Recombinant batroxobin, its preparation method and use

Abstract

This invention relates to the field of biochemical technology, specifically to a recombinant batroxobin, its preparation method, and its applications. The invention relates to a recombinant batroxobin that uses a short sequence of α-mating factor signal peptide and a short sequence of Bat as repeating units, and connects multiple repeating units through short sequences of tandem IRES elements. The tandem IRES element short sequences are selected from one or more of the following amino acid sequences: amino acid sequences expressed by any of the sequences shown in SEQ ID No. 1-4 and SEQ ID No. 21. The synergistic effect of IRES, stable sequences, and codon optimization overcomes multiple rate-limiting steps from gene integration to protein synthesis, resulting in a significant increase in the recombinant expression level of batroxobin.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological chemical industry, and particularly relates to a recombinant batroxobin, a preparation method and application thereof. BACKGROUND

[0002] Batroxobin is a serine protease isolated from the venom of the lancehead pit viper Bothrops atrox by Von Klobusitzky in 1963, and belongs to the family of coagulation factor Xa-like enzymes. As an important medicinal protein, batroxobin has been successfully developed into a hemostatic agent and a defibrinogen agent, and has important medical value.

[0003] However, although the batroxobin protein is only composed of one peptide chain, a complex disulfide bond network is formed in the molecule, and there is glycosylation modification. These complex post-translational modifications make it difficult to obtain recombinant proteins with natural conformation and activity by using a prokaryotic expression system (such as Escherichia coli).

[0004] Therefore, a eukaryotic expression system such as Pichia pastoris is usually selected for recombinant expression in order to obtain correctly folded proteins. However, even in the Pichia pastoris system, the yield of batroxobin is still low, which is difficult to meet the needs of industrial production, and this has become a major technical bottleneck restricting its wide application.

[0005] In order to overcome the problem of low expression of recombinant proteins in Pichia pastoris, an important strategy in the field of genetic engineering is to increase the copy number of the target gene. Studies have shown that multiple copy integration of recombinant genes in Pichia pastoris can effectively improve the expression level of target proteins. However, when Pichia pastoris integrates foreign genes into the genome through homologous recombination, high copy integration events are a spontaneous and low probability process, and the occurrence rate is usually only 1% to 10%, and most transformants only carry 1 to 10 gene copies. This low-efficiency spontaneous integration seriously limits the screening efficiency of high-yield strains.

[0006] In order to obtain higher copy number of engineering strains under the same transformation screening pressure, a feasible technical path is to pre-connect multiple gene expression units in vitro through molecular biology methods to construct a multi-copy expression cassette, and then integrate the whole. This "multi-copy" strategy can significantly increase the average gene copy number in the initial transformant, thereby laying a foundation for screening high-yield batroxobin engineering strains.

[0007] Currently, the common method for constructing multi-cistronic (i.e., a single mRNA translation produces multiple proteins) to achieve the co-expression of multiple genes in eukaryotic systems mainly relies on internal ribosome entry site (IRES) elements or self-cleaving polypeptides (such as 2A peptides). However, both of these two mainstream technologies have obvious limitations. The size of IRES element is large, and the translation efficiency of the downstream gene mediated by it is usually much lower than that of the upstream gene, resulting in a serious uneven expression of different proteins. Although 2A peptides can achieve the co-expression of multiple proteins, they often leave extra amino acid residues at the C-terminus or N-terminus of each protein during the self-cleavage process, and these additional peptide segments may affect the activity, purification and safety of the target protein (especially a pharmaceutical protein such as batroxobin with a delicate structure).

[0008] Therefore, developing a new multi-gene tandem and expression strategy that can avoid the defects of IRES and 2A peptides has important practical significance for efficiently constructing high-copy batroxobin expression vectors and further improving their yield.

[0009] In addition, on the one hand, there are differences in the understanding of those skilled in the art; on the other hand, the inventors have studied a large number of literatures and patents when making the present invention, but due to the limited space, all the details and contents are not listed in detail, which does not mean that the present invention does not have these prior art characteristics, on the contrary, the present invention has all the characteristics of the prior art, and the applicant reserves the right to add relevant prior art in the background art. SUMMARY

[0010] The present application relates to the technical field of biological chemical industry, in particular to a recombinant batroxobin and a preparation method and application thereof.

[0011] In view of the above technical problems, one of the purposes of the present application is to provide a recombinant batroxobin, which takes a short sequence of alpha mating factor signal peptide and a short sequence of Bat as a repeating unit, and connects a sequence of multiple repeating units through a tandem IRES element short sequence, wherein the tandem IRES element short sequence is selected from one or more of the following sequences:

[0012] the sequence shown in SEQ ID No. 1;

[0013] the sequence shown in SEQ ID No. 2;

[0014] the sequence shown in SEQ ID No. 3;

[0015] the sequence shown in SEQ ID No. 4;

[0016] the sequence shown in SEQ ID No. 21.

[0017] According to a preferred embodiment, the above-mentioned Bat short sequence has the nucleotide sequence shown in SEQ ID No. 7; or the amino acid sequence shown in SEQ ID No. 13.

[0018] According to a preferred embodiment, the nucleotide sequence of the short α-mating factor signal peptide is shown in SEQ ID No. 6.

[0019] According to a preferred embodiment, the repeating unit is provided with a short α-mating factor signal peptide sequence and a short Bat sequence in the order from the 5' end to the 3' end.

[0020] According to a preferred embodiment, the recombinant batroxobin described above is provided with three repeating units linked by short sequences of tandem IRES elements. Preferably, the recombinant batroxobin comprises the following expression sequence:

[0021] 3x-αMF-Bat-pPIC9K-IGG2 (SEQ ID No. 8);

[0022] 3x-αMF-Bat-pPIC9K-IGG4 (SEQ ID No. 9);

[0023] 3x-αMF-Bat-pPIC9K-IGG6 (SEQ ID No. 10);

[0024] 3x-αMF-Bat-pPIC9K-IGG10 (SEQ ID No. 11); or

[0025] 3x-αMF-Bat-pPIC9K-IGG11 (SEQ ID No. 12).

[0026] According to a preferred embodiment, a translation termination signal is provided between the repeat unit and the short sequence of the tandem IRES element. Preferably, the nucleotide sequence of the translation termination signal is TAA, TAG, or TGA.

[0027] One of the objectives of this invention is to provide a method for preparing the above-mentioned recombinant batroxobin, the method comprising the following steps: amplifying the nucleic acid molecule encoding the recombinant batroxobin, cloning it into a plasmid vector to obtain a recombinant vector, introducing the recombinant vector into a host strain for expression, and then isolating and purifying it to obtain the recombinant batroxobin.

[0028] According to a preferred embodiment, the host strain is Pichia pastoris. Preferably, Pichia pastoris is selected from one of GS115, X-33, KM71, KM71H, SMD1168, SMD1168H, Y11430, and MG1003.

[0029] According to a preferred embodiment, the fermentation conditions for the host strain are as follows:

[0030] The host strain was inoculated into YPD liquid medium;

[0031] Inoculate the bacterial culture into BMMY medium, and after the OD reaches 2, add 1% methanol by volume every 12 hours for induction.

[0032] Centrifuge and harvest the fermentation supernatant.

[0033] Preferably, the nucleic acid molecule encoding recombinant batroxobin is amplified by: synthesizing a batroxobin nucleic acid molecule, extracting it from a natural batroxobin product, or cloning and amplifying the recombinant batroxobin nucleic acid molecule from an engineered plasmid containing batroxobin (e.g., the Bat-His-P10 plasmid). More preferably, the amplification primers are upstream primer JD-P23 (SEQ ID No. 14) and downstream primer JD-P24 (SEQ ID No. 15).

[0034] Preferably, the recombinant vector is introduced into the host strain by mixing competent Pichia pastoris cells with a linearized recombinant vector, electroporating, and then transferring the mixture to a solid culture medium for culture.

[0035] Preferably, the conditions for electro-rotation are: 1800 V, 200 Ω, 25 μF.

[0036] Preferably, the solid culture medium is YPD solid culture medium (100 μg / mL G418).

[0037] Preferably, the method for amplifying the recombinant vector is as follows: the recombinant vector is transformed into competent DH5α cells. Specifically, DH5α cells are thawed on ice, and seamless cloning products are added in a clean bench. The cells are then incubated on ice for 30 min, heat-shocked at 42°C for 90 s, and incubated on ice for 5 min. 500 μL of LB liquid medium (antibiotic-free) is added to the transformation product and incubated at 37°C for 1 h. 250 μL of the culture medium is then plated on an LB agar plate (100 μg / mL ampicillin). The agar plate is then incubated upside down at 37°C overnight.

[0038] Preferably, the engineered plasmid αMF-Bat-pPIC9K is constructed by transforming a Bat gene fragment carrying a pPIC9K homologous arm into a pPIC9K linear vector.

[0039] Preferably, the recombinant vector is obtained by cloning into a plasmid vector by seamless cloning of the linearized pPIC9K vector and the batroxobin nucleic acid fragment Bat containing the homologous arm of the pPIC9K vector, followed by transformation. More preferably, the seamless cloning conditions include adding the required reaction mix and reacting at 37°C for at least 30 min. The mix is ​​2x Uniclone seamless cloning mix. The specific proportions of each component in the reaction system are shown in Table 3 for the amounts of gene fragment, linear vector, mix, and water used. The recombinant vector is then transformed into competent DH5α cells. Specifically, DH5α cells are thawed on ice, and the seamless cloning product is added in a clean bench, incubated on ice for 30 min, heat-shocked at 42°C for 90 s, and then incubated on ice for 5 min; 500 μL of LB liquid medium (antibiotic-free) is added to the transformation product, and the cells are incubated at 37°C for 1 h; 250 μL is then plated on an LB plate (100 μg / mL ampicillin); and the cells are incubated in an inverted incubator at 37°C overnight.

[0040] According to a preferred embodiment, the plasmid vector is selected from one of pPIC9K, pPIC9, pHIL-S1, pPICZα A, and pYAM75P.

[0041] One of the objectives of this invention is to provide the application of the above-mentioned recombinant batroxobin or the recombinant batroxobin prepared by the above-mentioned method in the preparation of hemostatic and fibrinolytic drugs.

[0042] The technical solution provided by this invention creatively and specifically assembles the IRES element, a short stable sequence derived from the α-mating factor signal peptide, and the batroxobin functional sequence, and simultaneously optimizes the codons of the batroxobin functional sequence. This achieves efficient co-expression of multiple genes while significantly improving the yield and quality of batroxobin in the Pichia pastoris system. Compared with existing technologies, this invention produces the following outstanding beneficial effects:

[0043] This invention effectively overcomes the inherent defects of traditional polycistronic construction strategies. By employing a composite regulatory unit containing a specific IRES element and a short sequence of α-crossing factor signal peptide, the translational purpose of multiple open reading frames on the same mRNA molecule is eliminated, directly avoiding the problem of low downstream gene translation levels caused by promoter or reading frame mismatch or excessive length. Simultaneously, this strategy successfully avoids the risk of leaving extra amino acid residues at the ends of the target protein due to the use of 2A peptide self-cleavage technology, thus ensuring that the final recombinant batroxobin protein has a precise terminal sequence completely identical to the natural protein. This is crucial for maintaining the biological activity of batroxobin and meeting the stringent quality control requirements for therapeutic drugs.

[0044] Simultaneously, this invention achieves a substantial leap in batroxobin expression levels through the synergistic effect of its components. The introduction of the short α-mating factor signal peptide sequence effectively enhances mRNA stability, reduces degradation, and provides a sufficient template basis for high-level translation. Furthermore, Pichia pastoris host preference optimization targeting the codons of the batroxobin gene itself significantly improves tRNA adaptation efficiency during transcriptomic translation, resolving the translation elongation delay problem that may be caused by differences in codon usage frequency. The synergistic effect of IRES, stable sequences, and codon optimization overcomes multiple rate-limiting stages from gene integration to protein synthesis, resulting in a significant improvement in batroxobin recombinant expression levels that surpasses conventional methods.

[0045] Furthermore, this technical solution provides a more universal optimization strategy for the efficient production of recombinant proteins with complex structures in Pichia pastoris. This method not only specifically solves the technical challenge of low expression levels of batroxobin due to numerous disulfide bonds and glycosylation modifications, but its element assembly and optimization approach also has significant reference value and broad application prospects for the efficient production of other difficult-to-express eukaryotic medicinal proteins. Attached Figure Description

[0046] Figure 1 The image shows the electrophoresis results after cloning the Bat functional gene. Lane M is the marker, and lane 1 is the result image, showing that a 732 bp fragment was successfully cloned.

[0047] Figure 2 The image shows the electrophoresis results after pPIC9K digestion. Lane M is the marker, and lane 1 is the result image, showing that the digestion successfully yielded a 9276 bp fragment.

[0048] Figure 3 The structure diagram of the plasmid after successful construction of aMF-Bat-pPIC9K;

[0049] Figure 4 The electrophoresis diagram of plasmid extraction after successful construction of aMF-Bat-pPIC9K is shown. Lane M is the marker and lane 1 is the result. It can be seen that the 9949 bp recombinant plasmid was successfully constructed.

[0050] Figure 5 This is a Western blotting image of the fermentation supernatant after successful electroporation of aMF-Bat-pPIC9K. Lane M is the marker, lane negative represents the recombinant batroxobin protein expression level of the GS115 negative control strain under the same treatment, and lanes 1-10 represent the recombinant batroxobin protein expression levels of the selected positive clone strains 1-10.

[0051] Figure 6The structure diagram of the plasmid after successful construction of 3x-aMF-Bat-pPIC9K-IGG2 / IGG4 / IGG6 / IGG10 / IGG11;

[0052] Figure 7 The above-mentioned plasmid electroporation results are shown in the Western Blot protein expression results of the fermentation supernatant. Lanes 1-5 show the recombinant batroxobin protein expression levels of strains GS115-3x-aMF-Bat-pPIC9K-IGG2, IGG4, IGG6, IGG10, and IGG11, respectively, while lane 6 shows the recombinant batroxobin protein expression level of strain GS115-aMF-Bat-pPIC9K.

[0053] Figure 8 The image shows the Western blotting results of protein expression in the fermentation supernatant after successful electroporation of plasmids 3x-αMF-Bat-IGG2-IGG6 and 3x-αMF-Bat-IGG6-IGG2. Lane M is a marker, and lanes 1 and 2 are the expression levels of recombinant batroxobin protein in strains GS115-3x-αMF-Bat-IGG2-IGG6 and GS115-3x-αMF-Bat-IGG6-IGG2, respectively. Detailed Implementation

[0054] In the description of this invention, terminology is used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. In the description of this invention, "a plurality of" means two or more, unless otherwise explicitly defined.

[0055] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials, reagents or instruments used, unless otherwise specified by the manufacturer, are all commercially available reagents and materials; the conditions not specified in the examples are all carried out according to conventional conditions or conditions recommended by the manufacturer. At the same time, the present invention does not limit the source of the raw materials used. Unless otherwise specified, the raw materials used in the present invention are all commercially available products in this technical field.

[0056] The amino acid sequence of natural batroxobin is (SEQ ID No. 13) VIGGDECDINEHPFLAFMYYSPRYFCGMTLINQEWVLTAAHCNRRFMRIHLGKHAGSVANYDEVVRYPKEKFICPNKKKNVITDKDIMLIRLDRPVKNSEHIAPLSLPSNPPSVGSVCRIMGWGAITTSEDTYPDVPHCANINLFNNTVCREAYNGLPAKTLCAGVLQGGIDTCGGDSGGPLICNGQFQGILSWGSDPCAEPRKPAFYTKVFDYLPWIQSIIAGNKTATCP.

[0057] Example 1

[0058] This embodiment relates to the construction of the engineered plasmid αMF-Bat-pPIC9K for efficient expression of batroxobin in Pichia pastoris, the preparation method of GS115 competent cells, the transfer of the engineered plasmid αMF-Bat-pPIC9K, and the fermentation of the engineered bacteria.

[0059] (I) Construction of engineered plasmid αMF-Bat-pPIC9K

[0060] 1. Target gene amplification

[0061] 1) Template preparation: The saved Bat-His-P10 plasmid was used as a template. The batroxobinase gene in this plasmid was optimized by codons to achieve higher translation efficiency in Pichia pastoris, while its amino acid sequence was consistent with the amino acid sequence of natural batroxobinase.

[0062] 2) The target gene was amplified by PCR reaction, and the reaction system is shown in the table below.

[0063] Table 1

[0064]

[0065] The reaction conditions are as follows:

[0066] 98℃ for 10 s; 98℃ for 10 s, 55℃ for 15 s, 68℃ for 1 min, for a total of 35 cycles; 68℃ for 5 min; keep warm at 25℃.

[0067] The primer sequences are as follows (the uppercase parts are the homologous arms of the pPIC9K vector):

[0068] JD-P23 (SEQ ID No.14):GTATCTCTCGAGAAAAGAgttattggtggtgatgagtgt;

[0069] JD-P24 (SEQ ID No. 15): TTCCGGCCGCCCTAGGGTTAtggacaagtagcagtcttgtt.

[0070] 3) The PCR products were subjected to 1% agarose gel electrophoresis, and the 732 bp fragment was purified and recovered using the Takara DNA Purification and Recovery Kit (Cat. 9762). The electrophoresis results are shown below. Figure 1 As shown.

[0071] 2. Vector enzyme digestion

[0072] 1) Prepare the enzyme digestion reaction system according to the table below, as shown in Table 2.

[0073] Table 2

[0074]

[0075] The reaction conditions were: metal bath at 37°C for 30 min.

[0076] 2) The enzyme digestion products were subjected to 1% agarose gel electrophoresis, and the 9276 bp fragment was purified and recovered using the Takara DNA Purification and Recovery Kit (Cat.9762). The electrophoresis results are shown below. Figure 2 As shown.

[0077] 3. Seamless cloning and conversion

[0078] 1) The aforementioned gene was cloned into the enzyme digestion vector using the Xinke Aoda seamless cloning enzyme (Cat.TQ111). The reaction system is shown in Table 3 below.

[0079] Table 3

[0080]

[0081] The reaction conditions were 37℃ for 30 min.

[0082] 2) Transformation

[0083] Melt DH5α on ice, add seamless cloning product to a clean bench, incubate on ice for 30 min, heat shock at 42℃ for 90 s, and incubate on ice for 5 min.

[0084] Take 500 μL of LB liquid medium (antibiotic-free), add it to the transformation product, and incubate at 37°C for 1 h. Take 250 μL and spread it on an LB plate (100 μg / mL ampicillin); incubate in an inverted incubator at 37°C overnight.

[0085] 3) After the transformant was expanded and the plasmid was extracted, it was sent for sequencing to confirm that the gene fragment was consistent with the above. The plasmid was named αMF-Bat-pPIC9K.

[0086] plasmid maps as follows Figure 3 As shown in the figure. The electrophoresis diagram of the plasmid extraction results is as follows. Figure 4 As shown.

[0087] (II) Preparation of GS115 competent cells

[0088] 1. Inoculation: Dip a sterile pipette tip into a small amount of GS115 bacterial suspension, streak it on a YPD plate, and incubate it upside down at 30°C.

[0089] 2. Seed culture: Pick a single colony and inoculate it into 20 mL of YPD liquid medium, and incubate at 30℃ and 180 rpm.

[0090] 3. Solution preparation

[0091] Preparation of SE1 solution:

[0092] 1) Prepare SE1 solution according to the formula in Table 4.

[0093] Table 4

[0094]

[0095] 2) After the solution is prepared, adjust the pH to 7.5 with 6 M HCl and filter it with a 0.22 μm filter membrane in a clean bench.

[0096] Preparation of SE2 solution:

[0097] 1) Prepare SE2 solution according to the formula in Table 5.

[0098] Table 5

[0099]

[0100] 2) After the solution is prepared, adjust the pH to 7.5 with 6 M HCl and filter it with a 0.22 μm filter membrane in a clean bench.

[0101] 4. Measure the OD of the overnight cultured bacterial solution. 600 Transfer the bacterial culture to 200 mL of YPD liquid medium to allow OD... 600 Incubate at 0.4, 30℃, and 180 rpm until the OD reaches 0.8-1.0.

[0102] 5. Centrifuge the bacterial culture at 3000 rpm for 5 min, discard the supernatant, and suspend the cells in 200 mL of pre-cooled pure water.

[0103] 6. Centrifuge at 3000 rpm for 5 min at 4℃, discard the supernatant, and resuspend the bacterial cells in 50 mL of pre-cooled SE2.

[0104] 7. Incubate in water at 37℃ for 15 minutes.

[0105] 8. Centrifuge at 3000 rpm for 5 min at 4℃, discard the supernatant, and resuspend the bacterial cells in 50 mL of pre-cooled SE1.

[0106] 9. Centrifuge at 3000 rpm for 5 min at 4℃, discard the supernatant, and resuspend the bacterial cells in 50 mL of pre-cooled SE1.

[0107] 10. Centrifuge at 3000 rpm for 5 min at 4℃, discard the supernatant, resuspend the cells in 1 mL of pre-cooled SE1, and aliquot into 100 μL tubes.

[0108] 11. Gradual cooling: let stand at 4℃ for 60 min, transfer to -20℃ and let stand for 60 min, then transfer to a freezer below -80℃ for freezing.

[0109] (III) Transformation

[0110] 1. Linearization of αMF-Bat-pPIC9k plasmid

[0111] The αMF-Bat-pPIC9k plasmid was digested with SacⅠ enzyme, and the reaction system is shown in Table 6.

[0112] Table 6

[0113]

[0114] The reaction conditions were a 37°C water bath for 30 min.

[0115] 2. The enzyme digestion products were subjected to 1% agarose gel electrophoresis and then purified and recovered using the Takara DNA purification kit.

[0116] 3. Electro-rotation

[0117] 1) Take GS115 competent cells and let them thaw on ice. Add the recovered enzyme digestion fragments, mix well, and place on ice for 5 min.

[0118] 2) Take the pre-cooled electrospinning cup, transfer the competent mixture into the electrospinning cup, and perform electrospinning under the following conditions: 1800 V, 200 Ω, 25 μF.

[0119] 3) After the electroporation is completed, add 900 μL of YPD liquid culture medium to each electroporation cup and place them in a 30℃ incubator for 3 h.

[0120] 4) Remove the electroporation cup, spread the bacterial culture onto a YPD plate (100 μg / mL G418), and incubate the plate upside down in a 30℃ incubator for 3-7 days.

[0121] 5) Select transformants for colony PCR identification. Primers are:

[0122] Upstream primer JD-P27 (SEQ ID No. 20): GACTGGTTCCAATTGACAAGC;

[0123] Downstream primer JD-P28 (SEQ ID No. 5): GGCAAATGGCATTCTGACAT.

[0124] The positive transformants were stored for later use.

[0125] (iv) Fermentation of GS115-αMF-Bat-pPIC9K engineered bacteria

[0126] 1. The preserved positive transformants were inoculated into 20 mL of YPD liquid medium (100 μg / mL G418) and cultured at 30℃ and 180 rpm for 24 h.

[0127] 2. Inoculate the bacterial culture into 20 mL of BMMY medium (1% methanol by volume) to achieve an initial OD of 2. Incubate at 28°C and 180 rpm, adding 1% methanol every 12 h for induction. After 48 h of culture, remove the yeast cells by centrifugation and harvest the fermentation supernatant.

[0128] 3. The fermentation supernatant was subjected to Western blotting to identify the target product.

[0129] The results of the identification are as follows Figure 5 As shown.

[0130] Example 2

[0131] This embodiment relates to the construction of GS115 engineered bacteria that highly express batroxobin. This embodiment also relates to the construction of the vector 3x-αMF-Bat-pPIC9K-IGG2 / IGG4 / IGG6 / IGG10 / IGG11.

[0132] The nucleotide sequence of the short tandem IRES element is as follows:

[0133] IGG2 (SEQ ID No.1):TTGAGTCAATCAAACACTCAA;

[0134] IGG4 (SEQ ID No.2): AGTCAATCAAACACT;

[0135] IGG11 (SEQ ID No.3):AAACACTCAACAG;

[0136] IGG10 (SEQ ID No.4):TGTTGAGTCAATC;

[0137] IGG6 (SEQ ID No. 21):CAATCAAAC.

[0138] The nucleotide sequence of αMF (SEQ ID No. 6) used in this embodiment is as follows:

[0139] ATGAGATTTCCTTCAATTTTTACTGCAGTTTTATTCGCAGCATCCTCCGCATTAGCTGCTCCAGTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGTTACTCAGA TTTAGAAGGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAATAACGGGTTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTATCTCTCGAGAAAAGA.

[0140] The functional sequence used in this embodiment, namely the nucleotide sequence of Bat (SEQ ID No. 7) after codon optimization, is as follows:

[0141] .

[0142] The following plasmids, 3x-αMF-Bat-pPIC9K-IGG2, 3x-αMF-Bat-pPIC9K-IGG4, 3x-αMF-Bat-pPIC9K-IGG6, 3x-αMF-Bat-pPIC9K-IGG10, and 3x-αMF-Bat-pPIC9K-IGG11, all contain αMF-Bat repeat units linked by different expression-promoting short sequences. A translation signal termination protein (nucleotide sequence denoted as TAA) is placed between each repeat unit and the expression-promoting short sequence.

[0143] The nucleotide sequence of 3x-αMF-Bat-IGG2 (SEQ ID No. 8) linked to the pPIC9K plasmid is as follows:

[0144]

[0145] The nucleotide sequence of 3x-αMF-Bat-IGG4 (SEQ ID No. 9) is as follows:

[0146]

[0147] The nucleotide sequence of 3x-αMF-Bat-IGG6 (SEQ ID No. 10) is as follows:

[0148]

[0149] The nucleotide sequence of 3x-αMF-Bat-IGG10 (SEQ ID No. 11) is as follows:

[0150]

[0151] The nucleotide sequence of 3x-αMF-Bat-IGG11 (SEQ ID No. 12) is as follows:

[0152]

[0153] In this embodiment, the plasmid vector contains three repeat units and two short expression-promoting sequences connecting the three repeat units.

[0154] (a) Ligation of batroxobin overexpression sequence with pPIC9K

[0155] 1) Prepare the enzyme digestion reaction system as shown in Table 7.

[0156] Table 7

[0157]

[0158] The reaction conditions were: metal bath at 37°C for 30 min.

[0159] 2) The enzyme digestion products were subjected to 1% agarose gel electrophoresis, and the 8892 bp fragment was purified and recovered using the Takara DNA Purification and Recovery Kit (Cat.9762).

[0160] 3) Seamless cloning and conversion

[0161] 1. Five synthetic gene fragments were cloned into the enzyme digestion vector using the Xinke Aoda seamless cloning enzyme (Cat.TQ111). The reaction system was the same as before, and the reaction conditions were 37℃ for 30 min.

[0162] 2. Transformation

[0163] Melt DH5α on ice, add seamless cloning product to a clean bench, incubate on ice for 30 min, heat shock at 42℃ for 90 s, and incubate on ice for 5 min.

[0164] Take 500 μL of LB liquid medium (antibiotic-free), add it to the transformation product, and incubate at 37℃ for 1 h. Take 250 μL and spread it on an LB plate (100 μg / mL ampicillin); incubate in an inverted incubator at 37℃ overnight.

[0165] 4) After the transformant was amplified and the plasmid was extracted, it was sent for sequencing to confirm that the gene fragment was consistent with the above. The plasmid was named 3x-αMF-Bat-pPIC9K-IGG2 / IGG4 / IGG6 / IGG10 / IGG11.

[0166] plasmid maps as follows Figure 6 As shown.

[0167] 2. Linearization of recombinant plasmids

[0168] 1) Linearization of 3x-αMF-Bat-pPIC9K-IGG2 / IGG4 / IGG6 / IGG10 / IGG11 plasmids

[0169] The plasmid was digested with SacⅠ enzyme, and the reaction system was the same as before. The reaction conditions were: 37℃ water bath for 30 min.

[0170] 2) The enzyme digestion products were subjected to 1% agarose gel electrophoresis and then purified and recovered using the Takara DNA purification kit.

[0171] 3. Electroconversion

[0172] 1) Take GS115 competent cells and let them thaw on ice. Add the recovered enzyme digestion fragments, mix well, and place on ice for 5 min.

[0173] 2) Take the pre-cooled electrospinning cup, transfer the competent mixture into the electrospinning cup, and perform electrospinning under the following conditions: 1800 V, 200 Ω, 25 μF.

[0174] 3) After the electroporation is completed, add 900 μL of YPD liquid culture medium to each electroporation cup and place them in a 30℃ incubator for 3 h.

[0175] 4) Remove the electroporation cup, spread the bacterial culture onto a YPD plate (100 μg / mL G418), and incubate the plate upside down in a 30℃ incubator for 3-7 days.

[0176] 5) Select a portion of the transformants for colony PCR identification, using the same primers as above: JD-P27 (SEQ ID No. 20) and JD-P28 (SEQ ID No. 5). Store the positive transformants for later use.

[0177] 4. Fermentation with engineered bacteria GS115-3x-aMF-Bat-pPIC9K-IGG2 / IGG4 / IGG6 / IGG10 / IGG11

[0178] 1) Identification of bacterial copy number

[0179] A. Yeast genome extraction: The genomes of GS115-aMF-Bat-pPIC9K and GS115-3x-aMF-Bat-pPIC9K-IGG2 / IGG4 / IGG6 / IGG10 / IGG11 positive transformants (50 strains each) were extracted using the Sangon Biotech Rapid Yeast Genomic DNA Isolation Kit (Cat.B518227-0100).

[0180] B. Batroxobin gene copy number identification

[0181] a) Prepare the fluorescence quantitative PCR reaction system

[0182] Prepare a quantitative PCR reaction system according to Table 8.

[0183] Table 8

[0184]

[0185] Internal reference GAPDH quantitative primer sequence:

[0186] JD-P35 (SEQ ID No.16):AAGGGTGAGGTTTCTGCCAGCG;

[0187] JD-P36 (SEQ ID No.17):TTCTTGGCACCGGCGTCGAT.

[0188] batroxobin quantitative primer sequence:

[0189] F (SEQ ID No.18):GAGAGGCTTACAACGGTTTGC;

[0190] R (SEQ ID No. 19): GCTGGCTTTCTAGGTTCAGCA.

[0191] b) The reaction conditions for quantitative real-time PCR are as follows:

[0192] 95℃ for 30 seconds; 95℃ for 15 seconds, 60℃ for 30 seconds, repeat for a total of 40 cycles.

[0193] After the reaction, the experimental data were saved and the copy number was calculated. According to the results, the maximum copy number of the GS115-aMF-Bat-pPIC9K strain was 6, while the maximum copy number of the tandem strain was 15. This method significantly increases the gene copy number under the same integration probability.

[0194] 2) Fermentation by strain

[0195] A. The preserved GS115-aMF-Bat-pPIC9K strain with a copy number of 6 and the GS115-3x-aMF-Bat-pPIC9K-IGG2 / IGG4 / IGG6 / IGG10 / IGG11 strain with a copy number of 15 were inoculated into 20 mL of YPD liquid medium (100 μg / mL G418) and cultured at 30℃ and 180 rpm for 24 h.

[0196] B. Inoculate the bacterial culture into 20 mL of BMMY medium (1% methanol) to achieve an initial OD of 2. Incubate at 28°C and 180 rpm, adding 1% methanol every 12 h to induce induction. After 48 h of incubation, remove the yeast cells by centrifugation and harvest the fermentation supernatant.

[0197] C. Western blot the fermentation supernatant to identify the target product.

[0198] like Figure 7 The results show that the expression levels of the five IGG tandem recombinant batroxobin strains were all no less than those of the GS115-aMF-Bat-pPIC9K strain, indicating that all five IGG fragments can mediate normal batroxobin expression. Furthermore, according to Western blotting results, the protein expression efficiency mediated was IGG2 > IGG10 > IGG11 > IGG4 > IGG6, suggesting that when these sequences are applied to Pichia pastoris or to batroxobin expression in Pichia pastoris, IGG2 has a significant impact on the expression efficiency of functional genes (especially the Bat functional gene).

[0199] Example 3

[0200] This embodiment is basically the same as Embodiment 2, except that the fragment linked to the pPIC9K plasmid is 3x-αMF-Bat-IGG10-IGG6. The short tandem IRES element sequences between the two repeating units in the fragment are IGG10 and IGG6, respectively. In this fragment, starting from the 5' end, the two repeating units are connected by IGG10 and then by IGG6.

[0201] Example 4

[0202] This embodiment is basically the same as Embodiment 2, except that the fragment linked to the pPIC9K plasmid is 3x-αMF-Bat-IGG4-IGG6. The short tandem IRES element sequences between the two repeating units in the fragment are IGG4 and IGG6, respectively. In this fragment, starting from the 5' end, the two repeating units are connected by IGG4 and then by IGG6.

[0203] Example 5

[0204] This embodiment is basically the same as Embodiment 2, except that the fragment linked to the pPIC9K plasmid is 3x-αMF-Bat-IGG2-IGG10. The short tandem IRES element sequences between the two repeating units in the fragment are IGG2 and IGG10, respectively. In this fragment, starting from the 5' end, the two repeating units are connected by IGG2 and then by IGG10.

[0205] Example 6

[0206] This embodiment is basically the same as Embodiment 2, except that the fragment linked to the pPIC9K plasmid is 3x-αMF-Bat-IGG11-IGG10. The short tandem IRES element sequences between the two repeating units in the fragment are IGG11 and IGG10, respectively. In this fragment, starting from the 5' end, the two repeating units are connected by IGG11 and then by IGG10.

[0207] Example 7

[0208] This embodiment is basically the same as Embodiment 2, except that the fragment linked to the pPIC9K plasmid is 3x-αMF-Bat-IGG2-IGG6. The short tandem IRES element sequences between the two repeating units in the fragment are IGG2 and IGG6, respectively. In this fragment, starting from the 5' end, the two repeating units are connected by IGG2, and then by IGG6.

[0209] Example 8

[0210] This embodiment is basically the same as Embodiment 2, except that the fragment linked to the pPIC9K plasmid is 3x-αMF-Bat-IGG6-IGG2. The short tandem IRES element sequences between the two repeating units in the fragment are IGG6 and IGG2, respectively. In this fragment, starting from the 5' end, the two repeating units are connected by IGG6 and then by IGG2.

[0211] Examples 3-8 selected strains with the same copy number for fermentation, and then performed WB analysis on the fermentation supernatant.

[0212] like Figure 8 As shown, the results indicate that the expression level of the strain with the 5'-IGG6-IGG2-3' linkage (Example 8) is lower than that of the strain with the 5'-IGG2-IGG6-3' linkage (Example 7).

[0213] Furthermore, WB expression results showed that the expression levels in Examples 7 and 8 were lower than those in the dual-linked IGG10 (Example 2).

[0214] WB expression results showed that, compared to the dual-linked IGG11 (Example 2), Example 7 had a higher expression level, while Example 8 had a lower expression level.

[0215] As can be seen from Examples 3-8, the specific arrangement of short sequences (including but not limited to short sequences derived from α-mating factor signal peptide short sequences) and the specific combination and stacking of multiple short sequences (such as elements with different functions) have a significant impact on the final expression level of batroxobin.

[0216] Example 9

[0217] This embodiment is basically the same as Embodiment 2, except that the vector is constructed using natural batroxobin (SEQ ID No. 13) nucleotides.

[0218] It should be noted that the specific embodiments described above are exemplary, and those skilled in the art can devise various solutions inspired by the disclosure of this invention. These solutions all fall within the scope of this invention and its protection. Those skilled in the art should understand that this specification and its accompanying drawings are illustrative and not intended to limit the scope of the claims. The scope of protection of this invention is defined by the claims and their equivalents.

Claims

1. A recombinant batroxobin, characterized in that, A sequence using short sequences of α-mating factor signal peptide and Bat as repeating units, and connecting at least three repeating units via tandem IRES element short sequences, wherein... The short sequence of tandem IRES elements is selected from one or more of the following sequences: The sequence represented by SEQ ID No. 1, namely IGG2; The sequence shown in SEQ ID No. 3, namely IGG11; The sequence shown in SEQ ID No. 4, namely IGG10, wherein, The encoding nucleotide sequence of the recombinant batroxobin is selected from the following sequences: Such as SEQ ID No. 8, 3x-αMF-Bat-pPIC9K-IGG2; Such as SEQ ID No. 11, 3x-αMF-Bat-pPIC9K-IGG10; or Such as SEQ ID No. 12, 3x-αMF-Bat-pPIC9K-IGG11.

2. A method for preparing the recombinant batroxobin according to claim 1, characterized in that, The nucleic acid molecule encoding the recombinant batroxobin is amplified, cloned into a plasmid vector to obtain a recombinant vector, introduced into a host strain for expression, and then isolated and purified to obtain the final product.

3. The method for preparing recombinant batroxobin according to claim 2, characterized in that, The host strain is Pichia pastoris.

4. The method for preparing recombinant batroxobin according to claim 2, characterized in that, The fermentation conditions for the host strain are as follows: The host strain was inoculated into YPD liquid medium; Inoculate the bacterial culture into BMMY medium, and after the OD reaches 2, add 1% methanol (v / v) for induction. Centrifuge and harvest the fermentation supernatant.

5. The method for preparing recombinant batroxobin according to claim 2, characterized in that, The plasmid vector is selected from one of pPIC9K, pPIC9, pHIL-S1, pPICZα A, and pYAM75P.

6. The use of the recombinant batroxobin according to claim 1 or the recombinant batroxobin prepared by any of the methods described in claims 2 to 5 in the preparation of drugs for hemostasis and fibrinolysis.

Images