CN118909083A - Pig B cell proliferation induction ligand protein and preparation method and application thereof - Google Patents

Abstract

The invention discloses a ligand protein for inducing proliferation of pig B cells, a preparation method and application thereof, and belongs to the field of genetic engineering. The recombinant protein amino acid sequence without the transmembrane region is obtained after the secondary structure, the functional domain and the transmembrane region of the proliferation-inducing ligand protein are predicted, then a recombinant vector for expressing the recombinant protein is constructed and expressed and purified in an escherichia coli expression system, and the recombinant protein can ensure the survival of the pig B cells through identification, so that the recombinant protein has important scientific significance and value in the aspects of researching the action mechanism of the proliferation-inducing ligand protein on the infection of the porcine epidemic diarrhea virus, preparing the mucous membrane vaccine for preventing and controlling the porcine enterovirus diseases and the like.

Description

A pig B cell proliferation inducing ligand protein and its preparation method and application

Technical Field

The present invention relates to the field of genetic engineering, and in particular to a porcine B cell proliferation inducing ligand protein and a preparation method and application thereof.

Background Art

Porcine enterovirus diseases cause huge economic losses to the development of the pig industry, mainly including transmissible gastroenteritis (TGE), porcine epidemic diarrhea (PED), porcine delta coronavirus (PDCo) and porcine acute diarrhea syndrome coronavirus (SADS-Co). Among them, porcine epidemic diarrhea (PED) is a common porcine enterovirus disease caused by porcine epidemic diarrhea virus (PEDV). It is a highly contact enteric infectious disease that is mainly infected through the oral and nasal route. It can colonize and multiply in large numbers in intestinal epithelial cells, causing damage and shedding of intestinal epithelial cells and intestinal villi, thereby leading to severe diarrhea symptoms and dehydration and death of pigs. It is one of the common diseases that has long endangered the healthy development of the pig industry. Vaccination is currently the most economical and effective measure to prevent PED, but due to the continuous emergence of PEDV variants, the immune protection effect of the vaccine is poor. Therefore, it is urgent to develop safer and more efficient vaccines to prevent PED.

Studies have shown that efficient stimulation of mucosal immunity is crucial for the prevention and treatment of PED. Research on pig mucosal immune response is of great significance for the design of mucosal vaccines for the prevention and control of porcine enterovirus diseases. Vaccines mainly work through humoral immunity and cellular immunity. There are few studies on pig mucosal immune response, and the role of various cytokines involved in pig mucosal immune response is still unclear. It is known that cytokines are important proteins that regulate cell functions and are involved in biological processes such as immune response, cell proliferation and differentiation. With the development of biotechnology, new cytokines are constantly being discovered, and the understanding of their mechanism of action and biological functions is also deepening.

Proliferation-inducing ligand (APRIL) is a member of the tumor necrosis factor family. In human medical research, APRIL was found to be involved in B cell maturation and stimulation of plasma cell formation. However, there is no research on the expression and preparation of porcine APRIL protein, and its role in porcine mucosal immune response is still unclear. Although there is no research on the effect of APRIL on PEDV, research results show that host cytokines may play an important role in the molecular mechanism of PEDV infection and immune response. In the future, there may be more studies revealing the potential role of APRIL in PEDV infection. Based on the above problems, the problem to be solved by the present invention is to prepare porcine B cell proliferation-inducing ligand and identify its basic functions.

Summary of the invention

The purpose of the present invention is to provide a porcine B cell proliferation inducing ligand protein and a preparation method and application thereof, so as to solve the problems existing in the above-mentioned prior art. The present invention removes the transmembrane region of the APRIL protein to obtain the amino acid sequence of the APRIL recombinant protein, then constructs a recombinant vector, expresses and purifies it in an Escherichia coli expression system, and it is identified that the recombinant protein can ensure the survival of porcine B cells.

To achieve the above object, the present invention provides the following solutions:

The invention provides a porcine B cell proliferation inducing ligand protein, the amino acid sequence of which is shown in SEQ ID NO:1.

The present invention also provides a biomaterial related to a porcine B cell proliferation inducing ligand protein, including any one of the following:

(1) A nucleic acid molecule encoding the porcine B cell proliferation-inducing ligand protein according to claim 1;

(2) An expression cassette, recombinant vector or recombinant bacteria containing the nucleic acid molecule.

Preferably, the sequence of the nucleic acid molecule is as shown in SEQ ID NO:2.

The present invention also provides a method for preparing a porcine B cell proliferation inducing ligand protein, comprising the following steps:

Obtaining a gene sequence encoding a porcine B cell proliferation-inducing ligand protein, and constructing a recombinant vector with the gene sequence and an expression vector;

Expressing the recombinant vector in a recipient bacterium to obtain a recombinant bacterium;

The recombinant bacteria are induced to culture, and the porcine B cell proliferation inducing ligand protein is separated and purified from the bacteria.

Preferably, the gene sequence is as shown in SEQ ID NO: 2:

caacaaacagaattacagactctaagaagggaggttacccgcctgcaacgtaccggtggtccatcggaaaaaggtgagggcgacccgtggcaaaacctgtggggagcaagagcaaagcccggacggcgcggaagcctgggaaaatggcgaac gttctcgccgtcgccgagctgtgttgacccgcaagcagaaaaagaagcgctccgtgttgcacctggttccgaccaacatcaccagcaaagaggatagcgatgtgacggaactgatgtggcagccggcgttaaaacgtggccgtggtctggag gcgcagggctacttcgttcgtgtttgggatgctggtgtatacctgctgtattcccaggttctcttccacgacgtcaccttcaccatgggtcaagttgtcagccgtgaaggccagggtcgtcaggagactctgtttcgttgtattcgctctat gccgagcaatccggactgggcatataacagctgctacagcgcaggcgtgtttcatttgcatcagggcgatattctgagcgttgtcatcccgagagcgcgtgcgaaattgtccctgtcaccgcatggtacgtttctgggtgtggtgaagctt.

Preferably, the recipient bacteria is Escherichia coli.

Preferably, the induction culture conditions of the recombinant bacteria are: induction with isopropylthiogalactoside (IPTG) at an induction concentration of 0.2 mmol/L-1.0 mmol/L; culturing at 16° C. and 200 rpm for 8-24 hours and harvesting the bacteria.

Preferably, the separation and purification comprises the following steps:

The collected bacteria were resuspended, disrupted by ultrasound, and the supernatant was collected;

After filtering the supernatant, a nickel column was added, and eluted with 20 mM-500 mM imidazole eluent, and eluents of different concentrations were collected.

The present invention also provides the use of the porcine B cell proliferation inducing ligand protein in any of the following items:

(1) Application in the preparation of products for improving the survival rate of pig B cells;

(2) Application in studying the mechanism of action of porcine mucosal immune response;

(3) Application in studying the mechanism of action of porcine B cell proliferation-inducing ligand protein in porcine epidemic diarrhea virus infection.

The present invention discloses the following technical effects:

The present invention obtains the amino acid sequence of the recombinant protein with the transmembrane region removed by predicting the secondary structure, functional domain and transmembrane region of the proliferation-inducing ligand protein. Based on genetic engineering technology, the present invention constructs a recombinant vector expressing the proliferation-inducing ligand recombinant protein with the transmembrane region removed, and expresses and purifies it in an Escherichia coli expression system. It is identified that the proliferation-inducing ligand recombinant protein with the transmembrane region removed inhibits the proliferation of IgM ⁺ B cells, but effectively ensures the survival of B cells, which has important scientific significance and research value for further studying the role of proliferation-inducing ligand in the infection and immune response of PEDV.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

Figure 1 shows the secondary structure of the APRIL protein;

FIG2 shows the functional domains of APRIL protein;

Figure 3 shows the results of double enzyme digestion electrophoresis of the recombinant plasmid; M: KB Ladder, 1: pCold-SUMO plasmid, 2: pCold-SUMO-APRIL plasmid after digestion with ECoRI and HindIII;

FIG4 is a schematic diagram of constructing the pCold-SUMO-APRIL plasmid;

Figure 5 shows the screening results of inducible expression conditions of soluble pCold-SUMO-APRIL expressed from E. coli BL21 (DE3); A: screening of different IPTGs, B: screening of different OD values, C: screening of different sampling times; where M: protein marker, s: supernatant after ultrasound, p: precipitate after ultrasound;

Figure 6 is the purification and identification results of the recombinant protein; A is the SDS-PAGE identification result of the purified recombinant protein, lane 1: protein marker, lane 2: whole bacteria after induction, lane 3: supernatant after induction, lane 4: precipitate after induction, lane 5: flow-through, lanes 6-10: 300mM elution collection solution, lanes 11-15: 500mM elution collection solution, lane 16: dialyzed concentrated protein; B is the Western blot identification result of the purified pCold-SUMO-APRIL, lane 1: protein marker, lane 2: empty load, lane 3: purified protein; C is the purified protein coated with different concentrations and reacted with anti-His tag antibodies to construct different protein concentration-His reaction curves;

FIG. 7 is the result of functional identification of the recombinant protein; A: Effects of different concentrations of APRIL protein on the expression of BCMA and CD40, B: Effects of different concentrations of APRIL protein on the proliferation and survival of B cells.

DETAILED DESCRIPTION

Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and/or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

Example 1

1. Materials

(1) Experimental Materials

The pCold-SUMO and pCold-SUMO-APRIL recombinant expression plasmids were preserved by the Veterinary Research Institute of Jiangsu Academy of Agricultural Sciences. Escherichia coli competent BL21 was purchased from Quanshijin Biotechnology Co., Ltd.; HisTrap HP was purchased from Cytiva; 10kd dialysis bag and dialysis clip, ampicillin, and red blood cell lysis buffer were purchased from Solarbio; porcine peripheral blood lymphocyte separation solution kit was purchased from Jinshi Haoyang Biological Products Technology Co., Ltd.; BCA protein concentration determination kit was purchased from Biyuntian; cell filter (40μm/100μm cell strainer) was purchased from Biologix; TRIzol was purchased from Takara; reverse transcriptase was purchased from Norvegian; flow tube was purchased from Labre; gauze, 15mL centrifuge tube and 50mL centrifuge tube were purchased from SAINING; MACS MultiStand, MACS ^TM Separator, Whole Blood Column Kit, Anti-Mouse IgG MicroBeads were purchased from Miltenyi; Anti-bovine sIgM MAb was purchased from Kingfisher Biotech; CD40 Antibody/anti-human/PE, CD269 (BCMA) Antibody, streptavidin-FITC, eBioscience ^TM CFSE Proliferation Dye were purchased from Invitrogen; 1640 culture medium was purchased from Yuanpei; PBS phosphate powder was purchased from Biosharp; chloroform was purchased from Keshi; isopropanol was purchased from Damao; glycerol was purchased from Komiou; ethanol was purchased from Hushi, IPTG was purchased from Biosharp; Fetal Bovine Serum was purchased from VivaCell. Fluorescence quantitative machines, flow cytometers and other instruments are all owned by the Jiangsu Academy of Agricultural Sciences, and sequencing was completed by Nanjing Qingke Biological Company.

(2) Culture medium

1L LB medium contains 10g NaCl, 10g tryptone and 5g yeast extract.

1L Coomassie Brilliant Blue staining solution contains 2.5g R250, 450mL methanol, 100mL glacial acetic acid and 450mL pure water.

1L of decolorizing solution contains 100mL of glacial acetic acid, 300mL of methanol and 600mL of pure water.

1 L of coating solution (carbonate buffer) contains 1.59 g of Na ₂ CO ₃ and 2.94 g of NaHCO ₃ .

1L Binding buffer contains 0.5M NaCl, 0.02MH ₂ PO ₄ .2H ₂ O, 20mM/100mM/500mM imidazole, pH=7.5-8.0.

2. Construction of truncated prokaryotic expression vector

After predicting the secondary structure, functional domain and transmembrane region of APRIL protein, the amino acid sequence without the transmembrane region was obtained, and the upstream and downstream restriction sites (KpnI, SalI) were designed according to the existing prokaryotic expression vector pCold-SUMO (purchased from Wuhan Miaoling Biotechnology Co., Ltd.) in the laboratory, and the sequence was optimized and synthesized and cloned into pCold-SUMO (GenScript Biotechnology Co., Ltd.) to construct the recombinant plasmid. The recombinant plasmid was transformed into BL21 (DE3) competent cells. Take the competent cells of Escherichia coli BL21 (DE3) and place them on ice to melt, add about 40 ng of the recombinant vector and mix evenly, and place them on ice for 20 minutes; heat shock the competent cells after adding the recombinant vector in a 42°C water bath for 90 seconds, and then transfer them to ice for 2 minutes. In the clean bench, add 600 μL LB culture medium (without resistance) to the competent cells containing the recombinant vector, and culture them at 37°C and 200 rpm for 1 hour. The cultured bacterial liquid is centrifuged at 12000 rpm for 10 minutes, the supernatant is discarded, and 100 μL of the culture liquid is retained for resuspending, and then added dropwise to the LB solid culture medium containing ampicillin resistance. Use a disposable sterile coating rod to evenly spread the culture liquid, invert it in a 37°C constant temperature incubator and culture it for 12-16 hours, pick a single colony, name it BL21 (pCold-SUMO-APRIL), and complete verification through bacterial liquid PCR, double enzyme digestion identification and sequencing.

3. Induced expression and identification of recombinant proteins

3.1 Screening of IPTG induction conditions

The positive strains identified correctly were inoculated into LB liquid medium containing ampicillin and cultured at 37°C and 200rpm for 6-8h. The culture was expanded at a volume ratio of 1:100. When the bacteria reached the logarithmic growth phase (OD _600nm ≈0.4-0.6), different concentrations of IPTG (0mmol/L, 0.2mmol/L, 0.4mmol/L, 0.6mmol/L, 0.8mmol/L, 1.0mmol/L) were added to induce BL21 (pCold-SUMO-APRIL) at 16°C for 30min, and the bacteria were harvested after culture at 16°C for 24h. After the culture is completed, centrifuge at 8000rpm for 6min at room temperature, discard the supernatant, wash, add 1/2PBS to resuspend the bacteria, centrifuge, discard the supernatant, concentrate 5 times to resuspend the bacteria; place on an ice-water mixture, use an ultrasonic disruptor for ultrasonic disruption, the ultrasonic power is 30%, ultrasonic for 3s and 5s, and ultrasonic for 1-2min; centrifuge at 4°C and 8000rpm for 6min, collect the supernatant and precipitate of the induced strain; add 5× Loading Buffer, denature in a water bath for 10min, and perform 10% SDS-PAGE analysis.

3.2 OD value screening

The culture was expanded at a volume ratio of 1:100, and cultured at 37°C, 200 rpm for 1.5 h, 2 h, 2.5 h, 3 h, 3.5 h, and 4 h (corresponding to OD _{600 nm} values ≈ 0.196, 0.325, 0.642, 1.023, 1.337, and 1.537), respectively. Expression was induced with the optimal IPTG concentration: 0.8 mM, and the bacteria were cultured at 16°C for 24 h, harvested, and ultrasonically disrupted. Samples were prepared for SDS-PAGE analysis.

3.3 Selection of sampling time

When the optimal induction IPTG concentration and OD value are known, the sampling time steps are as follows: expand the culture at a volume ratio of 1:100, culture at 37°C, 200rpm for 2h, then induce with 0.8mM IPTG, culture at 16°C for 8h, 12h, 16h, 20h and 24h, respectively, harvest the bacteria, ultrasonically disrupt them, prepare samples, and perform SDS-PAGE analysis.

4. Recombinant protein purification

When the soluble expression level of the recombinant bacteria was relatively stable, the recombinant protein was purified. After BL21 (pCold-SUMO-APRIL) induced expression, the bacteria were collected, resuspended in PBS (diluted to a concentration of 0.2 mg/mL), ultrasonically disrupted, and the supernatant was collected and filtered through a 0.45 μm filter. The nickel column and peristaltic pump were combined to pass through 5 times the column volume (5V) of 500mM Binding buffer (2.5mL/min), 5V _H2O , and 5V 20mM Binding buffer in sequence. The sample flow rate was (1mL/min). Gradient elution was used, i.e., 5V 20mM Binding buffer, 5V 50mM Binding buffer, 5V 100mM Binding buffer, 5V 200mM Binding buffer, 5V 300mM Binding buffer, 5V 500mM Binding buffer, 5V 20mM Binding buffer, 5V water, 5V 7V 100mM Binding buffer, 5V 200mM Binding buffer, 5V 300mM Binding buffer, 5V 500mM Binding buffer, 5V 20mM Binding buffer, 5V water, 5V 20mM Binding buffer, 5V 500mM Binding buffer, 5V 20mM Binding buffer, 5V 500mM Binding buffer, 5V 20mM Binding buffer, 5V water, 5V 70 ... 20% ethanol, collect different concentrations of eluate, prepare samples, and perform SDS-PAGE analysis. The eluted protein was dialyzed and concentrated, and then analyzed by SDS-PAGE, Western blot and ELISA. Western blot used primary His tag antibody (1:5000), secondary HRP-labeled goat anti-mouse IgG (1:5000), ELISA coated purified protein at different concentrations at 4°C overnight (4μg/mL, 2μg/mL, 1μg/mL, 0.5μg/mL, 0.25μg/mL, 0.125μg/mL, 0.063μg/mL, 0μg/mL), 5% skim milk powder was blocked at 37°C for 2h, primary His tag antibody (1:10000) at 37°C for 2h, secondary HRP-labeled goat anti-mouse IgG (1:10000) at 37°C for 1h, BCA measured protein concentration, labeled and packaged for freezing.

5. Functional identification of recombinant protein

5.1 Isolation of peripheral blood lymphocytes (PBMC)

Take 5mL of separation solution and put it into a 15mL centrifuge tube. Add 5mL of fresh porcine whole blood to the upper layer of separation solution. Centrifuge at 4℃ and 400g/min for 30min. Then, aspirate the mononuclear cell interface layer, wash with PBS containing triple antibody (containing penicillin, streptomycin and amphotericin mixed solution, item number: Solebo P7630; volume ratio 1:100), centrifuge at 4℃ and 400g/min for 10min, wash 3 times, add appropriate amount of red blood cell lysis solution and react at room temperature for 5min, stop lysis, centrifuge and discard the supernatant, wash 3 times, measure the cell number with a cell counting plate, plate at a density of 1×10 ⁷ /mL, and culture with 1640 complete medium containing 10% serum. IgM ⁺ B cells were separated by magnetic column separation, washed 3 times, resuspended, counted and plated for use.

5.2 Cell proliferation assay (CFSE)

The obtained lymphocytes were resuspended with 1 mL of PBS containing 0.2% serum, 1 μL of CFSE dye was added, and incubated at 37°C for 10 min. Five times the volume of 1640 medium containing 10% serum was used for ice bath for 5 min, and then centrifuged at 400 g/min at 4°C for 10 min. The supernatant was discarded, and the cells were washed with PBS containing 0.2% serum and plated. The experimental protein was added, and the medium was changed semi-quantitatively every 2-3 days. The samples were collected after 7 days, and the cell proliferation was detected by flow cytometry.

5.3 RT-qPCR

RNA was extracted using Trizol. Trizol was added at a volume ratio of 1:1 and allowed to stand for 5 minutes, and then the sample was collected by blowing. Chloroform was added at a volume ratio of 4:1, and vortexed and allowed to stand until stratification occurred. The sample was then centrifuged at 4°C and 12,000 rpm for 10 minutes. The supernatant was transferred and isopropanol was added at a volume ratio of 1:1 and mixed by inversion until the transparent flocs disappeared. The sample was then precipitated at -20°C for 30-60 minutes, and then centrifuged at 4°C and 12,000 rpm for 10 minutes. The supernatant was discarded, and pre-cooled 75% ethanol was added. After washing twice, the supernatant was discarded, and 25 μL H ₂ O was added for re-suspending and centrifugation to obtain total RNA. According to the reverse transcriptase and fluorescence quantitative operation instructions of Novozyme, the expression of the transcription level of downstream factors was detected by relative quantitative method, and statistical analysis was performed. The primers were synthesized by Nanjing Qingke Biological Company. The specific primer sequences are shown in Table 1.

Table 1 Fluorescence quantitative PCR primers

5.4 Flow cytometry

The cell samples were transferred to flow cytometers, incubated with primary antibody BCMA ⁺ CD40 ⁺ for 30 min at 4°C in the dark, washed and centrifuged twice with 1 mL of PBS containing 0.2% BSA, incubated with secondary antibody streptavidin-FITC for 30 min at 4°C in the dark, washed and centrifuged twice with 1 mL of PBS containing 0.2% BSA, tested on the flow cytometer according to the operating instructions, and data processed by Flowjo software.

6. Results

6.1 Construction and identification of recombinant expression plasmids

As shown in Figures 1 to 2 and Table 2, the results show that the APRIL protein has multiple helical and folded structures, its functional domain is located at amino acids 117-251aa, and the transmembrane region is located at 27-49aa. The amino acid sequence of the truncated APRIL protein (SEQ ID NO: 1) obtained by removing the sequence of the transmembrane region (50-251aa) is:

QQTELQTLRREVTRLQRTGGPSEKGEGDPWQNLWEQEQSPDGAEAWENGERSRRRRAVLTRKQKKKRSVLHLVPTNITSKEDSDVTELMWQPALKRGRGLE AQGYFVRVWDAGVYLLYSQVLFHDVTFTMGQVVSREGQGRQETLFRCIRSMPSNPDWAYNSCYSAGVFHLHQGDILSVVIPRARAKLSLSPHGTFLGVVKL.

As shown in Figure 3, the constructed recombinant plasmid was successfully verified by double enzyme digestion, the band size was consistent with the expectation, and the DNA sequencing results showed that the sequence was correct. As shown in Figure 4, it shows a schematic diagram of the construction of pCold-SUMO-APRIL.

Table 2 APRIL protein structure

name Starting amino acid position End amino acid position E-value Transmembrane domain 27 49 N/A Functional domain (TNF) 117 251 0.00246

6.2 Screening of inducible expression conditions for recombinant proteins

As shown in Figure 5 AC, the results showed that pCold-SUMO-APRIL was stably expressed in soluble form at OD ₆₀₀ ≈0.5-0.6, induced by 0.8 mM IPTG, and cultured at 16°C and 200 rpm for 24 h, with a size of about 40 ku, which was consistent with the expected size.

6.3 Purification and identification of recombinant protein

As shown in Figure 6A, the results showed that the eluate obtained by eluting the protein in 500mM eluent was dialyzed and concentrated, and the concentration measured by BCA was 0.1mg/mL; as shown in Figure 6B, the Western blot identification results showed that pCold-SUMO-APRIL reacted with the anti-His tag antibody, and a band appeared at 41.6ku, which was consistent with the expected size; as shown in Figure 6C, the ELISA identification results showed that pCold-SUMO-APRIL reacted with the anti-His tag antibody, and different protein concentration-His reaction curves were constructed.

6.4 Functional identification of recombinant proteins

As shown in Figure 7A, 10 ng/mL and 100 ng/mL of APRIL protein both effectively upregulated the expression of BCMA and slightly upregulated the expression of CD40. Figure 7B shows that APRIL protein inhibited the proliferation of IgM ⁺ B cells, but effectively ensured the survival of B cells.

The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims (9)

1. A pig B cell proliferation-inducing ligand protein, which has an amino acid sequence as shown in SEQ ID NO: 1.

2. A biological material associated with a porcine B cell proliferation-inducing ligand protein comprising any one of the following:

(1) A nucleic acid molecule encoding the porcine B cell proliferation-inducing ligand protein of claim 1;

(2) An expression cassette, a recombinant vector or a recombinant bacterium containing the nucleic acid molecule.

3. The biomaterial of claim 2, wherein the nucleic acid molecule has the sequence set forth in SEQ ID NO: 2.

4. A method for preparing a pig B cell proliferation-inducing ligand protein, comprising the steps of:

obtaining a gene sequence for encoding a pig B cell proliferation induction ligand protein, and constructing a recombinant vector by the gene sequence and an expression vector;

expressing the recombinant vector in a recipient bacterium to obtain a recombinant bacterium;

and (3) carrying out induction culture on the recombinant bacteria, and separating and purifying from the bacteria to obtain the pig B cell proliferation induction ligand protein.

5. The method of claim 4, wherein the gene sequence is as set forth in SEQ ID NO: 2.

6. The method according to claim 4, wherein the recipient bacterium is Escherichia coli.

7. The method according to claim 4, wherein the conditions for the induction culture of the recombinant bacteria are as follows: inducing with isopropyl thiogalactoside at an induction concentration of 0.2-1.0 mmol/L; culturing at 16deg.C and 200rpm for 8-24 hr for bacterial recovery.

8. The method of claim 4, wherein the separation and purification comprises the steps of:

suspending the collected bacteria again, performing ultrasonic crushing, and collecting supernatant;

Filtering the supernatant, adding the filtered supernatant into a nickel column, eluting with 20mM-500mM imidazole eluent, and collecting eluents with different concentrations.

9. Use of a porcine B cell proliferation-inducing ligand protein according to claim 1 in any of the following:

(1) The application in preparing the product for improving the survival rate of the B cells of the pigs;

(2) The application in researching the action mechanism of the pig mucous membrane immune response;

(3) The application of the ligand protein for inducing the proliferation of the pig B cells in researching the action mechanism of the ligand protein in the infection of the porcine epidemic diarrhea virus is disclosed.