliferation of MDBK cells more than 50 ng / mL IFNα-FSA (P<0.5), and the protein's anti-cell proliferation activity showed a dose-dependent relationship.

[0146] 2.3 Antiviral activity assay

[0147] The antiviral activity of proteins IFNα and IFNα-FSA was detected on the MDBK-PRV system using the cytopathic effect inhibition (CPE) assay. Both proteins were serially diluted 4-fold. Blank control and virus control groups were also included. Cells were observed 24 hours after inoculation, and varying degrees of cytopathic effect were observed. Cells were observed until the virus control wells (without protein) showed complete cytopathic effect, and the results were recorded. The highest interferon dilution that protected half of the cells from viral damage was defined as one unit of interferon activity. Antiviral activity was calculated, and the results are shown in Table 5. Both IFNα and IFNα-FSA showed antiviral activity on the MDBK-PRV system. The antiviral activity of IFNα-FSA was slightly higher than that of IFNα, but the difference was not significant.

[0148] Table 5. Detection of antiviral activity of IFNα and IFNα-FSA

[0149]

[0150] 2.4 Half-life determination

[0151] (1) In vitro half-life determination

[0152] Proteins IFNα and IFNα-FSA were mixed with 50% plasma from SPF-grade KM mice and incubated in a 37°C water bath for 0–24 h. Antiviral activity of interferon in the plasma was measured at different time points. The antiviral activity detected in plasma at 0 h was set as 100%. Results are as follows: Figure 19 In the A group, the negative plasma of mice showed no antiviral activity. With prolonged incubation, the antiviral activity of IFNα and IFNα-FSA gradually decreased. The in vitro half-life of IFNα was between 6 and 12 hours, while IFNα-FSA retained more than 50% of its activity after incubation at 37°C.

[0153] (2) In vivo half-life determination

[0154] SPF-grade KM mice were subcutaneously injected with IFNα 10 μg / kg, IFNα 100 μg / kg, IFNα-FSA 10 μg / kg, and IFNα-FSA 100 μg / kg, respectively. A negative control group was also included. Anticoagulated blood was collected from the tail at different time points, and the antiviral activity of residual interferon in the plasma was detected. The results are as follows: Figure 19In the B group, the antiviral activity of residual interferon in plasma peaked 6 hours after injection and then gradually decreased. IFNα injected at a dose of 10 μg / kg lost its activity after 36 hours, while IFNα injected at a dose of 100 μg / kg lost its activity after 48 hours. After injections of IFNα-FSA at doses of 10 μg / kg and 100 μg / kg, no antiviral activity of interferon was observed in residual plasma after 96 hours, demonstrating that increasing the injection concentration prolongs the duration of interferon presence in the body, and that the half-life of IFNα-FSA in vivo is longer than that of IFNα.

[0155] In summary, IFNα-FSA has a certain inhibitory effect on the proliferation of MDBK cells, and the inhibitory effect decreases with decreasing dosage. IFNα-FSA is safe for use in mice in vivo. Both IFNα-FSA and IFNα have antiviral activity, but the half-life of IFNα-FSA is longer than that of IFNα.

[0156] The above specific embodiments are merely explanations of the present invention and are not intended to limit the present invention. After reading this specification, those skilled in the art can make modifications to these embodiments without contributing any inventive step, but as long as they are within the scope of the claims of the present invention, they are protected by patent law.

Claims

1. A long-acting forest musk deer interferon fusion protein, characterized in that, The amino acid sequence of the fusion protein is shown in SEQ ID NO.2, and it is formed by linking the IFNα protein (amino acid sequence shown in SEQ ID NO.3) and the FSA protein (amino acid sequence shown in SEQ ID NO.4) via a flexible linker.

2. A gene encoding the amino acid sequence of the fusion protein of claim 1, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

3. A recombinant vector comprising the gene sequence of SEQ ID NO.1 as described in claim 2.

4. An engineered bacterium comprising the recombinant vector of claim 3.

5. A method for preparing the fusion protein of claim 1, characterized in that, Includes the following steps: S1, Construction of recombinant plasmid pMD19-T-IFNα-FSA; S2. Construction of recombinant Pichia pastoris strain with exogenous expression of fusion protein IFNα-FSA; S3, induced expression of the fusion protein IFNα-FSA; S4. Purification and refolding.

6. The method according to claim 5, characterized in that, Step S1 includes the following steps: S11. Design overlapping PCR primers to amplify the FSA gene and IFNα gene; The amplification primers for the FSA gene include pf1 with the sequence shown in SEQ ID NO.5 and pf2 with the sequence shown in SEQ ID NO.6; the amplification primers for the IFNα gene include pf3 with the sequence shown in SEQ ID NO.7 and pf4 with the sequence shown in SEQ ID NO.

8. S12. The FSA gene and IFNα gene obtained in step S11 are amplified using overlap PCR technology to obtain the gene of the fusion protein. S13. Ligate the recombinant plasmid with the pMD19-T vector to construct a cloning plasmid. Digest the recombinant plasmid with two enzymes to obtain the target fragment. Ligate the recombinant plasmid with a eukaryotic expression vector to construct the recombinant plasmid pPICZαA-IFNα-FSA.

7. The method according to claim 5, characterized in that, Step S2 includes: digesting the recombinant plasmid pPICZαA-IFNα-FSA with a single SacⅠ restriction endonuclease, then electroporating the linearized plasmid into Pichia pastoris to construct the recombinant strain X33-pPICZαA-IFNα-FSA.

8. The method according to claim 5, characterized in that, Step S3 includes: the fusion protein IFNα-FSA is induced to express under the following conditions: 0.5% methanol induction concentration for 96 h.

9. The use of the long-acting musk deer interferon fusion protein according to claim 1 in the preparation of antiviral drugs for musk deer.

10. The use of the long-acting musk deer interferon fusion protein according to claim 1 in the preparation of antiviral biological agents of musk deer.

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