Snake venom toxin signal peptide for improving secretion expression of recombinant antibody, product and application thereof

Abstract

The application discloses a kind of recombinant antibody secretion expression quantity improving snake venom toxin signal peptide and its product and application, belong to genetic engineering antibody technical field, the signal peptide involved in the application includes snake venom toxin signal peptide and the new signal peptide obtained by mutation thereof, by PCR, the original signal peptide carried by recombinant antibody itself is replaced with the signal peptide described in the application, compared with the original signal peptide of recombinant antibody, the signal peptide described in the application guides more efficient secretion of recombinant antibody;Compared with the signal peptide of antibody itself and other signal peptides commonly used for antibody secretion expression, the expression level of antibody containing the signal peptide of the application is significantly improved, the production cost of target antibody is reduced, and it has great application prospect in the biomedical field with great demand for antibody.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering antibody technology, specifically relating to a class of snake venom signal peptides that enhance the secretion and expression of recombinant antibodies, their products, and applications. Background Technology

[0002] Recombinant antibodies, also known as genetically engineered antibodies, are antibodies obtained by modifying and recombining the corresponding gene sequences of antibodies using DNA recombination technology, constructing them on plasmids, and then transfecting / transforming the constructed plasmids into suitable host cells for expression using exogenous protein expression technology. Recombinant antibodies achieve antibody humanization, effectively solving the problem of rejection reactions in humans caused by animal-derived antibodies. They are now widely used in biomedical research, as well as disease diagnosis and treatment, and the demand is high. Recombinant antibodies can be successfully expressed in various systems, among which mammalian cells are currently the most mature and widely used expression system. Mammalian cell expression systems can be used for the expression of various recombinant antibodies; therefore, the production of therapeutic monoclonal antibodies currently primarily uses mammalian cell expression systems. However, these systems also have disadvantages such as relatively low expression yields and high production costs. Therefore, optimizing antibody expression to increase antibody expression levels and reduce production costs plays a crucial role in meeting the ever-increasing demand for antibodies in the future.

[0003] In protein (such as antibody) production, expression levels are influenced by many factors, with efficient secretion being crucial. Most secretory proteins are expressed via a signal peptide that initiates the classic endoplasmic reticulum-Golgi pathway. The signal peptide is a unique amino acid sequence at the N-terminus of a secretory protein, consisting of approximately 30 amino acids. It significantly impacts the transmembrane secretion efficiency of the protein, guiding the precursor peptide into the rough endoplasmic reticulum and facilitating its secretion extracellularly. It can localize the protein to the cell membrane or allow it to cross the membrane and reach the extracellular space. Signal peptide sequences generally do not exhibit significant amino acid sequence conservation and typically consist of a positively charged N-domain, a hydrophobic amino acid-rich H-domain, and a negatively charged C-domain. It is evident that signal peptides play a crucial role in the secretion of recombinant antibodies. In actual antibody production, problems such as the constructed vector's antibody protein not being secreted or secreting at low levels are frequently encountered. When the recombinant antibody's gene sequence carries its own signal peptide sequence for secretion, if the reconstructed expression vector cannot or inefficiently utilizes the antibody's own signal peptide sequence to obtain high expression and secretion of the target antibody, the signal peptide for high-expression protein can be re-screened to replace the original signal peptide, or the original signal peptide can be optimized. By improving the secretion efficiency of the recombinant antibody, the expression level of the target antibody can be increased. When the recombinant antibody does not carry its own signal peptide, a suitable signal peptide sequence can be added to guide antibody secretion and thus increase the expression level of the target antibody.

[0004] Current reports indicate that replacing the signal peptide can significantly increase protein secretion expression levels. Therefore, adding or replacing a suitable signal peptide with the gene for the target antibody is an effective method to increase the secretion yield of the target protein. This not only improves secretion efficiency and simplifies downstream purification processes but also has a positive effect on antibody stability and activity. Therefore, the screening and optimization of signal peptides are of great significance for improving antibody secretion expression levels and reducing production costs.

[0005] Currently, there are no reports on snake venom signal peptides increasing the secretion and expression levels of recombinant antibodies or recombinant proteins. Summary of the Invention

[0006] The purpose of this invention is to provide a class of snake venom signal peptides that enhance the secretion and expression of recombinant antibodies, as well as their products and applications.

[0007] To achieve the above objectives, the present invention employs the following technical solution:

[0008] This invention discloses the application of snake venom signal peptides in increasing the secretory expression of recombinant antibodies. The snake venom signal peptides include snake venom signal peptides and their mutant signal peptides, which can guide the secretory expression of recombinant antibodies.

[0009] Preferably, the snake venom signal peptide is a silver ring snake venom signal peptide, the amino acid sequence of which is shown in SEQ ID NO.2, the nucleotide sequence encoding which is shown in SEQ ID NO.6, and other nucleotide sequences that can encode which are also included.

[0010] More preferably, the mutant signal peptide based on the banded krait venom signal peptide is obtained by mutating the 3rd and 13th amino acids of the banded krait venom signal peptide, and its amino acid sequence is shown in SEQ ID NO.1.

[0011] Furthermore, it is obtained by mutating both the third amino acid T and the thirteenth amino acid I of the banded krait venom signal peptide to amino acid L. The nucleotide sequence encoding it is shown in SEQ ID NO.5, and also includes other nucleotide sequences that can encode it.

[0012] Preferably, the snake venom signal peptide is used to replace the original signal peptide of the antibody itself in order to increase the secretory expression level of the target antibody.

[0013] More preferably, the application can be when the recombinant antibody is not secreted or secreted at a low level, that is, when it cannot utilize or can not utilize its own natural signal peptide to obtain a high expression of the target antibody, by replacing the original signal peptide of the antibody itself with the signal peptide of the present invention to increase the secretion expression of the target antibody.

[0014] Preferably, the snake venom signal peptide is added to a recombinant antibody that does not contain a signal peptide to guide antibody secretion and increase the secretion expression of the target antibody.

[0015] Preferably, the recombinant antibody that does not contain a signal peptide is prepared using degenerate primers.

[0016] This invention also discloses a type of snake venom signal peptide that enhances the secretion and expression of recombinant antibodies, obtained by mutation of the silver ring snake venom signal peptide, the amino acid sequence of which is shown in SEQ ID NO.1.

[0017] Preferably, the recombinant antibody is a monoclonal antibody, a bispecific antibody, a single-chain antibody, a chimeric antibody, Fab, Fv, scFv, SdAb, a microantibody, or a nanoantibody.

[0018] The present invention also discloses a recombinant expression vector comprising a nucleic acid molecule encoding the snake venom signal peptide that enhances the secretion and expression of the recombinant antibody. In construction, the nucleic acid molecule is linked to the front end of a polynucleotide encoding the target antibody and then linked to the expression vector.

[0019] The expression vectors include pcDNA3.1, pIRES, pTT3, pCEP4, and pATX1.

[0020] The present invention also provides a method for constructing the above-mentioned recombinant expression vector, comprising the following steps:

[0021] 1) Synthesize the nucleotide sequence of the antibody to be expressed onto the vector, and design primers for the signal peptide and the nucleotide sequence of the antibody to be expressed according to the present invention;

[0022] 2) Using the recombinant vector containing the antibody to be expressed as a template, PCR amplification was performed using the corresponding primers;

[0023] 3) Demethylate the PCR product using Dpn1 restriction enzyme to remove the original template plasmid;

[0024] 4) The PCR product was subjected to agarose gel electrophoresis, and the band was successfully amplified; the gel was cut and the product was recovered; the recovered product was treated with T4 polynucleotide kinase; and then T4 ligase was added to obtain a recombinant expression vector containing the signal peptide described in this invention linked to the antibody to be expressed.

[0025] Preferably, the PCR amplification system is 50 μL, comprising: 10 ng template, 2 μL primer F, 2 μL primer R, 1 μL Fast Pfu DNA Polymerase, 25 μL 2× Fast Pfu buffer, 4 μL 2.5 mM dNTP, and the remainder being nuclease-free water;

[0026] Preferably, the PCR amplification program is as follows: 98℃ pre-denaturation for 5 min; 98℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 4 min 30 s, denaturation to extension for 25 cycles; 72℃ extension for 5 min; storage at 4℃.

[0027] The present invention also discloses a host cell comprising the above-mentioned recombinant expression vector, wherein the host cell includes HEK293, CHO, BHK and SP2 / 0.

[0028] The present invention also provides a method for producing recombinant antibodies, comprising the following steps: culturing host cells, transfecting the above-mentioned recombinant expression vector into the cells, and then isolating the recombinant antibody from the culture.

[0029] The cells may be HEK293 (human embryonic kidney cells), CHO (Chinese hamster ovary cells), BHK (juvenile hamster kidney cells), SP2 / 0 (mouse myeloma cells), etc.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] This invention discloses the application of a snake venom signal peptide to enhance the secretory expression of recombinant antibodies. The signal peptides involved include a snake venom signal peptide and a novel signal peptide obtained by mutating it. By using PCR, the original signal peptide carried by the recombinant antibody is replaced with the signal peptide described in this invention. Compared to the original signal peptide of the recombinant antibody, the signal peptide described in this invention guides the recombinant antibody to secrete more efficiently. Compared with the antibody's own signal peptide and other signal peptides commonly used for antibody secretory expression, the antibody expression level containing the signal peptide of this invention is significantly improved, reducing the production cost of the target antibody. This has great application potential in the biomedical field, where antibody demand is high. Attached Figure Description

[0032] Figure 1 To detect the secretory expression levels of recombinant antibodies 1A9, TIML-1A9, abgtx-1A9, HSA-1A9, and IL2-1A9 using ELISA;

[0033] Figure 2 Immunofluorescence staining results of recombinant antibodies 1A9, TIML-1A9, and abgtx-1A9 on CHI3L1-overexpressing cell smears and control pcDNA3.1 cell smears;

[0034] Figure 3 The images show the results of Western blotting verification of the secretion expression levels of recombinant antibodies 1A9, TIML-1A9, abgtx-1A9, HSA-1A9, and IL2-1A9 in this invention; where (a) is the result of the gapdh internal control and (b) is the result of the target protein.

[0035] Figure 4 The secretory expression levels of recombinant antibodies 6D9, TIML-6D9, abgtx-6D9, HSA-6D9, and IL2-6D9 were detected by ELISA.

[0036] Figure 5 Immunofluorescence staining results of recombinant antibodies 6D9, TIML-6D9, and abgtx-6D9 on CHI3L1-overexpressing cell smears and control pcDNA3.1 cell smears;

[0037] Figure 6 The following graphs show the results of Western blotting verification of the secretion expression levels of recombinant antibodies 6D9, TIML-6D9, abgtx-6D9, HSA-6D9, and IL2-6D9 in this invention; where (a) is the result of the gapdh internal control and (b) is the result of the target protein.

[0038] Figure 7 The secretory expression levels of recombinant antibodies 11D7, TIML-11D7, abgtx-11D7, mmp9-11D7, pelb-11D7 and mog-11D7 were detected by ELISA. Detailed Implementation

[0039] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0040] It should be noted that the terms "first," "second," etc., in the specification, claims, and accompanying drawings of this invention are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that such data can be interchanged where appropriate so that the embodiments of the invention described herein can be implemented in orders other than those illustrated or described herein. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover a non-exclusive inclusion; for example, a process, method, system, product, or apparatus that comprises a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to such processes, methods, products, or apparatus.

[0041] The present invention will now be described in further detail with reference to the accompanying drawings:

[0042] This invention provides signal peptides for recombinant antibody expression, including the krait venom mutant signal peptide TIML (hereinafter referred to as signal peptide TIML) and the krait venom signal peptide abgtx (hereinafter referred to as signal peptide abgtx). By replacing the signal peptides carried by the antibody itself with the signal peptides TIML and abgtx of this invention, as well as two other natural signal peptides commonly used to guide antibody secretion expression, recombinant antibodies containing different signal peptides are obtained. The secretion expression levels of antibodies with different signal peptides are verified by ELISA, WB, and CBA, respectively.

[0043] The two natural signal peptides commonly used to guide antibody secretion expression are the signal peptide of human serum albumin (HSA) and the signal peptide of recombinant human interleukin-2 (IL2).

[0044] The amino acid sequences of the above four signal peptides are as follows:

[0045] TIML: MKLLLLLTLVVVTLVCLDLGYT(SEQ ID NO.1)

[0046] abgtx:MKTLLLLTLVVVTIVCLDLGYT(SEQ ID NO.2)

[0047] HSA: MKWVTFISLLFLFSSAYS(SEQ ID NO.3)

[0048] IL2:MYRMQLLSCIALSLALVTNS(SEQ ID NO.4)

[0049] The nucleotide sequences encoding the above four signal peptides are as follows:

[0050] TIML:

[0051] ATGAAACTTCTGCTGCTGACCTTGGTGGTGGTGACACTCGTGTGCCT GGACTTAGGATATACC(SEQID NO.5)

[0052] Abgtx:

[0053] ATGAAAACTCTGCTGCTGACCTTGGGTGGTGGTGACAATCGTGTGCCT GGACTTAGGATATACC(SEQID NO.6)

[0054] HSA:

[0055] ATGAAGTGGGTAACCTTCATTTCCCTACTATTCCTATTCAGCTCGGCTT ATTCC(SEQ ID NO.7)

[0056] IL2:

[0057] ATGTACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTGCACTT GTCACGAATTCG(SEQ IDNO.8)

[0058] Example 1: Comparison of antibody 1A9 secretion expression levels

[0059] I. Construction of Recombinant Expression Vectors

[0060] 1. The mouse-derived CHI3L1 monoclonal antibody 1A9 prepared in our laboratory has the heavy chain sequence of SEQ ID NO.9 and the light chain sequence of SEQ ID NO.10. The heavy chain of antibody 1A9 was synthesized into the pcDNA3.1 vector and designated as pcDNA3.1-1A9-m2a. The light chain of antibody 1A9 was synthesized into the pcDNA3.1 vector and designated as pcDNA3.1-1A9-mk. The constructed recombinant vectors pcDNA3.1-1A9-m2a and pcDNA3.1-1A9-mk were subjected to plasmid extraction and reserved for later use. They were named 1A9-m2a and 1A9-mk, respectively.

[0061] 2. Design primers as shown in Table 1. Replace the signal peptide of the heavy chain of monoclonal antibody 1A9 (as shown in SEQ ID NO. 15) with the four signal peptides (TIML, abgtx, HSA, IL2) mentioned above using PCR. Name the resulting recombinant plasmids TIML-1A9-m2a, abgtx-1A9-m2a, HSA-1A9-m2a, and IL2-1A9-m2a, respectively. The specific operation steps are as follows:

[0062] (1) Using the recombinant vector pcDNA3.1-1A9-m2a as a template, PCR was performed using the four primer pairs in Table 1 as primers. The PCR amplification system (50 μl) consisted of: 10 ng template, 2 μl upstream primer (0.2 μM), 2 μl downstream primer (0.2 μM), 1 μl FastPfuDNA Polymerase (2.5 units), 25 μl 2×FastPfu buffer, 4 μl 2.5 mM dNTPs, and the remainder Nuclease-free Water. The PCR amplification program was as follows: 98℃ pre-denaturation for 5 min; 98℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 4 min 30 s, denaturation to extension for 25 cycles, 72℃ extension for another 5 min, and storage at 4℃.

[0063] (2) The PCR product was demethylated with DpnI restriction enzyme and treated at 37℃ for 1 h to remove the original template plasmid.

[0064] (3) The PCR product was subjected to agarose gel electrophoresis, and the bands were successfully amplified; the gel was cut and recovered; about 500-1000 μg of the recovered product was added to 1 μl of T4 polynucleotide kinase (500 units), 2 μl of T4 ligase buffer and 20 μL of Nuclease-free Water, and treated at 37°C for at least 2 h; then T4 ligase was added and treated at room temperature for at least 2 h to obtain 4 recombinant vectors, namely pcDNA3.1-TIML-1A9-m2a, pcDNA3.1-abgtx-1A9-m2a, pcDNA3.1-HSA-1A9-m2a and pcDNA3.1-IL2-1A9-m2a.

[0065] (4) Perform plasmid extraction on the four recombinant vectors in step (3) above, and set them aside for later use.

[0066] Table 1

[0067]

[0068]

[0069] 2. Design primers as shown in Table 2. Replace the signal peptide of the light chain of antibody 1A9 (as shown in SEQ ID NO. 16) with the four signal peptides mentioned above (TIML, abgtx, HSA, IL2), and name the resulting recombinant plasmids TIML-1A9-mk, abgtx-1A9-mk, HSA-1A9-mk, and IL2-1A9-mk, respectively. The specific operation steps are as follows: Use the recombinant vector pcDNA3.1-1A9-mk as a template, and use the four primer pairs in Table 2 as primers for PCR. Except for the different template and primer sequences, the system, program, and operation steps are the same as step 1 of this embodiment.

[0070] Table 2

[0071]

[0072]

[0073] II. ELISA verification of recombinant antibody secretion expression level

[0074] Following the PEI transfection method (transfection reagents were purchased from Thermo Biotech, and transfection was performed according to the instructions), two groups of 1A9 plasmids with the same signal peptide were co-transfected into HEK293 cells. The specific steps are as follows:

[0075] (1) Transfection was performed according to the PEI instructions. 1.5 μg each of plasmids 1A9-m2a and 1A9-mk were co-transfected into HEK293 cells grown on cell smears in a 6-well plate. The cells were cultured in a 37°C, 5% CO2 cell culture incubator. After 48 hours, the culture medium was collected. The collected culture medium was centrifuged (4500 r, 5 min), the precipitate was discarded, and the supernatant was collected and labeled as 1A9. The supernatant collected from HEK293 cells transfected with 3 μg pcDNA3.1 plasmid was used as a blank control and labeled as pcDNA3.1.

[0076] Using the method described above, the heavy and light chain plasmids of the same signal peptide after the signal peptide was replaced were co-transformed sequentially, and the supernatant was collected. Among them, TIML-1A9-m2a and TIML-1A9-mk were co-transformed as TIML-1A9, abgtx-1A9-m2a and abgtx-1A9-mk were co-transformed as abgtx-1A9, HSA-1A9-m2a and HSA-1A9-mk were co-transformed as HSA-1A9, and IL2-1A9-m2a and IL2-1A9-mk were co-transformed as IL2-1A9.

[0077] (2) Collected supernatant 1A9, TIML-1A9, abgtx-1A9, HSA-1A9, IL2-1A9 and pcDNA3.1 were serially diluted threefold to eight dilutions (1:3, 1:9, 1:27, 1:81, 1:243, 1:729, 1:2187 and 1:6561, respectively), and added to ELISA plates coated with CHI3L1 protein, 100 μl per well, and incubated at 37°C for 1 h; washed 3 times with PBST, and patted dry each time; then incubated with HRP-labeled secondary antibody at 37°C for 30 min, washed 3 times with PBST, and patted dry each time; added TMB for color development for 10 min, stopped with 2M H2SO4, and the absorbance was measured at 450 nm. Each dilution was replicated in four wells.

[0078] The secretory expression levels of recombinant antibodies 1A9, TIML-1A9, abgtx-1A9, HSA-1A9, and IL2-1A9 were detected using ELISA. The average value of four replicate wells was taken. The results are shown in Table 3. Figure 1 As shown.

[0079] Table 3

[0080]

[0081] From Table 3 and Figure 1 It can be seen that, under the same conditions, the secretion expression levels of signal peptides TIML, abgtx, HSA, and IL2 are all increased compared with the original antibody signal peptides. Among them, the signal peptides TIML and abgtx described in this invention are more efficient than other signal peptides HSA and IL2 commonly used for antibody secretion expression.

[0082] III. CBA Validation of Recombinant Antibody Secretion Expression Level

[0083] 1. Preparation of cell spreads: The CHI3L1 gene was synthesized into the pcDNA3.1 vector. The large-scale plasmid and pcDNA3.1 plasmid were transfected into HEK293 cells grown on cell spreads according to the PEI transfection method (transfection reagent was purchased from Thermo Biotech, and transfection was performed according to the instructions). After 48 hours, the cells were washed twice with PBS, fixed with acetone for 30 minutes, washed twice with PBS, and dried to obtain cell spreads overexpressing CHI3L1 and control pcDNA3.1 cell spreads for later use.

[0084] 2. Immunofluorescence staining: Take 1 mL each of the supernatant 1A9, TIML-1A9, and abgtx-1A9 collected in step 2 of this embodiment, add 2 μL of Tween-20, mix well, and incubate separately on the cell smears overexpressing CHI3L1 from step 1 and the control pcDNA3.1 cell smears. Incubate at room temperature for 1 h, wash 3 times with PBST, 5 min each time; then add FITC-labeled secondary antibody (mouse), incubate at room temperature for 30 min, wash 3 times with PBST, 5 min each time; observe the results under a microscope and take pictures. The results are as follows. Figure 2 As shown.

[0085] Depend on Figure 2 It can be seen that after the original signal peptides of the light and heavy chains of the 1A9 antibody were replaced with the signal peptides TIML and abgtx described in this invention, the secretion expression levels were significantly increased, which is consistent with the ELISA results.

[0086] IV. Western blot verification of recombinant antibody secretion expression level

[0087] 1. Sample preparation:

[0088] (1) Transfection was performed according to the PEI instructions. 1.5 μg of each of 1A9-m2a and 1A9-mk plasmids were co-transfected into HEK293 cells cultured in 6-well plates and cultured in a 37℃, 5% CO2 cell culture incubator. The cells were labeled 1A9. HEK293 cells transfected with 3 μg of pcDNA3.1 plasmid were used as a blank control and labeled pcDNA3.1.

[0089] Using the method described above, the heavy and light chain plasmids of the same signal peptide after the signal peptide was replaced were co-transformed sequentially. Specifically, TIML-1A9-m2a and TIML-1A9-mk were co-transformed into TIML-1A9, abgtx-1A9-m2a and abgtx-1A9-mk were co-transformed into abgtx-1A9, HSA-1A9-m2a and HSA-1A9-mk were co-transformed into HSA-1A9, and IL2-1A9-m2a and IL2-1A9-mk were co-transformed into IL2-1A9.

[0090] (2) After 48 hours, the cell culture medium from step (1) was collected separately. After centrifuging the collected culture medium (4℃, 4500r, 5min), the precipitate was discarded and the supernatant was collected. 40μL of supernatant was taken, 10μL of 5×protein Loading was added, and the mixture was boiled at 90℃ for 10min. Supernatant samples of 1A9, TIML-1A9, abgtx-1A9, HSA-1A9, IL2-1A9 and pcDNA3.1 were obtained respectively.

[0091] (3) Wash the cells from step (1) twice with PBS, add 500 μL of RIPA cell lysis buffer, collect the cells, sonicate them, centrifuge (4℃, 9500 rpm, 20 min), discard the precipitate, and collect the supernatant; take 40 μL of supernatant, add 10 μL of 5× protein loading, and boil at 90℃ for 10 min; obtain 1A9, TIML-1A9, abgtx-1A9, HSA-1A9, IL2-1A9 and pcDNA3.1 cell samples respectively;

[0092] 3. After sample preparation, transfer to a PVDF membrane:

[0093] Prepare an SDS-PAGE gel. Load 10 μL of the boiled sample (loading order from left to right: pcDNA3.1 supernatant, pcDNA3.1 cells, IL2-1A9 supernatant, IL2-1A9 cells, HSA-1A9 supernatant, HSA-1A9 cells, abgtx-1A9 supernatant, abgtx-1A9 cells, TIML-1A9 supernatant, TIML-1A9 cells, 1A9 supernatant, 1A9 cells, Marker). Run the gel at 120V until the blue loading indicator reaches the bottom. Transfer to a PVDF membrane and block with 5% skim milk powder for 2 hours.

[0094] 2. Sample incubation:

[0095] (1) Take a membrane sheet and incubate it with the gapdh internal control antibody (rabbit-derived) for 2 hours, wash it three times with TBST, then add HRP-labeled anti-rabbit secondary antibody and incubate for 1 hour, wash it three times with TBST, and expose it to the light. The results are as follows: Figure 3 As shown in (a).

[0096] (2) Take a membrane sheet and incubate it directly with HRP-labeled anti-mouse secondary antibody for 1 hour, wash three times with TBST, and expose it to sunlight. The results are as follows: Figure 3 As shown in (b).

[0097] Depend on Figure 3 (a) and Figure 3 As shown in Figure (b), under the same cell mass conditions, the secretory function of the 1A9 antibody varied after changing different signal peptides, and the secretion level of the original 1A9 signal peptide was very low. Compared with the original signal peptide, TIML and abgtx showed slightly lower intracellular expression levels but significantly higher secretion levels. In contrast, HSA and IL2 showed significantly lower intracellular expression levels but slightly higher secretion levels compared with the original signal peptide, consistent with the ELISA results. Therefore, replacing the original antibody signal peptide with the signal peptides TIML and abgtx described in this invention can improve the antibody secretion expression level.

[0098] Example 2: Comparison of antibody 6D9 secretion expression levels

[0099] I. Construction of Recombinant Expression Vectors

[0100] 1. The mouse-derived CHI3L1 monoclonal antibody 6D9 prepared in our laboratory has the heavy chain sequence of SEQ ID NO.11 and the light chain sequence of SEQ ID NO.12. The heavy chain of antibody 6D9 was synthesized into the pcDNA3.1 vector and designated as pcDNA3.1-6D9-m2a, and the light chain of antibody 6D9 was synthesized into the pcDNA3.1 vector and designated as pcDNA3.1-6D9-mk. The constructed recombinant vectors pcDNA3.1-6D9-m2a and pcDNA3.1-6D9-mk were subjected to plasmid extraction and reserved for later use, and were named 6D9-m2a and 6D9-mk, respectively.

[0101] 2. Design primers as shown in Table 4. Replace the original signal peptide of the heavy chain of monoclonal antibody 6D9 (as shown in SEQ ID NO. 17) with the above four signal peptides (TIML, abgtx, HSA, IL2) by PCR. Name the obtained recombinant plasmids TIML-6D9-m2a, abgtx-6D9-m2a, HSA-6D9-m2a, and IL2-6D9-m2a, respectively. The specific operation steps are as follows: Use the recombinant vector pcDNA3.1-6D9-mk as a template and the four pairs of primer sequences in Table 4 as primers for PCR. Except for the different template and primer sequences, the system, program and operation steps are the same as in Example 1.

[0102] Table 4

[0103]

[0104]

[0105] The primers were designed as shown in Table 5. The signal peptide of the light chain of antibody 6D9 (as shown in SEQ ID NO. 18) was replaced with the four signal peptides (TIML, abgtx, HSA, IL2) mentioned above. The resulting recombinant plasmids were named TIML-6D9-mk, abgtx-6D9-mk, HSA-6D9-mk, and IL2-6D9-mk, respectively. The specific operation steps are as follows: Using the recombinant vector pcDNA3.1-6D9-mk as a template, PCR was performed using the four pairs of primers in Table 5. Except for the different template and primer sequences, the system, program, and operation steps were the same as in Example 1.

[0106] Table 5

[0107]

[0108]

[0109] II. ELISA verification of recombinant antibody secretion expression level

[0110] Following the PEI transfection method (transfection reagents purchased from Thermo Biotech, transfection performed according to the instructions), two groups of 6D9 plasmids with the same signal peptide were co-transfected into HEK293 cells. The specific operational steps were the same as in Example 1. The secretory expression levels of recombinant antibodies 6D9, TIML-6D9, abgtx-6D9, HSA-6D9, and IL2-6D9 were detected using ELISA. The results are shown in Table 6. Figure 4 As shown.

[0111] Table 6

[0112]

[0113] From Table 6 and Figure 4 It can be seen that, under the same conditions, the secretion expression levels of the signal peptides TIML and abgtx described in this invention are all increased compared with the original antibody signal peptide, and the antibody titer is increased by about 3 times. However, the titer of other signal peptides commonly used for antibody secretion expression, such as IL2, is slightly lower than that of the original antibody signal peptide, and the titer of HSA signal peptide is significantly lower than that of the original signal peptide.

[0114] III. CBA Validation of Recombinant Antibody Secretion Expression Level

[0115] Take 1 mL each of the supernatant collected in step two of this embodiment (6D9, TIML-6D9, and abgtx-6D9), add 2 μL of Tween-20, mix well, and incubate with the control pcDNA3.1 cell smears overexpressing CHI3L1 prepared in Example 1. Incubate at room temperature for 1 h, wash three times with PBST for 5 min each time; then add FITC-labeled secondary antibody (mouse), incubate at room temperature for 30 min, wash three times with PBST for 5 min each time; observe the results under a microscope and take pictures. The results are as follows. Figure 5 As shown.

[0116] Depend on Figure 5 It can be seen that after the original signal peptides of the light and heavy chains of the 6D9 antibody were replaced with the signal peptides TIML and abgtx described in the invention, the secretion expression levels were significantly increased, which is consistent with the ELISA results.

[0117] IV. Western blot verification of recombinant antibody secretion expression level

[0118] The experimental procedure was the same as in Example 2. The loading order from left to right was: pcDNA3.1 supernatant, pcDNA3.1 cells, HSA-6D9 supernatant, HSA-6D9 cells, IL2-6D9 supernatant, IL2-6D9 cells, abgtx-6D9 supernatant, abgtx-6D9 cells, TIML-6D9 supernatant, TIML-6D9 cells, 6D9 supernatant, 6D9 cells, and marker. The results are as follows: Figure 6 (a) and Figure 6 As shown in (b).

[0119] Depend on Figure 6 (a) and Figure 6 As shown in Figure (b), under the same cell quantity conditions, the secretory functions of different signal peptides of the 6D9 antibody are different. The intracellular expression levels of TIML and abgtx are not significantly different from those of the original signal peptide, but the secretion levels are significantly different. The intracellular expression levels of HSA and IL2 are significantly reduced, and the secretion levels are also significantly reduced, which is consistent with the results of ELISA.

[0120] Comparative Example 1: Comparison of secretion levels of other signal peptides

[0121] Three natural signal peptides, including pelb (pectate lyase B), mmp9 (matrix metalloproteinase 9), and mog (myelin oligodendrocyte glycoprotein), were randomly selected and their secretion expression levels were compared with those of the antibody's own original signal peptide.

[0122] The amino acid sequences of the three signal peptides mentioned above are as follows:

[0123] pelb:MKYLLPTAAAGLLLLAAQPAMA(SEQ ID NO.41)

[0124] mmp9:MSLWQPLVLVLLVLGCCFA(SEQ ID NO.42)

[0125] mog: MASLLSRPSLPSCLCSFLLLLLLQVSSSYA(SEQ ID NO.43)

[0126] The nucleotide sequences encoding the above three signal peptides are as follows:

[0127] pelb:

[0128] ATGAAATACCTGCTGCCGACCGCTGCTGCTGGTCTGCTGCTCCTCGCT GCCCAGCCGGCGATGGCC(SEQ ID NO.44)

[0129] mmp9:

[0130] ATGAGCCTCTGGCAGCCCCTGGTCCTGGTGCTCCTGGTGCTGGGCTG CTGCTTTGCT(SEQ IDNO.45)

[0131] mog:

[0132] ATGGCAAGCTTATCAAGACCCTCTCTGCCCAGCTGCCTCTGCTCCTTC CTCCTCCTCCTCCTCCTCCAAGTGTCTTCCAGCTATGCA (SEQ ID NO.46)

[0133] I. Construction of Recombinant Expression Vectors

[0134] 1. The mouse-derived ENO2 monoclonal antibody 11D7 prepared in our laboratory has the heavy chain sequence of SEQ ID NO.13 and the light chain sequence of SEQ ID NO.14. The heavy chain of antibody 11D7 was synthesized into the pcDNA3.1 vector and designated as pcDNA3.1-11D7-mg1. The light chain of antibody 11D7 was synthesized into the pcDNA3.1 vector and designated as pcDNA3.1-11D7-mk. The constructed recombinant vectors pcDNA3.1-11D7-mg1 and pcDNA3.1-11D7-mk were subjected to plasmid extraction and reserved for later use. They were named 11D7-mg1 and 11D7-mk, respectively.

[0135] 2. Design primers as shown in Table 7. Replace the signal peptide of the heavy chain of monoclonal antibody 11D7 (as shown in SEQ ID NO. 19) with signal peptides TIML, abgtx, mmp9, pelb, and mog, respectively, using PCR. Name the resulting recombinant plasmids TIML-11D7-mg1, abgtx-11D7-mg1, mmp9-11D7-mg1, pelb-11D7-mg1, and mog-11D7-mg1, respectively. The specific operation steps are as follows: Use the recombinant vector pcDNA3.1-11D7-mg1 as a template and the five pairs of primer sequences in Table 7 as primers for PCR. Except for the different template and primer sequences, the system, program, and operation steps are the same as in Example 1.

[0136] Table 7

[0137]

[0138]

[0139] 3. Design primers as shown in Table 8. Replace the signal peptide of the light chain of antibody 11D7 (as shown in SEQ ID NO.20) with signal peptides TIML, abgtx, mmp9, pelb, and mog, respectively. Name the resulting recombinant plasmids TIML-11D7-mk, abgtx-11D7-mk, mmp9-11D7-mk, pelb-11D7-mk, and mog-11D7-mk, respectively. The specific operation steps are as follows: Use the recombinant vector pcDNA3.1-11D7-mk as a template, and use the five pairs of primer sequences in Table 8 as primers for PCR. Except for the different template and primer sequences, the system, program, and operation steps are the same as in Example 1.

[0140] Table 8

[0141]

[0142]

[0143] II. ELISA detection of recombinant antibody secretion expression level

[0144] Following the PEI transfection method (transfection reagents purchased from Thermo Biotech, transfection performed according to the instructions), two groups of 11D7 plasmids with the same signal peptide were co-transfected into HEK293 cells. The specific operational steps were the same as in Example 1, except that the protein coated on the ELISA plate was ENO2. The secretory expression levels of recombinant antibodies 11D7, TIML-11D7, abgtx-11D7, mmp9-11D7, pelb-11D7, and mog-11D7 were detected using ELISA. The results are shown in Table 9. Figure 7 As shown.

[0145] Table 9

[0146]

[0147] From Table 9 and Figure 7 It can be seen that, under the same conditions, compared with the original antibody signal peptide, the secretion expression level of signal peptide TIML is increased, the secretion expression level of abgtx is comparable, while the other three signal peptides, mmp9, pelb and mog, are all reduced to varying degrees.

[0148] in conclusion:

[0149] Based on the above experimental results, the signal peptides TIML and abgtx described in this invention can guide the correct expression of target antibodies. Compared with the commonly used signal peptides HSA and IL2 currently used to guide antibody secretion expression, the signal peptides described in this invention significantly increase the secretion expression of target antibodies in cells, thereby reducing antibody production costs. This has great application prospects in the biomedical field where antibody demand is high.

[0150] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. The application of a mutant signal peptide from snake venom in increasing the secretory expression level of recombinant antibodies, characterized in that, The amino acid sequence of the snake venom mutant signal peptide is shown in SEQ ID NO.

1. This snake venom mutant signal peptide can guide the secretion and expression of recombinant antibodies. The recombinant antibody is 1A9, 6D9, or 11D7; Among them, the heavy chain sequence of 1A9 is shown in SEQ ID NO.9, and the light chain sequence is shown in SEQ ID NO.10; the heavy chain sequence of 6D9 is shown in SEQ ID NO.11, and the light chain sequence is shown in SEQ ID NO.12; the heavy chain sequence of 11D7 is shown in SEQ ID NO.13, and the light chain sequence is shown in SEQ ID NO.

14.

2. The application as described in claim 1, characterized in that, The original signal peptide of the antibody was replaced by a mutant snake venom signal peptide to increase the secretory expression of the recombinant antibody.

3. The application as described in claim 1, characterized in that, A mutant signal peptide from snake venom was added to a recombinant antibody that did not contain a signal peptide to guide antibody secretion and increase the secretory expression level of the recombinant antibody.

4. A snake venom mutant signal peptide that enhances the secretory expression of recombinant antibodies, characterized in that, Its amino acid sequence is shown in SEQ ID NO.

1.

5. The snake venom mutant signal peptide for enhancing the secretory expression of recombinant antibodies according to claim 4, characterized in that, The recombinant antibody is 1A9, 6D9, or 11D7; Among them, the heavy chain sequence of 1A9 is shown in SEQ ID NO.9, and the light chain sequence is shown in SEQ ID NO.10; the heavy chain sequence of 6D9 is shown in SEQ ID NO.11, and the light chain sequence is shown in SEQ ID NO.12; the heavy chain sequence of 11D7 is shown in SEQ ID NO.13, and the light chain sequence is shown in SEQ ID NO.

14.

6. A recombinant expression vector, characterized in that, The recombinant expression vector contains a nucleic acid molecule encoding a snake venom mutant signal peptide that enhances the secretion and expression of the recombinant antibody as described in claim 4. During construction, the above nucleic acid molecule is linked to the front end of a polynucleotide encoding the target antibody and then linked to the expression vector. The expression vectors include pcDNA3.1, pIRES, pTT3, pCEP4, and pATX1.

7. A host cell, characterized in that, The recombinant expression vector of claim 6 is included, and the host cells include HEK293, CHO, BHK, and SP2 / 0.

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