VNP6-Sortase A7plus fusion protein as well as preparation method and application thereof
By introducing VNP6 tags at the N-terminal of Sortase A7plus and combining a low-temperature culture strategy, the intracellular molecular crowding problem of Sortase A7plus protein in E. coli was solved, its expression amount and enzyme activity were improved, and efficient and stable recombinant protein production was achieved.
Patent Information
- Application Number
- CN202510679219.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-26
- Publication Date
- 2025-08-29
AI Technical Summary
In the E. coli expression system, the high level of Sortase A7plus protein expression is affected by intracellular molecular crowding, resulting in misfolding of proteins and reduced enzyme activity, limiting its industrial application.
VNP6 tag was introduced at the N-terminal of Sortase A7plus, and combined with a low-temperature culture strategy, it regulates intracellular vesicle concentration to alleviate molecular crowding problems and optimizes the protein folding environment.
It significantly improves the expression amount and enzyme activity of Sortase A7plus, reduces the risk of protein misfolding and aggregation, and achieves efficient and stable recombinant protein production.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biomedicine, and specifically relates to a VNP6-Sortase A7plus fusion protein and a preparation method and application thereof. Background Art
[0002] In recent years, with the continuous expansion of the application of protein engineering and recombinant proteins in biomedicine, industrial catalysis, and basic research, various protein expression systems (such as mammalian cells, yeast, and Escherichia coli) have become increasingly mature. Among them, Escherichia coli has become the preferred platform for large-scale production of recombinant proteins due to its short culture cycle, low raw material cost, fast expression speed, and simple operation. However, as a prokaryotic microorganism, Escherichia coli has a significant difference in its intracellular biosynthetic environment from that of eukaryotic cells. Especially during the efficient expression process, proteins often cause misfolding, nonspecific aggregation, and even the formation of inclusion bodies due to the crowding effect between high-concentration macromolecules in the cell, which seriously affects the correct folding and functional display of the target protein.
[0003] Sortase A7plus (SRT7+), a novel transpeptidase (sortase) protein variant, possesses specialized protein cleavage and recombinant ligation capabilities and is widely used in protein engineering and biomaterials. However, when SRT7+ is expressed at high levels in Escherichia coli, intracellular molecular crowding can affect proper protein folding and enzyme activity, limiting its widespread industrial application.
[0004] Existing literature lacks research on how to enhance SRT7+ expression by regulating intracellular vesicle concentration to overcome molecular crowding in prokaryotic cells. Therefore, it is urgent to develop a novel strategy to screen for suitable sequences that induce the formation of vesicle structures that are conducive to protein folding and stability, thereby alleviating the molecular crowding effect and improving the expression yield and activity of SRT7+. Summary of the Invention
[0005] In response to the shortcomings of the prior art, the present invention aims to provide a VNP6-Sortase A7plus fusion protein, its preparation method, and its application. The present invention improves the SRT7+ expression system by introducing a VNP6 tag at the N-terminus of the SRT7+ protein. Combined with optimized culture strategies, this protein induces the concentration of intracellular vesicles in E. coli, effectively alleviating the molecular crowding problem caused by high expression load, thereby achieving the effect of increasing SRT7+ expression.
[0006] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:
[0007] In the first aspect, the present invention provides a VNP6-Sortase A7plus (VNP6-transpeptidase 7+, VNP6-SRT7+) fusion protein, which includes a VNP6 sequence and a Sortase A7plus sequence connected in sequence from N-terminus to C-terminus; the amino acid sequence after the VNP6 sequence and the Sortase A7plus sequence are connected is shown in SEQ ID NO:5.
[0008] The present invention utilizes the VNP6 tag to induce the formation of a vesicle structure in cells that is conducive to protein folding and stability, thereby alleviating the molecular crowding effect and increasing the expression yield and activity of SRT7+.
[0009] Preferably, the fusion protein is further connected to a His6 tag sequence; the amino acid sequence of the fusion protein is shown in SEQ ID NO: 1.
[0010] In the present invention, a VNP6-Sortase A7plus fusion protein expression construct was constructed, the design of which included: adding a VNP6 tag coding sequence at the N-terminus to regulate the concentration of Escherichia coli intracellular vesicles and alleviate molecular crowding; for the Sortase A7plus coding sequence, sequence optimization was performed to ensure efficient expression and correct folding; optional purification tags (such as His6, His10 or StrepII tags) were added to facilitate subsequent protein purification and functional testing.
[0011] In the present invention, the amino acid sequence of VNP6 is shown in SEQ ID NO:3.
[0012] SEQ ID NO:3: MDVFKKGFSIADEGVVGAVEKTDQGVTEAAEKTKEGV.
[0013] VNP6 peptide sequences are widely used to enhance the expression efficiency and secretion levels of recombinant proteins, primarily by promoting extracellular vesicle formation. In eukaryotic cells, the VNP6 tag can modulate cell signaling and vesicle secretion mechanisms, enhancing the extracellular distribution and stability of proteins by activating the exocytic pathway. However, existing research strategies primarily focus on how VNP6 activates cellular protein secretion mechanisms, and the research subjects are limited to cell systems with complex secretion regulatory capabilities.
[0014] In the present invention, the amino acid sequence of Sortase A7plus is shown in SEQ ID NO:4.
[0015] In a second aspect, the present invention provides a nucleic acid molecule encoding the VNP6-Sortase A7plus fusion protein described in the first aspect.
[0016] Preferably, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO: 2.
[0017] In a third aspect, the present invention provides an expression vector comprising the nucleic acid molecule described in the second aspect.
[0018] In a fourth aspect, the present invention provides a recombinant cell, comprising at least one copy of the expression vector described in the third aspect, or the nucleic acid molecule described in the second aspect is integrated into the genome of the recombinant cell.
[0019] Preferably, the recombinant cell is Escherichia coli.
[0020] In a fifth aspect, the present invention provides a method for preparing VNP6-Sortase A7plus fusion protein, the method comprising: culturing the recombinant cells described in the fourth aspect, inducing expression with IPTG, collecting the induced cells, and purifying the protein using Ni-NTA affinity chromatography to obtain the VNP6-Sortase A7plus fusion protein.
[0021] Preferably, the IPTG-induced expression is carried out at a low temperature of 10-20°C, for example, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C or 20°C.
[0022] When the present invention is expressed in Escherichia coli, low temperature culture conditions (e.g., 10-20°C, preferably 15-16°C) are used to promote the concentration of intracellular vesicles without significantly inducing extracellular vesicle secretion, thereby reducing the crowding effect between protein molecules and improving the protein folding environment.
[0023] In the present invention, if the temperature for inducing expression is too high (over 25°C), the VNP6 tag will promote the secretion of the protein outside the cell, causing most of the fusion protein to be secreted into the culture medium rather than retained in the intracellular vesicle structure, which reduces the intracellular concentration effect of the target protein. When the temperature is too low (below 10°C), the efficiency of the protein synthesis machinery of E. coli decreases significantly, the activity of the ribosome is limited, and the transcription and translation processes become extremely slow, resulting in a serious lack of target protein expression.
[0024] Preferably, the conditions for culturing the recombinant cells are as follows: culturing the recombinant Escherichia coli cell monoclonal colony into which the recombinant vector has been introduced in LB liquid culture medium at a culture temperature of 35-40°C (for example, 35°C, 36°C, 37°C, 38°C, 39°C or 40°C, etc.), and waiting for the bacterial solution OD to reach 0. 600 When the pH value is 0.6-0.8 (for example, it can be 0.6, 0.7 or 0.8), IPTG is added to induce expression.
[0025] Preferably, the conditions for inducing expression are: a final IPTG concentration of 0.4-0.5 mM (for example, 0.4 mM or 0.5 mM, etc.), and inducing expression for 16-18 h (for example, 16 h, 17 h or 18 h, etc.).
[0026] In the sixth aspect, the present invention provides the use of the VNP6-Sortase A7plus fusion protein described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, the recombinant cell described in the fourth aspect, or the method for preparing the VNP6-Sortase A7plus fusion protein described in the fifth aspect in the preparation of biological drugs.
[0027] The VNP6-Sortase A7plus fusion protein of the present invention has broad application prospects in the field of biopharmaceuticals, especially for the preparation of antibody-drug conjugates (ADCs) and site-specific modification of therapeutic proteins. This fusion protein can be used for protein labeling and tracing, structural modification, and functional research in protein engineering, significantly improving the efficiency of related technologies. In addition, this technology also provides a more efficient enzyme source for industrial biocatalysis and provides new ideas for optimizing the production of difficult-to-express proteins. It also shows practical value in the development of biological detection and diagnostic reagents.
[0028] The numerical range described in the present invention includes not only the point values listed above, but also any point values between the above numerical ranges that are not listed. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values included in the range.
[0029] Compared with the prior art, the present invention has the following beneficial effects:
[0030] (1) When SRT7+ is expressed in Escherichia coli, VNP6 induces intracellular vesicle concentration, effectively alleviating the molecular crowding phenomenon caused by high-density proteins, thereby reducing the risk of protein misfolding and aggregation degradation.
[0031] (2) The improved expression system can significantly increase the expression yield of recombinant protein under low temperature culture conditions (from Figure 1 It can be seen that the expression of VNP6-SRT7+ is 10 to 20 times higher than that of traditional expression systems, and the enzymatic activity of VNP6-SRT7+ is kept intact. BRIEF DESCRIPTION OF THE DRAWINGS
[0032] Figure 1 This is the effect of VNP6 on intracellular SRT7+ expression. In the figure, VNP6-SRT7+ refers to VNP6-SortaseA7plus-His6; SRT7+ refers to Sortase A7plus-His6.
[0033] Figure 2 This is VNP6-SRT7+IMAC purification. In the figure, VNP6-SRT7+ refers to VNP6-Sortase A7 plus-His6.
[0034] Figure 3 This is a fluorescent gel image of VNP6-SRT7+-mediated small molecule conjugation. In the image, VNP6-SRT7+ refers to VNP6-Sortase A7plus-His6.
[0035] Figure 4 This is a Coomassie Brilliant Blue-stained SDS-PAGE of VNP6-SRT7+-mediated protein coupling. In the figure, VNP6-SRT7+ refers to VNP6-Sortase A7plus-His6. DETAILED DESCRIPTION
[0036] The technical solution of the present invention is further described below by way of specific embodiments. It should be understood by those skilled in the art that the embodiments are merely to help understand the present invention and should not be regarded as specific limitations of the present invention.
[0037] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.
[0038] Example 1
[0039] 1. Build the design
[0040] Gene recombination technology was used to prepare an SRT7+ fusion protein expression vector containing a VNP6 tag. The coding sequence of this construct was designed as follows:
[0041] (1) VNP6 tag coding sequence: Selecting an effective VNP6 sequence that promotes the concentration of intracellular vesicles helps reduce the effect of intracellular molecular crowding during protein expression.
[0042] (2) Sortase A7plus coding sequence: The coding sequence of the optimized and modified SRT7+ gene ensures high expression and correct protein folding.
[0043] (3) His6 purification tag coding sequence: used for subsequent protein purification using Ni-NTA affinity chromatography.
[0044] The final arrangement order of the recombinant protein coding sequence is: N-terminal-VNP6-Sortase A7plus-His6 tag.
[0045] The amino acid sequence of the VNP6-SRT7+ exemplary sequence is shown below, SEQ ID NO:1.
[0046] MDVFKKGFSIADEGVVGAVEKTDQGVTEAAEKTKEGVMSGGGSGDYDIPTTENLYFQSIGGGSQAKPQIPKDKSKVAGYIEIPDADIKEPVYPGPATREQLNRGVSFAKENQSLDDQNISIAGHTFIGRPNYQFTNLKAAKKGSMVYFKVGNETRKYKMTSIRNVKPTAVEVLDEQKGKDKQLTLITCDDLNRETGVWETRKIFVATEVKLEHHHHHH.
[0047] The nucleotide sequence of the VNP6-SRT7+ exemplary sequence, SEQ ID NO:2:
[0048] ATGGATGTATTTAAAAAGGGATTCTCAATAGCGGATGAAGGCGTTGTTGGCGCTGTGGAAAAGACCGATCAAGGCGTAACCGAGGCTGCTGAAAAGACCAAAGAGGGCGTGATGAGCGGTGGAGGCTCCGGTCAGGCGAAGCCGCAGATTCCGAAGGACAAGTCGAAAGTGGCGGGTTATATTGAAATTCCAGATGCGGACATCAAGGAGCCGGTTTACCCGGGTCCGGCAACCCGTGAGCAGTTGAATAGAGGCGTCAGCTTCGCCAAGGAGAACCAAAGCCTGGACGACCAAAACATCAGCATCGCCGGTCATACCTTCATCGGCCGTCCGAATTATCAGTTTACCAACCTGAAAGCAGCGAAGAAAGGTTCCATGGTTTACTTCAAAGTTGGTAATGAAACGCGTAAATACAAAATGACCTCTATCCGTAACGTGAAACCGACTGCGGTGGAGGTCCTGGATGAGCAAAAAGGCAAGGACAAACAGTTGACCCTGATTACCTGCGATGACCTCAACCGCGAAACGGGTGTGTGGGAAACGCGCAAGATCTTTGTTGCGACCGAGGTTAAACTGGAACATCACCACCACCATCACTAA.
[0049] The amino acid sequence of VNP6, SEQ ID NO: 3:
[0050] MDVFKKGFSIADEGVVGAVEKTDQGVTEAAEKTKEGV.
[0051] The amino acid sequence of Sortase A7plus, SEQ ID NO: 4:
[0052] MSGGGSGDYDIPTTENLYFQSIGGGSQAKPQIPKDKSKVAGYIEIPDADIKEPVYPGPATREQLNRGVSFAKENQSLDDQNISIAGHTFIGRPNYQFTNLKAAKKGSMVYFKVGNETRKYKMTSIRNVKPTAVEVLDEQKGKDKQLTLITCDDLNRETGVWETRKIFVATEVKLE.
[0053] The sequence obtained by connecting the amino acid sequence of VNP6 and the amino acid sequence of Sortase A7plus is SEQ ID NO: 5:
[0054] MDVFKKGFSIADEGVVGAVEKTDQGVTEAAEKTKEGVMSGGGSGDYDIPTTENLYFQSIGGGSQAKPQIPKDKSKVAGYIEIPDADIKEPVYPGPATREQLNRGVS FAKENQSLDDQNISIAGHTFIGRPNYQFTNLKAAKKGSMVYFKVGNETRKYKMTSIRNVKPTAVEVLDEQKGKDKQLTLITCDDLNRETGVWETRKIFVATEVKLE.
[0055] The steps for constructing the expression vector are as follows:
[0056] In this example, a VNP6-Sortase A7plus-His6 protein expression construct was constructed, which included, from the 5' end to the 3' end: a VNP6 tag coding sequence, a Sortase A7plus protein coding sequence, and a His6 tag coding sequence.
[0057] 2. Protein expression and purification
[0058] (1) Transform the construct into the modified Escherichia coli strain, pick a single clone and place it in LB liquid medium, culture at 37°C, and wait until the OD 600 When the pH value was 0.8, IPTG was added to a final concentration of 0.4 mM and the expression was induced at 16°C for 16 h.
[0059] (2) After induction, the cells were collected and the cell pellet was resuspended in lysis buffer (lysis buffer composition: 50 mM Na HEPES, 0.1 M NaCl, 1 mM EDTA, 5% glycerol, 5 mM β-mercaptoethanol, 0.1% TritonX-100, pH 7.5). The cell suspension was sonicated using an ultrasonic processor at 40% amplitude for 6 cycles (on 5 seconds / off 30 seconds). The crude cell lysate was centrifuged at 12000g for 20 minutes at 4°C. The supernatant was then filtered through a filter. The recombinant protein expression level was detected by SDS-PAGE ( Figure 1 ).
[0060] from Figure 1 It can be seen that the expression level of the recombinant protein VNP6-Sortase A7plus-His6 in the cells was significantly increased. Under the same culture conditions, the recombinant protein expression level of the Sortase A7plus-His6 construct control group was significantly lower than that of the group with the VNP6 tag added, further verifying the effect of the VNP6 tag in the method of the present invention on the concentration of intracellular vesicles and the enhancement of protein expression.
[0061] (3) Protein purification was performed using Ni-NTA affinity chromatography by gradient elution.
[0062] The clarified lysate was incubated with pre-equilibrated Ni-NTA resin on a roller at 4°C for 8 hours. The resin was then transferred to a gravity column and washed five times with 10 column volumes (10CV) of buffer containing 30mM imidazole (buffer composition: 10mM Na2HPO4, 137mM NaCl, 5mM β-mercaptoethanol, pH 7.4). The target protein was then eluted with elution buffer containing 250mM, 350mM, 450mM, and 500mM imidazole (buffer composition: 10mM Na2HPO4, 137mM NaCl, 5mM β-mercaptoethanol, pH 7.4) for a total of 10 column volumes. All collected elution fractions were analyzed by SDS-PAGE.
[0063] The results are as follows Figure 2 As shown, from Figure 2 It can be seen that 30 mM imidazole was used to wash away proteins non-specifically bound to the Ni-NTA resin, and the target protein with high purity was successfully eluted by increasing the imidazole concentration from 250 mM (elution 1), 350 mM (elution 2), 450 mM (elution 3) to 500 mM (elution 4).
[0064] 3. Enzyme activity test
[0065] In order to verify whether the SRT7+ protein maintains the expected enzymatic activity after adding the VNP6 tag, the following detection system was established.
[0066] (1) Detect the protein cleavage and recombinant ligation functional activities of the protease using a reporter system containing an SRT7+ specific substrate (e.g., containing an LPXTG cleavage site).
[0067] In this example, in order to verify whether the enzymatic activity of the VNP6-Sortase A7plus-His6 protein is retained during the in vivo vesicle concentration process, a small molecule reporter system containing a Sortase A7plus-specific substrate, namely IL1Ra-LPETGGH and a peptide fluorescent probe (GGRSKK(FAM)-NH2), was constructed.
[0068] First, an enzymatic cleavage experiment was carried out using the purified VNP6-Sortase A7plus-His6 protein. In the reaction system, the VNP6-Sortase A7plus-His6 protein specifically recognized the LPXTG sequence in the reporter substrate, cut between T and G, and recognized the N-terminal G sequence in the reporter substrate for recombinant connection.
[0069] Figure 3 This is a fluorescent gel image of VNP6-SRT7+ mediated small molecule coupling. Figure 3 As can be seen, SDS-PAGE electrophoresis detection of the fluorescent reaction products revealed the expected cleavage fragments and recombinant ligation to form IL1Ra-LPETGGRSKK(FAM)-NH2. The overall test results indicate that the expressed VNP6-Sortase A7plus-His6 protein possesses the expected enzymatic cleavage and recombinant ligation functions.
[0070] (2) After in vitro protein cleavage and recombinant ligation functional reaction, the ligation product was analyzed by SDS-PAGE to confirm its cleavage specificity and ligation reaction efficiency.
[0071] In order to verify whether the enzymatic activity of VNP6-Sortase A7plus-His6 protein is retained during the vesicle concentration process in vivo, this example constructed a macromolecular reporter system containing a Sortase A7plus specific substrate, namely IL1Ra-LPETGGH and a protein probe (GGG-mNG3A). First, an enzymatic cleavage experiment was carried out using the purified VNP6-Sortase A7plus-His6 protein. In the reaction system, the Sortase A7plus protein specifically recognized the LPXTG sequence in the reporter substrate and cut between T and G, and recognized the N-terminal G sequence in the reporter substrate for recombination and connection. Figure 4As can be seen, SDS-PAGE electrophoresis analysis of the reaction products revealed the expected cleavage fragments and recombinant ligation into IL1Ra-LPETGGG-mNG3A. Overall, the test results indicate that the expressed VNP6-Sortase A7plus-His6 protein possesses the expected enzymatic cleavage and recombinant ligation functions.
[0072] The experimental results showed that compared with the SRT7+ control group without VNP6 tag, the SRT7+ modified with VNP6 not only significantly alleviated the problem of intracellular molecular crowding, but also achieved high expression levels under low-temperature culture conditions and maintained good enzyme catalytic activity.
[0073] in, Figure 3 It was demonstrated that VNP6-SRT7+ could conjugate IL-1Ra-LPETGGH protein with GGRSKK(FAM)-NH2 peptide to generate IL1-Ra-LPETGGRSKK(FAM)-NH2 protein; Figure 4 It shows that it can interact with GGG-mNGA protein to generate IL1-Ra-LPETGGG-mNGA protein.
[0074] The present invention significantly optimizes the intracellular microenvironment of Escherichia coli by introducing a VNP6 tag at the N-terminus of SRT7+ and combining it with a low-temperature culture strategy, effectively reducing the crowding effect between protein molecules and promoting a more reasonable spatial distribution and correct folding of the SRT7+ protein.
[0075] In summary, the present invention proposes an innovative expression system that utilizes a VNP6 tag to regulate intracellular vesicle concentration to increase the expression yield and enzyme activity of SortaseA7plus. By modifying the VNP6 tag at the N-terminus of Sortase A7plus and supplementing it with a low-temperature culture strategy, this system effectively alleviates the protein misfolding and degradation problems caused by molecular crowding within Escherichia coli cells, thereby achieving efficient and stable recombinant protein production. While maintaining the function of the Sortase A7plus protein, this method significantly improves the expression efficiency, providing a new technical solution with practical production application value, suitable for the large-scale preparation of related proteins in the fields of biomedicine and industrial biotechnology.
[0076] The applicant declares that the above is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention fall within the scope of protection and disclosure of the present invention.
Claims
1. A VNP6-Sortase A7plus fusion protein, characterized in that The fusion protein includes a VNP6 sequence and a Sortase A7plus sequence connected in sequence from the N-terminus to the C-terminus; the amino acid sequence after the VNP6 sequence and the Sortase A7plus sequence are connected is shown in SEQ ID NO:
5.
2. The VNP6-Sortase A7plus fusion protein according to claim 1, characterized in that The fusion protein is also connected to a His6 tag sequence; the amino acid sequence of the fusion protein is shown in SEQ ID NO:
1.
3. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the VNP6-SortaseA7plus fusion protein according to claim 1 or 2; Preferably, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:
2.
4. An expression vector, characterized in that The expression vector contains the nucleic acid molecule according to claim 3.
5. A recombinant cell, characterized in that The recombinant cell contains at least one copy of the expression vector according to claim 4, or the nucleic acid molecule according to claim 3 is integrated into the genome of the recombinant cell.
6. A method for preparing VNP6-Sortase A7plus fusion protein, characterized in that: The method comprises: culturing the recombinant cell according to claim 5, inducing expression with IPTG, collecting the induced cells, and purifying the protein using Ni-NTA affinity chromatography to obtain the VNP6-Sortase A7plus fusion protein.
7. The method for preparing VNP6-Sortase A7plus fusion protein according to claim 6, characterized in that: The IPTG-induced expression is carried out at a low temperature of 10-20°C.
8. The method for preparing VNP6-Sortase A7plus fusion protein according to claim 6 or 7, characterized in that: The conditions for culturing the recombinant cells are as follows: culturing the recombinant E. coli monoclonal colonies into which the recombinant vector has been transferred in LB liquid culture medium at a culture temperature of 35-40° C. and waiting for the bacterial solution OD 600 When the pH value is 0.6-0.8, IPTG is added to induce expression.
9. The method for preparing VNP6-Sortase A7plus fusion protein according to any one of claims 6 to 8, characterized in that: The induction expression conditions are: the final IPTG concentration is 0.4-0.5 mM, and the induction expression is 16-18 hours.
10. Use of the VNP6-Sortase A7plus fusion protein according to claim 1 or 2, the nucleic acid molecule according to claim 3, the expression vector according to claim 4, the recombinant cell according to claim 5, or the method for preparing the VNP6-Sortase A7plus fusion protein according to any one of claims 6 to 9 in the preparation of biopharmaceuticals.