An in vitro expression production method of LTα1β2

Abstract

This patent relates to an in vitro expression method of a secreted LTα1β2 cytokine with low cost and high activity. By removing the signal peptide sequence of the LTα subunit, the transmembrane domain and signal peptide sequence of the LTβ subunit, and connecting one LTα subunit and two LTβ subunits together through a G4S flexible linker, a 6×his tag is added to the C-terminus of this protein complex, and then it is constructed onto the pPIC9K plasmid. The secreted LTα1β2 is expressed using the Pichia pastoris expression system. Subsequently, it is purified by nickel column and ultrafiltrated and dialyzed into 1×PBS. Subsequently, its high activity is verified through experimental methods such as Q-PCR, WB, and flow cytometry.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology and relates to a method for in vitro expression and production of LTα1β2. Background Art

[0002] Lymphotoxin α (LTα), also known as TNFβ, was initially described in the 1960s as a cytotoxic factor produced by activated lymphocytes that can kill transformed cell lines. LTα forms a soluble homotrimer (LTα3), which can bind to tumor necrosis factor receptor 1 (TNFR1), TNFR2, and HVEM (Herpesvirus entry mediator).

[0003] When co-expressed with lymphotoxin β (LTβ) having a transmembrane domain, LTα forms a cell surface-binding heterotrimer (LTα1β2), which can specifically bind to the LTβ receptor (LTβR). LTα1β2 is expressed by lymphocytes, such as activated T lymphocytes, B cells, natural killer cells (NK), and type 3 innate lymphoid cells (ILC3). LTβR is mainly expressed by stromal cells, including endothelial cells, mesenchymal cells, and epithelial cells, as well as myeloid cells, such as dendritic cells (DC) and macrophages. This specific expression pattern means that the LTα1β2 / LTβR interaction serves as a communication signal between lymphocytes and stromal cells. Existing studies also show that LTα1β2 plays a leading role in lymphoid organ development.

[0004] In addition to lymphoid organogenesis, related studies have also shown that lymphotoxin members control the development and homeostasis of different immune cells, as well as many aspects of the immune response. For example, LTβR promotes the development of NK and NKT cells. LT members are crucial for the formation of germinal centers and humoral immunity. After viral infection, LTβR signaling also controls the production of type I interferon in stromal cells and macrophages of lymphoid organs. In addition, other studies have shown that in subcutaneous tumors derived from the established CT26 cell line, monoclonal antibody (mAb)-mediated LTβR activation leads to T cell infiltration (possibly mediated by pro-inflammatory chemokines) and tumor necrosis. Recombinant LTα1β2 and LIGHT proteins or agonist LTβR mAb can inhibit the in vitro growth of human colon cancer and soft tissue sarcoma cell lines, which supports the view that immune cells interact with tumor cells through LTβR to inhibit spontaneous tumor development. Similarly, Yang et al. previously demonstrated that LTβR is a direct effector of CTL-mediated tumor rejection in vivo using a syngeneic mouse model of sarcoma metastasis to the lung and adoptive transfer of tumor-specific cytotoxic T lymphocytes (CTLs). Regarding the mechanism, agonist mAb stimulation of LTβR induces caspase- and mitochondria-dependent apoptosis and activates the classical and alternative NF-κB pathways in human cancer cell lines. In addition, NF-κB inhibition can promote the in vivo metastatic potential of CT26 colon cancer cells, indicating that in this context, LTβR-mediated apoptosis and activation of the NF-κB signaling pathway may act synergistically to inhibit tumor development.

[0005] In addition, other studies have shown that LTα1β2 and LIGHT ligands are expressed in immune cells such as T cells, and NK cells or DCs may bind to LTβR on tumor cells, thereby triggering anti-tumor cytotoxicity. However, tumor cell death in these studies was induced by recombinant ligands and / or LTβR agonist antibodies, which may not reflect the physiological levels and activities of ligands expressed on the surface of immune cells. LTβR activation and downstream signaling pathways induced by recombinant ligands or agonist antibodies in vitro may depend on the duration and extent of receptor oligomerization. Nevertheless, LTβR-mediated tumor suppression, whether through agonist mAb or adoptively transferred tumor-specific CTLs, has been proposed as a therapeutic approach to prevent tumor growth and overcome chemoresistance and radioresistance in colon cancer and soft tissue sarcoma.

[0006] The LTβ protein consists of a shorter N-terminal CD, TMD, and C-terminal ECD, and LTα lacks the TMD. Thus, when expressed in the absence of LTβ, LTα forms soluble LTα3 homotrimers, which are mainly stabilized by interactions between hydrophobic and aromatic side chains. When LTα is co-expressed with LTβ, these proteins oligomerize to produce cell surface LTα1β2 heterotrimers. LTα2β1 heterotrimers can also form, but these heterotrimers are only detectable in vitro and account for less than 10% of the total LTαβ heterotrimers. The LTα subunit mainly contributes to the conformation of the heterotrimer, while the LTβ subunit provides a membrane anchor for LTα1β2 and confers LTβR binding specificity. LTB expression in lymphocytes is constitutive, but LTA expression is not, although both are induced by cell stimulation. Mouse LTα cDNA encodes a secreted soluble protein of 202 amino acids (aa) with a 33-aa signal sequence. Mature mouse LTα has 75% aa sequence identity with human LTα. Mouse LTβ cDNA encodes a type II membrane protein of 306 aa with a 27-aa N-terminal cytoplasmic domain, a 21-aa transmembrane region, and a 258-aa extracellular domain. It has 73% aa sequence identity with human LTβ in the common region of its extracellular domain. Secreted LTα assembles into soluble homotrimers of LTα3. In addition, secreted LTα also complexes with membrane-associated LTβ to generate two heterotrimers, LTα1 / β2 and LTα2 / β1. Soluble LTα3 can bind to TNFRI (p55) and TNFRII (p75). In contrast, the major membrane-bound heterotrimer LTα1 / β2 binds only to the lymphotoxin β receptor (LTβR). LTα2 / β1 is capable of binding to LTβR, TNFRI (p55), and TNFRII (p75). LT plays a role in normal lymphoid organogenesis.

[0007] Although the commercially available LTα1β2 on the market currently performs excellently in terms of its functions and applications, however, its high price limits its use by the majority of researchers. This makes it impossible for many laboratories and research institutions to fully utilize this reagent for innovative research and carry out relevant experiments. Especially for in vivo animal experiments, the required dose of LTα1β2 is larger and the cost is higher, which greatly restricts the smooth progress of the experiments. Therefore, it is necessary to provide an in vitro expression production method of LTα1β2 that reduces the cost of LTα1β2 while maintaining excellent performance. Summary of the Invention

[0008] In response to this problem, this patent provides an economically efficient alternative method aimed at reducing the cost of LTα1β2 while maintaining excellent performance. By adopting the new technology disclosed in this patent, researchers can obtain high-quality LTα1β2 under more economical conditions, thus effectively promoting the progress of scientific research. Therefore, the advantage of this patent is that it makes up for the price limitation of existing commercial LTα1β2, provides a more economically viable option for researchers, and does not reduce the quality and performance of LTα1β2.

[0009] The object of the present invention is to provide a simple, efficient, low-cost and highly active production method for LTα1β2, so as to reduce the research cost of lymphoid tissue development and related diseases, including various cancer tumors.

[0010] The object of the present invention is achieved by the following technical solutions:

[0011] An in vitro expression and production method for the cytokine LTα1β2, the method comprising the following steps:

[0012] S1. Construct the pPIC9K-mLTα1β2-His plasmid: Delete the signal peptide sequence of the LTα amino acid sequence, and at the same time delete the transmembrane domain of LTβ. Connect one LTα and two LTβs with a G4S flexible linker, and add a 6×His tag at the C-terminus to obtain the mLTα1β2-His fragment. Construct the nucleotide sequence encoding the mLTα1β2-His fragment into the pPIC9K plasmid to obtain the pPIC9K-mLTα1β2-His plasmid, and optimize the codons;

[0013] S2. Remove the endotoxin of the pPIC9K-mLTα1β2-His plasmid;

[0014] S3. Linearize the pPIC9K-mLTα1β2-His plasmid obtained in S2 by single digestion with SalⅠ and recover it by gel extraction;

[0015] S4. Prepare Pichia pastoris competent cells;

[0016] S5. Electroporate the linearized pPIC9K-mLTα1β2-His plasmid obtained in S3 into Pichia pastoris competent cells;

[0017] S6. Screen out electrotransformation-positive and high-copy strains through MD medium and G418 solid medium;

[0018] S7. Extract yeast genomic DNA;

[0019] S8. Detect by QRT-PCR and select strains with relatively high copy numbers for sequencing, and select strains with correct sequencing results for use as subsequent expression yeast strains;

[0020] S9. Ferment the strains screened in S8 and collect the supernatant of the bacterial liquid.

[0021] S10. Purify with nickel column, ultrafilter and dialyze the nickel column eluate to obtain LTα1β2.

[0022] Furthermore, the signal peptide sequence of the LTα amino acid sequence described in S1 is located at positions 1 - 58 of the LTα amino acid sequence shown in SEQ ID NO.1, and the transmembrane domain of LTβ is located at positions 1 - 152 of the LTβ amino acid sequence shown in SEQ ID NO.2.

[0023] The nucleotide sequence encoding LTα described in S1 is as shown in SEQ ID NO.5, and the signal peptide sequence is located at positions 1 - 174 of the LTα nucleotide sequence; the nucleotide sequence encoding LTβ is as shown in SEQ ID NO.6, and the transmembrane domain is located at positions 1 - 456 of the LTβ nucleotide sequence shown in SEQ ID NO.6.

[0024] Furthermore, the nucleotide sequence encoding the mLTα1β2 - His fragment described in S1 is constructed between the EcoRⅠ and NotⅠ restriction enzyme cleavage sites of the pPIC9K plasmid.

[0025] Furthermore, the amino acid sequence of the mLTα1β2 - His fragment described in S1 is as shown in SEQ ID NO.3.

[0026] Furthermore, the nucleotide sequence encoding the mLTα1β2 - His fragment described in S1 is as shown in SEQ ID NO.4 (8 bases after the stop codon of the sequence shown in SEQ ID NO.4 are the restriction enzyme cleavage sites).

[0027] Furthermore, the Pichia pastoris described in S4 is GS115.

[0028] Furthermore, the fermentation in S9 is to inoculate the strains into BMGY medium, shake the bacteria at 28 °C and 250 rpm until OD600 = 5, centrifuge at 4 °C and 1500 g for 3 min, resuspend with 100 ml of BMMY medium to OD600 = 1, add methanol every 24 h to make the final concentration of methanol 1%, after fermenting for 96 hours, centrifuge the bacterial liquid, centrifuge at 4 °C and 12000 rpm for 10 min, and collect the supernatant of the bacterial liquid.

[0029] Furthermore, the molecular cut - off of the ultrafiltration in S10 is 10 KD.

[0030] Furthermore, the method further includes S11. Activity detection.

[0031] The beneficial effects of the present invention are as follows:

[0032] The present invention discloses for the first time a method for in vitro expression and purification of secreted LTα1β2 cytokine, and successfully expresses LTα1β2 cytokine with high activity, greatly reducing the cost of research on secondary lymphoid organ development, non-classical signaling pathways, and related tumor diseases, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 is the plasmid construction map of pPIC9K-mLTα1β2-His;

[0034] Figure 2 is the sequencing result of multi-copy strains;

[0035] Figure 3 is the western blot result (His-tag antibody) of purified LTα1β2;

[0036] Figure 4 is the western blot result of p100 / p52 after CT-26, Bend.3 and MEF cells were stimulated with different concentrations of LTα1β2 for 24 h. Among them, Figure 4 A is CT-26 cells, Figure 4 B is Bend.3 cells, Figure 4 C is MEF cells;

[0037] Figure 5 is the Q-PCR result of the mRNA expression level of MAdCAM-1 after Bend.3 cells were stimulated with different concentrations of LTα1β2 for 24 h;

[0038] Figure 6 is the flow cytometry analysis result of MAdCAM-1 after Bend.3 cells were stimulated with different concentrations of LTα1β2 for 48 h;

[0039] Figure 7 is the flow cytometry analysis result of LTβR after Bend.3 cells were stimulated with different concentrations of LTα1β2 for 48 h. DETAILED DESCRIPTION OF THE INVENTION

[0040] The following examples are used to further explain the present invention, but the examples do not limit the present invention in any form.

[0041] Example 1

[0042] 1. Plasmid construction (yeast codon optimization)

[0043] Delete the signal peptide sequence of LTα (amino acid positions 1 - 58 of the amino acid sequence shown in SEQ ID NO.1), and at the same time delete the signal peptide sequence and transmembrane domain of LTβ (amino acid positions 1 - 152 of the amino acid sequence shown in SEQ ID NO.2). Connect one LTα and two LTβ with a G4S flexible linker (a total of three subunits) for co-expression, which greatly increases the combination probability of LTα1β2, and add a 6×His tag at the C-terminus to obtain the amino acid fragment of mLTα1β2-His (SEQ ID NO.3). Construct the nucleotide sequence encoding the mLTα1β2-His fragment (SEQ ID NO.4) between the EcoRⅠ and NotⅠ restriction enzyme sites of the pPIC9K plasmid and optimize the codons. The plasmid map is as shown in Figure 1 (This plasmid contains the signal peptide required for Pichia pastoris to express and secrete proteins). Send the constructed pPIC9K-mLTα1β2-His plasmid to the company for sequencing to detect whether mLTα1β2 is correct (see Figure 2 )

[0044] 2. Plasmid endotoxin removal and extraction

[0045] This step involves the Endotoxin-free Plasmid Mega Kit (DP117) from Tiangen

[0046] (1) Add 100 ml of overnight cultured bacterial solution to a centrifuge tube and centrifuge at 8,000 rpm for 3 min at room temperature. Collect the bacterial pellet.

[0047] (2) Aspirate the supernatant as much as possible. To ensure that all the supernatant is aspirated, use a clean absorbent paper to blot the water droplets on the bottle wall.

[0048] (3) Add 8 ml of solution P1 to the centrifuge tube containing the bacterial pellet and use a vortex oscillator to thoroughly suspend the bacterial cell pellet.

[0049] (4) Add 8 ml of solution P2 to the centrifuge tube and immediately gently invert it 6 - 8 times to fully lyse the bacteria, and let it stand at room temperature for 5 min.

[0050] (5) Add 8 ml of solution P4 to the centrifuge tube and immediately gently invert it 6 - 8 times to mix well until a white dispersed flocculent precipitate appears in the solution. Then let it stand at room temperature for about 10 min. Centrifuge at 8,000 rpm for 5 - 10 min to sediment the white precipitate to the bottom of the tube (the centrifugation time can be appropriately increased). Carefully pour all the solution into the filter CS1 and slowly push the push handle to filter. Collect the filtrate in a clean 50 ml tube.

[0051] (6) Add 2.5 ml of equilibration buffer BL8 to adsorption column CP6 (the adsorption column is placed in a 50 ml collection tube), centrifuge at 8,000 rpm for 2 min, pour out the waste liquid in the collection tube, and place the adsorption column back into the collection tube.

[0052] (7) Add isopropanol with a volume 0.3 times that of the filtrate to the filtrate (adding too much isopropanol easily causes RNA contamination), invert the tube up and down, mix well, and transfer it to adsorption column CP6 (the adsorption column is placed in a 50 ml collection tube).

[0053] (8) Centrifuge at 8,000 rpm for 2 min at room temperature, pour out the waste liquid in the collection tube, and place adsorption column CP6 back into the collection tube.

[0054] (9) Add 10 ml of washing buffer PW to adsorption column CP6, centrifuge at 8,000 rpm for 2 min, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.

[0055] (10) Repeat operation step 9.

[0056] (11) Add 3 ml of absolute ethanol to adsorption column CP6, centrifuge at 8,000 rpm for 2 min at room temperature, and pour out the waste liquid.

[0057] (12) Place adsorption column CP6 back into the collection tube, centrifuge at 8,000 rpm for 5 min to remove the residual washing buffer in the adsorption column.

[0058] (13) Place adsorption column CP6 in a clean 50 ml collection tube, suspend and add 1 - 2 ml of ddH2O dropwise to the middle part of the adsorption membrane, let it stand at room temperature for 5 min, and then centrifuge at 8,000 rpm for 2 min at room temperature. Transfer all the eluate in the 50 ml centrifuge tube to a clean 1.5 ml centrifuge tube, store it at -20°C, or directly perform downstream experiments.

[0059] 3. Linearization of pPIC9K - mLTα1β2 - His plasmid

[0060] Linearization system:

[0061] pPIC9K plasmid: 20 μg

[0062] SalⅠ: 20 μl

[0063] 10X Buffer CutC: 100 μl

[0064] ddH2O: Make up to 1000 μl

[0065] Digest at 37°C for 2 h, recover and purify the linearized plasmid using a gel extraction kit, and measure the concentration. Take a small portion of the digested product for gel analysis to determine whether the digestion is complete. If the digestion is incomplete, the transformation number and targeting efficiency will both decrease. 4. Preparation of Pichia pastoris GS115 competent cells

[0066] (1) Streak plate by the four-zone streaking method: Dip an inoculation loop in a small amount of Pichia pastoris GS115 yeast and streak it on a YPD solid medium containing ampicillin resistance in four zones to obtain monoclonal strains.

[0067] (2) Incubate at a constant temperature: Invert the streaked plate and incubate at 28 - 30°C for 2 days to obtain dispersed monoclonal colonies (the yeast culture temperature should not exceed 32°C).

[0068] (3) Pick a single colony from the YPD plate and transfer it to a 250 mL Erlenmeyer flask containing 20 mL of YPD. Incubate overnight at 30°C with shaking at 250 r / min;

[0069] (4) Take 200 μL of the above Pichia pastoris culture solution and add it to a 500 mL conical flask containing 100 mL of YPD medium with double antibiotics. Incubate on a shaker at 250 r / min and 30°C until OD600 = 1.3 - 1.5;

[0070] (5) Collect the culture solution and centrifuge at 1500 g for 5 min at 4°C;

[0071] (6) Resuspend with 500 mL of pre-cooled sterile water. Centrifuge at 1500 g for 5 min at 4°C;

[0072] (7) Resuspend with 250 mL of pre-cooled sterile water. Centrifuge at 1500 g for 5 min at 4°C;

[0073] (8) Resuspend with 20 mL of pre-cooled 1 M sorbitol. Centrifuge at 1500 g for 5 min at 4°C;

[0074] (9) Resuspend with 200 μL of pre-cooled 1 M sorbitol to obtain Pichia pastoris GS115 competent cells.

[0075] 5. Electroporation of GS115 Pichia pastoris:

[0076] (1) Take 80 μL of the above Pichia pastoris GS115 competent cells and mix with 10 μg of linearized DNA. Transfer them into a pre-cooled 0.2 cm electroporation cuvette on ice;

[0077] (2) Place on ice for 5 min;

[0078] (3) Turn on the electroporator and perform electroporation with a capacitance of 25 μF, PC200 hm, voltage of 2000 V, and resistance of 200 Ω;

[0079] 6. Screen for electrotransformation-positive and high-copy strains through MD and G418 solid media.

[0080] (1) Immediately after electrotransformation, add 1 mL of pre-cooled 1 M sorbitol solution to the cup. After taking it out, incubate it on a shaker at 28 °C for 1 hour, then take 200 μL and spread it on an MD plate.

[0081] (2) Incubate the plate at 30 °C until colonies are clearly formed (about 3 - 4 days).

[0082] (3) Scrape the colonies on the MD plate with sterile water, dilute them 1:1000, and take 200 μL and spread it on a 4 mg / mL G418 plate.

[0083] (4) Incubate the plate at 30 °C until colonies are clearly formed (about 3 - 4 days) until monoclonal colonies are obtained on the 4 mg / mL G418 plate.

[0084] (5) Pick single colonies from the 4 mg / mL G418 plate into 5 mL of YPD (containing ampicillin and G418) medium and shake the bacteria overnight.

[0085] 7. Extract yeast genomic DNA

[0086] Take a sample of the bacterial solution and use a yeast genomic DNA extraction kit to extract yeast genomic DNA according to the instructions. Dilute the DNA concentration to 50 ng / μL with ddH2O.

[0087] 8. QRT-PCR

[0088] Use QRT-PCR to identify the copy number of the target gene in yeast. GAPDH is used as an internal reference. Select the one with the highest copy number and send it for sequencing. After the sequencing result is correct, use it as the subsequent expression yeast strain.

[0089] 9. Fermentation

[0090] (1) Add 25 mL of BMGY medium to 7 250-mL conical flasks respectively and inoculate 200 μL of the bacterial strain. Shake the bacteria at 28 °C and 250 rpm until OD600 = 5. Centrifuge at 4 °C and 1500 g for 3 min, and resuspend with 100 mL of BMMY medium to OD600 = 1. Transfer them into 7 500-mL conical flasks respectively, and add methanol to a final concentration of 1% (using 100% methanol, add 1 mL) every 24 h.

[0091] (2) After 96 hours of expression, centrifuge the bacterial solution at 4 °C and 12000 rpm for 10 min to collect the supernatant of the bacterial solution.

[0092] 10. Nickel column purification, ultrafiltration and dialysis

[0093] (1) Filter the obtained bacterial supernatant at 4°C using a 0.22 μm filter to avoid clogging the purification column. Use Ni-NTA affinity chromatography medium (GenScript, Cat. No. L00250) for column purification.

[0094] (2) The nickel column eluate was ultrafiltered using a 10KD 15 ml ultrafiltration tube (millipore) and dialyzed in 1× PBS to obtain LTα1β2.

[0095] 11. BCA method for concentration measurement

[0096] (1) Prepare the standard and test sample (LTα1β2) as shown in the table below.

[0097]

[0098]

[0099] Prepare the corresponding standard and sample dilutions in a 96-well plate according to the table above, and then add 200ul of Reagent A and Reagent B mixture to each well (Reagent A: Reagent B = 50:1)

[0100] (2) Incubate at 37°C for 30 min;

[0101] (3) Measure absorbance using an ELISA reader (562 nm);

[0102] (4) Preparation of standard curve equation (R 2 >0.99), and the mLTα1β2 absorbance was substituted into the equation to calculate the mLTα1β2 concentration;

[0103] 11. Take a portion for SDS-PAGE or WB (His tag antibody) detection:

[0104] (1) Take a certain amount of sample and add the corresponding 5× protein loading buffer, boil at 95°C for 5 minutes;

[0105] (2) Loading the sample;

[0106] (3) Concentrated gel: 80V, about 50min;

[0107] (4) Separation gel: 120V, about 60min;

[0108] (5) Transfer: 100V 90min;

[0109] (6) Blocking: 5% milk powder, blocking for 1.5 h (shaking speed 40 rpm);

[0110] (7) Wash three times with 1×TBST for 8 min each time (shaker speed 90 rpm);

[0111] (8) Incubate with primary antibody: his-tag antibody, incubate overnight at 4°C (shaker speed 20 rpm);

[0112] (9) Wash three times with 1×TBST for 10 min each time (shaker speed 90 rpm);

[0113] (10) Incubate with secondary antibody: incubate at room temperature for 1.5 h (shaker speed 40 rpm);

[0114] (11) Wash three times with 1×TBST for 10 min each time (shaker speed 90 rpm);

[0115] (12) ECL development.

[0116] For the purified and ultrafiltered LTα1β2, take a part to verify whether the relevant protein is obtained by western blot, and use HIS-tag antibody for verification (see Figure 3 ).

[0117] 11. Activity detection

[0118] To determine the activity of LTα1β2 prepared by the method of the present invention, an activity detection experiment was carried out. The experimental principle is as follows: CT-26, Bend.3 and MEF contain LTβR receptors. After this receptor is stimulated by LTα1β2, it will activate the non-canonical NF-κB signaling pathway. One of the characteristics of the activation of the non-canonical signaling pathway is that part of p100 is processed into p52. However, since this receptor can also activate the canonical NF-κB signaling pathway, after the activation of the canonical signaling pathway, it further activates the downstream signaling pathway, promoting the cell to express p100. Therefore, p100 may not decrease after being stimulated, but the expression level of p52 will increase. At the same time, after the activation of the non-canonical signaling pathway, it will cause the activation of the downstream signaling pathway, and then promote the increase in the expression of MAdCAM-1. MAdCAM-1 is a cell surface protein and can also be analyzed by flow cytometry. In addition, the LTβR receptor will undergo endocytosis after being stimulated by LTα1β2, so its expression level can also be analyzed by flow cytometry as an index for activity detection.

[0119] Experimental example 1:

[0120] Seed CT-26, Bend.3 and MEF cells into six-well plates, and stimulate them with 0 ng / ml (control), 10 ng / ml, and 100 ng / ml of LTα1β2 for 24 h respectively. Collect the cells, lyse them with RIPA, collect the total protein, and perform BCA quantification. Detect the expression of p100 / p52 by western blot. The WB results show (see Figure 4) After stimulation with LTα1β2, p100 will be partially processed into p52, resulting in an increase in the expression level of p52.

[0121] Experimental Example 2:

[0122] Bend.3 cells were seeded in six-well plates and stimulated with 0 ng / ml (control), 10 ng / ml, and 100 ng / ml of LTα1β2 for 24 h. The cells were collected, and total RNA was collected using an RNA extraction kit and reverse-transcribed into cDNA using a reverse transcription kit. The expression level of MAdCAM-1 mRNA was detected by Q-PCR. The Q-PCR results showed (see Figure 5 ) that after stimulation with LTα1β2, the expression level of MAdCAM-1 mRNA increased, and the higher the concentration, the higher the expression level.

[0123] Experimental Example 3:

[0124] Bend.3 cells were seeded in six-well plates and stimulated with 0 ng / ml (control), 1 μg / ml, and 10 μg / ml of LTα1β2 for 48 h. The cells were collected, stained, and analyzed by flow cytometry. The results showed (see Figure 6 、 Figure 7 ) that after stimulation with LTα1β2, the expression level of MAdCAM-1 protein increased, and the higher the stimulation concentration, the higher the expression level of MAdCAM-1 protein. At the same time, the LTβR receptor was endocytosed with the stimulation of LTα1β2, resulting in a decrease in its surface level, and the higher the stimulation concentration, the higher the decrease level of the LTβR receptor.

[0125] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for in vitro expression and production of cytokine LTα1β2, characterized in that, The method includes the following steps: S1. Construct the pPIC9K-mLTα1β2-His plasmid: Delete the signal peptide sequence of the LTα amino acid sequence, and at the same time delete the transmembrane domain of LTβ. Connect one LTα with the signal peptide sequence deleted and two LTβs with the transmembrane domains deleted using a G4S flexible linker, and add a 6×His tag at the C-terminus to obtain the mLTα1β2-His fragment. Construct the nucleotide sequence encoding the mLTα1β2-His fragment into the pPIC9K plasmid to obtain the pPIC9K-mLTα1β2-His plasmid, and optimize the codons. S2. Remove the endotoxin of the pPIC9K-mLTα1β2-His plasmid. S3. Linearize the pPIC9K-mLTα1β2-His plasmid obtained in S2 by single digestion with SalⅠ and recover it by gel extraction. S4. Prepare Pichia pastoris competent cells. S5. Electrotransform the linearized pPIC9K-mLTα1β2-His plasmid obtained in S3 into the Pichia pastoris competent cells. S6. Screen out the electrotransformation-positive and high-copy-number strains through MD medium and G418 solid medium. S7. Extract yeast genomic DNA. S8. Detect by QRT-PCR and select the strains with relatively higher copy numbers for sequencing. Select the strains with correct sequencing results and use them as subsequent expression yeast strains. S9. Ferment the strains screened in S8 and collect the supernatant of the bacterial liquid. S10. Purify with a nickel column, perform ultrafiltration dialysis on the nickel column eluate to obtain LTα1β2S1. The signal peptide sequence of the LTα amino acid sequence is located at positions 1-58 of the LTα amino acid sequence shown in SEQ ID NO.1, and the transmembrane domain of LTβ is located at positions 1-152 of the LTβ amino acid sequence shown in SEQ ID NO.

2.

2. The method according to claim 1, wherein In S1, the nucleotide sequence encoding the mLTα1β2-His fragment is constructed between the EcoRⅠ and NotⅠ restriction enzyme sites of the pPIC9K plasmid.

3. The method according to claim 1, characterized in that, The amino acid sequence of the mLTα1β2-His fragment in S1 is as shown in SEQ ID NO.

3.

4. The method according to claim 1, wherein The nucleotide sequence encoding the mLTα1β2-His fragment in S1 is as shown in SEQ ID NO.

4.

5. The method according to claim 1, wherein The Pichia pastoris in S4 is GS115.

6. The method according to claim 1, characterized in that The fermentation in S9 is to inoculate the strains into BMGY medium, shake the bacteria at 28 °C and 250 rpm until OD600 = 5, centrifuge at 4 °C and 1500 g for 3 min, resuspend with 100 ml of BMMY medium to OD600 = 1, add methanol every 24 h to make the final concentration of methanol 1%, after fermenting for 96 hours, centrifuge the bacterial liquid, centrifuge at 4 °C and 12000 rpm for 10 min, and collect the supernatant of the bacterial liquid.

7. The method according to claim 1, wherein The molecular cut-off of the ultrafiltration in S10 is 10KD.

8. The method according to claim 1, characterized in that, The method further includes S11. Activity detection.

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