LT alpha1beta2 in-vitro expression production method

Abstract

The invention relates to an in-vitro expression method of a secreting type LT alpha1beta2 cell factor with low cost and high activity. A signal peptide sequence of an LT alpha subunit is removed, a transmembrane structural domain of an LT beta subunit and the signal peptide sequence are removed, the subunits are connected together through a G4S flexible linker, a 6 * his tag is added to the C end of a protein compound, then the protein compound is constructed to a pPIC9K plasmid, and the secreting type LT alpha 1 beta 2 is expressed by using a pichia pastoris expression system. And purifying through a nickel column, and carrying out ultrafiltration dialysis to obtain 1 * PBS. Subsequent Q-PCR, WB and flow cytometry experimental methods verify that the compound has relatively high activity.

Description

Technical Field

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

[0002] Lymphotoxin alpha (LTα), also known as TNFβ, was first described in the 1960s as a cytotoxic factor produced by activated lymphocytes that kills transformed cell lines. LTα forms a soluble homotrimer (LTα3) that binds to tumor necrosis factor receptor 1 (TNFR1), TNFR2, and HVEM (Herpes virus entry mediator).

[0003] When co-expressed with lymphotoxin β (LTβ) with a transmembrane domain, LTα forms a cell surface-bound 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 acts as a communication signal between lymphocytes and stromal cells. Existing studies have also shown that LTα1β2 plays a dominant role in the development of lymphoid organs.

[0004] In addition to lymphoid organogenesis, studies have shown that lymphotoxin members control the development and homeostasis of different immune cells and many aspects of the immune response. For example, LTβR promotes the development of NK and NKT cells. LT members are essential for the formation of germinal centers and humoral immunity. After viral infection, LTβR signaling also controls the production of type I interferons in stromal cells and macrophages in lymphoid organs. In addition, other studies have shown that monoclonal antibody (mAb)-mediated activation of LTβR in established CT26 cell line-derived subcutaneous tumors leads to T cell infiltration (possibly mediated by proinflammatory chemokines) and tumor necrosis. Recombinant LTα1β2 and LIGHT proteins or agonist LTβRmAb 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., using a syngeneic mouse model of sarcoma metastasis to the lung and adoptive transfer of tumor-specific cytotoxic T lymphocytes (CTLs), previously demonstrated that LTβR is a direct effector of CTL-mediated tumor rejection in vivo. Mechanistically, agonist mAb stimulation of LTβR induced caspase- and mitochondria-dependent apoptosis and activated both the classical and alternative NF-κB pathways in human cancer cell lines. Furthermore, NF-κB inhibition promoted the metastatic potential of CT26 colon cancer cells in vivo, suggesting that LTβR-mediated apoptosis and activation of the NF-κB signaling pathway may work synergistically to inhibit tumor development in this setting.

[0005] In addition, other studies have shown that LTα1β2 and LIGHT ligands are expressed in immune cells such as T cells, NK cells or DCs, which may bind to LTβR on tumor cells to induce antitumor 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 mAbs or adoptively transferred tumor-specific CTLs, has been proposed as a therapeutic approach to block tumor growth and overcome chemotherapy and radiotherapy resistance in colon cancer and soft tissue sarcomas.

[0006] The LTβ protein consists of a shorter N-terminal CD, a TMD, and a C-terminal ECD, while LTα lacks the TMD. Thus, when expressed in the absence of LTβ, LTα forms a soluble LTα3 homotrimer stabilized primarily by interactions between hydrophobic and aromatic side chains, and when LTα is expressed together 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 contributes primarily 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, but both are induced by cell stimulation. The mouse LTα cDNA encodes a secreted, soluble protein of 202 amino acids (aa) with a 33 aa signal sequence. Mature mouse LTα shares 75% aa sequence identity with human LTα. Mouse LTβ cDNA encodes a 306aa type II membrane protein with a 27aa N-terminal cytoplasmic domain, a 21aa transmembrane region, and a 258aa extracellular domain. It shares 73% aa sequence identity with human LTβ in the common region of its extracellular domain. Secreted LTα assembles into a soluble homotrimer, 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 exclusively to the lymphotoxin beta receptor (LTβR). LTα2 / β1 is able to bind to LTβR, TNFRI(p55), and TNFRII(p75). LT plays a role in normal lymphoid organogenesis.

[0007] Although the commercialized LTα1β2 currently on the market performs well in its functions and applications, its high price limits the use of researchers. This makes it impossible for many laboratories and research institutions to fully utilize the reagent for innovative research and related experiments. Especially for in vivo animal experiments, the required LTα1β2 dose is larger and the cost is higher, which greatly limits the smooth progress of the experiment. Therefore, it is necessary to provide a method for in vitro expression and production 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 economical and efficient alternative method that aims to reduce 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, thereby effectively promoting the progress of scientific research. Therefore, the advantage of this patent is that it makes up for the price limit of existing commercialized LTα1β2, providing researchers with a more economical and feasible option without reducing the quality and performance of LTα1β2.

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

[0010] The purpose of the present invention is achieved through the following technical solutions:

[0011] A method for expressing and producing cytokine LTα1β2 in vitro, the method comprising the following steps:

[0012] S1. Construction of pPIC9K-mLTα1β2-His plasmid: Delete the signal peptide sequence of the LTα amino acid sequence, delete the transmembrane domain of LTβ, connect one LTα and two LTβ with a G4S flexible linker, 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, obtain the pPIC9K-mLTα1β2-His plasmid, and optimize the codons;

[0013] S2, removal of endotoxin from pPIC9K-mLTα1β2-His plasmid;

[0014] S3, linearize the pPIC9K-mLTα1β2-His plasmid obtained in S2 by single restriction digestion with SalⅠ, and recover it by gel;

[0015] S4, preparing Pichia pastoris competent cells;

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

[0017] S6. Using MD medium and G418 solid medium, screen out the strains that are positive for electroporation and have high copies;

[0018] S7, extracting yeast genomic DNA;

[0019] S8. QRT-PCR detection and selection of strains with relatively high copy numbers for sequencing. The strains with correct sequencing results were selected as the subsequent expression yeast strains.

[0020] S9, fermenting the strain screened in S8, and collecting the supernatant of the bacterial solution;

[0021] S10, nickel column purification, ultrafiltration and dialysis of the nickel column eluate to obtain LTα1β2.

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

[0023] The nucleotide sequence encoding the LTα described in S1 is 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 the LTβ is 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 was constructed between the EcoRⅠ and NotⅠ restriction sites of the pPIC9K plasmid.

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

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

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

[0028] Furthermore, the fermentation described in S9 is as follows: the strain is inoculated into BMGY medium, shaken at 28°C and 250rpm to OD600=5, centrifuged at 4°C and 1500g for 3min, resuspended to OD600=1 using 100ml BMMY medium, and methanol is added every 24h to make the final concentration of methanol 1%. After fermentation for 96 hours, the bacterial solution is centrifuged at 4°C and 12000rpm for 10min, and the supernatant of the bacterial solution is collected.

[0029] Furthermore, the molecular cutoff of the ultrafiltration in S10 is 10KD.

[0030] Furthermore, the method also includes S11, activity detection.

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

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

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

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

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

[0036] Figure 4 The western blot results of p100 / p52 in CT-26, Bend.3 and MEF cells after stimulation with different concentrations of LTα1β2 for 24 hours are shown in Figure 2. Figure 4 A is CT-26 cells, Figure 4 B is Bend.3 cell, Figure 4 C is MEF cells;

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

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

[0039] Figure 7 This is the flow cytometry analysis result of LTβR in Bend.3 cells stimulated by different concentrations of LTα1β2 for 48 hours. DETAILED DESCRIPTION

[0040] The present invention is further explained below with reference to the examples, 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 (positions 1-58 of the amino acid sequence shown in SEQ ID NO.1), and delete the signal peptide sequence and transmembrane domain of LTβ (positions 1-152 of the amino acid sequence shown in SEQ ID NO.2). Use a G4S flexible linker to connect one LTα and two LTβ (three subunits in total) to express them simultaneously, greatly increasing the combination probability of LTα1β2. 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 sites of the pPIC9K plasmid, and optimize the codons. The plasmid map is shown in the figure below. Figure 1 As shown (this plasmid contains the signal peptide required for Pichia pastoris to express secretory proteins). The constructed pPIC9K-mLTα1β2-His plasmid was sent to the company for sequencing to detect whether mLTα1β2 was correct (see Figure 2 )

[0044] 2. Plasmid endotoxin removal and extraction

[0045] This step involves Tiangen's Endotoxin Plasmid Extraction Kit (DP117)

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

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

[0048] (3) Add 8 ml of solution P1 to the centrifuge tube containing the bacterial cell 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 and gently invert the tube 6-8 times to fully lyse the bacteria. Leave the tube at room temperature for 5 min.

[0050] (5) Add 8 ml of solution P4 to the centrifuge tube, and immediately and gently turn it upside down 6-8 times to mix thoroughly until white dispersed flocculent precipitates appear in the solution. Then leave it at room temperature for about 10 minutes. Centrifuge it at 8,000 rpm for 5-10 minutes to allow the white precipitate to settle to the bottom of the tube (the centrifugation time can be increased appropriately). Pour all the solution carefully into the filter CS1, slowly push the push handle to filter, and collect the filtrate in a clean 50 ml tube.

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

[0052] (7) Add 0.3 times the volume of isopropanol to the filtrate (adding too much isopropanol can easily lead to RNA contamination), invert and mix well, and then transfer to the 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, discard the waste liquid in the collection tube, and put the adsorption column CP6 back into the collection tube.

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

[0055] (10) Repeat step 9.

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

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

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

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

[0060] Linearized system:

[0061] pPIC9K plasmid: 20ug

[0062] SalⅠ:20ul

[0063] 10X Buffer CutC: 100ul

[0064] ddH2O: make up to 1000ul

[0065] Digest at 37℃ for 2h, recover and purify the linearized plasmid with gel recovery kit, and measure the concentration. Take a small part of the digestion product gel. Analyze to determine whether the digestion product is complete. If the digestion is not complete, the transformation number and targeting efficiency will be reduced. 4. Preparation of Pichia GS115 competent cells

[0066] (1) Four-zone streak method: Dip a small amount of Pichia pastoris GS115 yeast into an inoculation loop and streak it into four zones on a YPD solid medium containing ampicillin resistance to obtain a single clone strain.

[0067] (2) Constant temperature culture: Invert the streaked plate and culture at 28-30°C for 2 days to obtain dispersed single clones (the yeast culture temperature does not exceed 32°C).

[0068] (3) Pick a single colony from the YPD plate and place it in a 250 mL Erlenmeyer flask containing 20 mL YPD at 250 rpm and 30 °C overnight;

[0069] (4) Take 200ul of the above Pichia culture solution and add it to a 500ml conical flask containing 100ml YPD medium containing double antibodies. Cultivate in a shaking incubator at 250r / min and 30°C until OD600 = 1.3-1.5;

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

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

[0072] (7) Resuspend in 250 ml of pre-cooled sterile water and centrifuge at 1500 g for 5 min at 4°C;

[0073] (8) Resuspend in 20 ml of pre-cooled 1 M sorbitol and centrifuge at 1500 g for 5 min at 4°C;

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

[0075] 5. GS115 Pichia pastoris electroporation:

[0076] (1) Take 80uL of the above-mentioned Pichia GS115 competent cells and mix with 10ug of linearized DNA, and transfer to a 0.2cm electroporation cuvette pre-cooled 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, a PC of 200 hm, a voltage of 2000 V, and a resistance of 200 Ω;

[0079] 6. Use MD and G418 solid culture medium to screen out electroporation-positive and high-copy strains

[0080] (1) After electroporation, add 1 mL of pre-cooled 1 M sorbitol solution to the cup immediately. Incubate on a shaker at 28°C for 1 hour, then take 200 μL of the solution and apply it to the MD plate.

[0081] (2) Incubate the plates at 30°C until colonies are clearly formed (approximately 3 to 4 days).

[0082] (3) Scrape the colonies from the MD plate with sterile water, dilute 1:1000, and apply 200ul on a 4mg / ml G418 plate.

[0083] (4) Incubate the plates at 30°C until colonies are clearly formed (approximately 3 to 4 days) and until a single clone is obtained on a 4 mg / ml G418 plate.

[0084] (5) Pick a single colony from the 4 mg / ml G418 plate and transfer it to 5 ml YPD medium (containing ampicillin and G418) and shake overnight.

[0085] 7. Extraction of yeast genomic DNA

[0086] The bacterial liquid was sampled, and the yeast genome was extracted using a yeast genomic DNA extraction kit according to the instructions, and the DNA concentration was diluted to 50 ng / ul using ddH2O.

[0087] 8. QRT-PCR

[0088] QRT-PCR was used to identify the copy number of the target gene in yeast, GAPDH was used as an internal reference, and the one with the highest copy number was selected for sequencing. After the sequencing results were correct, it was used as the subsequent expression yeast strain.

[0089] 9. Fermentation

[0090] (1) Add 25 ml of BMGY medium to each of seven 250 ml conical flasks and inoculate 200 ul of the bacterial strain. Shake the culture at 28°C, 250 rpm until OD600 = 5. Centrifuge at 1500 g for 3 min at 4°C. Resuspend the culture in 100 ml of BMMY medium until OD600 = 1. Transfer the culture to seven 500 ml conical flasks. Add methanol every 24 h to a final concentration of 1% (use 100% methanol, add 1 ml).

[0091] (2) After 96 hours of expression, the bacterial solution was centrifuged at 4°C, 12,000 rpm for 10 min, and the supernatant was collected.

[0092] 10. Nickel column purification, ultrafiltration 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, 8 min each time (shaking speed 90 rpm);

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

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

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

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

[0115] (12) ECL development.

[0116] After purification and ultrafiltration, a portion of LTα1β2 was taken for western blot to verify whether the relevant protein was obtained, and HIS tag antibody was used for verification (see Figure 3 ).

[0117] 11. Activity detection

[0118] In order to determine the activity of LTα1β2 prepared by the method of the present invention, an activity detection experiment was performed. The experimental principle is that CT-26, Bend.3 and MEF contain LTβR receptors, which will activate the non-classical NF-κB signaling pathway after being stimulated by LTα1β2. One of the characteristics of the activation of the non-classical signaling pathway is that p100 is partially processed into p52, but because the receptor can also activate the classical NF-κB signaling pathway, and after the classical signaling pathway is activated, the downstream signaling pathway is further activated, prompting the cell to express p100. Therefore, p100 may not decrease after being stimulated, but the expression of p52 will increase. At the same time, after the non-classical signaling pathway is activated, the downstream signaling pathway will be activated, thereby prompting 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, after the LTβR receptor is stimulated by LTα1β2, it will be internalized, so its expression level can also be analyzed by flow cytometry as an indicator of activity detection.

[0119] Test Example 1:

[0120] CT-26, Bend.3 and MEF cells were plated in six-well plates and stimulated with 0 ng / ml (control), 10 ng / ml and 100 ng / ml of LTα1β2 for 24 h, respectively. The cells were collected and lysed with RIPA, and the total protein was collected and quantified by BCA method. The expression of p100 / p52 was detected by western blot. WB results showed (see Figure 4), after LTα1β2 stimulation, p100 will be partially processed into p52, resulting in increased p52 expression.

[0121] Test Example 2:

[0122] Bend.3 cells were plated in six-well plates and stimulated with 0 ng / ml (control), 10 ng / ml, and 100 ng / ml of LTα1β2 for 24 h, respectively. 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 ), after LTα1β2 stimulation, the expression level of MAdCAM-1 mRNA increased, and the higher the concentration, the higher the expression level.

[0123] Test Example 3:

[0124] Bend.3 cells were plated in six-well plates and stimulated with 0 ng / ml (control), 1 ug / ml, and 10 ug / ml of LTα1β2 for 48 h. The cells were collected, stained, and analyzed by flow cytometry (see Figure 6 , Figure 7 )After LTα1β2 stimulation, the expression level of MAdCAM-1 protein increased, and as the stimulation concentration increased, the expression level of MAdCAM-1 protein also increased. At the same time, LTβR receptors were internalized with LTα1β2 stimulation, causing their surface levels to decrease, and the higher the stimulation concentration, the higher the decrease in LTβR receptor levels.

[0125] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for in vitro expression and production of cytokine LTα1β2, characterized in that: The method comprises the following steps: S1. Construction of pPIC9K-mLTα1β2-His plasmid: Delete the signal peptide sequence of the LTα amino acid sequence, delete the transmembrane domain of LTβ, connect one LTα and two LTβ with a G4S flexible linker, 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, obtain the pPIC9K-mLTα1β2-His plasmid, and optimize the codons; S2, removal of endotoxin from pPIC9K-mLTα1β2-His plasmid; S3, linearize the pPIC9K-mLTα1β2-His plasmid obtained in S2 by single restriction digestion with SalⅠ, and recover it by gel; S4, preparing Pichia pastoris competent cells; S5, electroporate the linearized pPIC9K-mLTα1β2-His plasmid obtained in S3 into Pichia pastoris competent cells; S6. Using MD medium and G418 solid medium, screen out the strains that are positive for electroporation and have high copies; S7, extracting yeast genomic DNA; S8. QRT-PCR detection and selection of strains with relatively high copy numbers for sequencing. The strains with correct sequencing results were selected as the subsequent expression yeast strains. S9, fermenting the strain screened in S8, and collecting the supernatant of the bacterial solution; S10, nickel column purification, ultrafiltration and dialysis of the nickel column eluate to obtain LTα1β2.

2. The method according to claim 1, characterized in that: The signal peptide sequence of the LTα amino acid sequence S1 is located at positions 1-58 of the LTα amino acid sequence shown in SEQ ID NO.1, and the transmembrane domain of the LTβ is located at positions 1-152 of the LTβ amino acid sequence shown in SEQ ID NO.

2.

3. The method according to claim 1, characterized in that The nucleotide sequence encoding mLTα1β2-His fragment described in S1 was constructed between the EcoRⅠ and NotⅠ restriction sites of the pPIC9K plasmid.

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

3.

5. The method according to claim 1, characterized in that The nucleotide sequence encoding the mLTα1β2-His fragment described in S1 is shown in SEQ ID NO.

4.

6. The method according to claim 1, characterized in that The Pichia yeast described in S4 is GS115.

7. The method according to claim 1, characterized in that The fermentation of S9 is as follows: the strain is inoculated into BMGY medium, shaken at 28°C and 250rpm to OD600=5, centrifuged at 4°C and 1500g for 3min, resuspended in 100ml BMMY medium to OD600=1, and methanol is added every 24h to make the final concentration of methanol 1%. After fermentation for 96 hours, the bacterial solution is centrifuged at 4°C and 12000rpm for 10min, and the supernatant of the bacterial solution is collected.

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

9. The method according to claim 1, characterized in that: The method further comprises S11, activity detection.

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