Polyclonal antibody of macrophage migration inhibition factor of sea urchin globosus, antigen protein and preparation method of polyclonal antibody of macrophage migration inhibition factor of sea urchin globosus

Abstract

The invention provides a sea urchin globosus macrophage migration inhibition factor polyclonal antibody, an antigen protein and a preparation method thereof, and belongs to the technical field of antibody preparation. According to the invention, the coding gene and the recombinant expression vector of the sea urchin MIF antigen protein are successfully obtained, and the sea urchin MIF antigen protein can be prepared by the method provided by the invention; after Kunming mice are immunized with the obtained sea urchin globosus MIF antigen protein, the sea urchin globosus MIF polyclonal antibody is successfully obtained, the problem that in the prior art, a sea urchin globosus MIF antibody is lacked is solved, and a meaningful tool and experimental support are provided for biological function research of sea urchin MIF. The invention further provides a gene sequence for coding the sea urchin globosus MIF, the complete sequence of the gene coding region of the MIF can be successfully amplified through the primer provided by the invention, and the blank of related research is filled.

Description

Technical Field

[0001] The invention relates to the technical field of antibody preparation, and in particular to a polyclonal antibody and antigen protein of a macrophage migration inhibitory factor of an echinococcus cyanobacterium. Background Art

[0002] Macrophage Migration Inhibitory Factor (MIF) is a cytokine with multiple biological functions, closely related to immune regulation, inflammatory response, tumorigenesis, etc. It exists in a unique trimer structure, which gives it strong biological activity and stability. It regulates cells and microenvironment through autocrine or paracrine pathways, binds to different receptors on the cell membrane as a signal molecule, and thus plays a corresponding role in different cells.

[0003] In higher vertebrates, the functions and related regulatory mechanisms of macrophage migration inhibitory factor have been studied more maturely. However, for more primitive biological groups, such as sea urchins, research on MIF is relatively scarce. In particular, basic research on the MIF protein of Echinococcus ilustica, especially the exploration of MIF antibody preparation, is still blank. Therefore, the preparation of polyclonal antibodies against MIF of Echinococcus ilustica will undoubtedly fill the gap in this field. Such antibodies can not only be used as specific probes to track and identify the expression and distribution of MIF in Echinococcus ilustica, but also provide strong experimental support for in-depth research on the mechanism of action of MIF in the growth and development, immune response, disease resistance and other aspects of Echinococcus ilustica. Summary of the invention

[0004] The purpose of the present invention is to provide a polyclonal antibody and antigen protein of macrophage migration inhibitory factor (MIF) of sea urchin, and a preparation method thereof, so as to solve the problem of lack of MIF antibody of sea urchin in the prior art.

[0005] As a primary purpose, the present invention protects an Echinococcus sphaeroides macrophage migration inhibitory factor MIF antigen protein, the amino acid sequence of which is as shown in SEQ ID NO.1; or the amino acid sequence of the antigen protein formed by substituting, deleting or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO:1.

[0006] At the same time, the present invention also requests protection for a gene encoding the MIF antigen protein of the sea urchin Echinococcus leucoderma, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0007] The invention also provides a recombinant expression vector of the Echinococcus glabrata MIF antigen protein, comprising the above gene and an initial vector pColdⅡ, wherein the gene is inserted between the NdeI and HindIII restriction sites of the initial vector.

[0008] The present invention also provides a method for preparing the MIF antigen protein of Echinococcus glabrata, comprising the following steps:

[0009] The recombinant expression vector is transformed into Escherichia coli for screening to obtain a strain containing the recombinant plasmid.

[0010] The above strains are inoculated into the culture medium for growth and induction to express the target protein, the bacteria are collected and the cells are lysed to release the target protein.

[0011] The protein mixture was purified using a nickel ion affinity purification column, and the concentration of the purified protein was measured using the BSA grayscale analysis method.

[0012] The present invention also provides a polyclonal antibody against Echinococcus glabrata MIF, wherein the polyclonal antibody is obtained by immunizing mice with the antigen protein.

[0013] The present invention also provides a complete sequence of the coding region of the MIF gene of the Echinococcus glabrata, as shown in SEQ ID NO.3.

[0014] The present invention also provides a primer pair for amplifying the full sequence of the MIF gene coding region. The sequence of the primer pair is shown in SEQ ID NOs. 4-5.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0016] (1) The present invention successfully developed and isolated and purified a macrophage migration inhibitory factor antigen protein from the sea urchin Echinococcus leucoderma. The protein exhibited excellent immunogenicity. By immunizing mice with it, we successfully induced the production of highly specific polyclonal antibodies against the sea urchin MIF. The MIF polyclonal antibody provides a valuable research tool for in-depth exploration of the biological function and mechanism of action of sea urchin MIF at the protein level, greatly promoting research progress in related fields.

[0017] (2) The present invention also successfully cloned and identified the gene sequence encoding the MIF antigen protein of the Echinococcus ilonicus, and further constructed a corresponding recombinant expression vector system. The system can efficiently and stably express and purify the MIF antigen protein of the Echinococcus ilonicus, laying a solid foundation for the large-scale preparation of the protein to meet the needs of scientific research and potential applications.

[0018] (3) More importantly, the present invention also analyzed and obtained the complete coding region sequence of the Echinococcus glabrata MIF gene for the first time, and designed and synthesized a pair of specific primers for efficient amplification of the complete coding region of the MIF gene. This study not only fills the gap in the information of the sea urchin MIF gene sequence, but also provides an indispensable key resource for the subsequent analysis of the sea urchin MIF gene function and the exploration of its expression regulation mechanism. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 for amino acid sequence alignment;

[0020] Figure 2 IPTG was used to induce the expression of recombinant proteins;

[0021] Figure 3 Western blotting of recombinant protein samples (histone tag antibody is the primary antibody);

[0022] Figure 4 ELISA was used to detect antibody titer;

[0023] Figure 5 The polyclonal antibody prepared was used to detect the expression of MIF protein in the coelomocytes of Echinococcus glabrata by western blotting. DETAILED DESCRIPTION

[0024] In order to more clearly explain the purpose, technical solutions and advantages of the present disclosure, the embodiments of the present disclosure will be described in detail below in conjunction with the accompanying drawings. It should be understood that the following description of the embodiments is intended to explain and illustrate the overall concept of the present disclosure, and should not be understood as limiting the present disclosure.

[0025] Example 1 Amplification of the complete sequence of the coding region of the Echinococcus glabrata MIF gene and codon optimization

[0026] The total RNA of the coelomocytes of the sea urchin Echinococcus leucoderma was extracted by the Trizol method, and the purity and integrity of the RNA were observed by agarose gel electrophoresis and ultra-micro spectrophotometer, and transcriptome sequencing and annotation were performed (transcriptome sequencing and annotation were completed by Beijing Novogene Technology Co., Ltd.). Primer pairs were designed according to the MIF transcript sequence in the transcriptome sequencing results (Table 1), and the cDNA obtained by reverse transcription was used as a template to perform PCR amplification of the target fragment and sequence it. The amino acid sequence deduced from the sequencing results was consistent with the amino acid sequence deduced from the transcriptome MIF transcript sequence. The nucleotide sequence (417 bp) of the coding region of the sea urchin macrophage migration inhibitory factor (MIF) gene of the sea urchin Echinococcus leucoderma was obtained as follows:

[0027] ATGCCGCTTGCAGTATTTGTAACGAATGTGGATATGAGCTCGACAATGGAAGAATTTGCCACAGGCCTCACCCACATATTATCGGACAGACTGCATCGGGAAGAGGAGAAAATAACTGTTCACATTCAGCCGAATCAGTTCATGCTTCGTGGGGGTTCCACTGACCCTGCTGGGTACGTCTCATTGTGCACATCCCGTGGCTTTGGAGACG TAGAGCACAGGAGAGACACTTCTCAAAAGATCTTGGACTTCATCAAGGAACAACTTAAACTTAAAAATCCTTCCAGGTTCATGGTATATATGCACACGATGTCAGCAGATGATATCGGCATCGATGGCGGTCTGGTATCCGATAGGTTTGGTTGCATGGGCCAAGGAGGACCGTTTGATGTCAGATGGCACAAATTGACCAAATAA (as in SEQ ID NO.3 shown). The deduced amino acid residue sequence of this sequence ( Figure 1 The upper row) was subjected to BLAST with the NCBI gene database data, and the amino acid residue sequence of MIF of purple sea urchin [Strongylocentrotuspurpuratus] (XP_799963.1) with the highest similarity ( Figure 1 The comparison between the two is 94% similar (such as Figure 1 shown).

[0028] The primer sequences in Example 1 are shown in Table 1 below:

[0029] Table 1 Primer sequences

[0030]

[0031]

[0032] The rare codons contained in the coding region of the above-mentioned sea urchin macrophage migration inhibitory factor gene were optimized. The optimized coding sequence: (417 bp, nucleotide sequence:

[0033] ATGCCGCTGGCGGTTTTCGTTACCAACGTTGATATGAGCAGCACCATGGAAGAATTCGCGACCGGCCTGACCCACATCCTGAGCGATCGTCTGCACCGTGAAGAAGAAAAAATCACCGTTCACATCCAGCCGAACCAGTTCATGCTGCGTGGCGGCAGCACCGATCCGGCGGGCTACGTTAGCCTGTGCACCAGCCGTGGCTTCGGTGATGTTGAACACCGTCGTGATACCAGCCAGAAAATCCTGGATTTCATCAAAGAACAGCTGAAACTGAAAAACCCGAGCCGTTTCATGGTTTACATGCACACCATGAGCGCGGATGATATCGGCATCGATGGCGGCCTGGTTAGCGATCGTTTCGGTTGCATGGGTCAGGGCGGCCCGTTCGATGTTCGTTGGCACAAACTGACCAAATAA (as shown in SEQ ID NO.2). The in vitro synthesized fragment was completed by Shanghai Sangon Biotechnology Co., Ltd.

[0034] Example 2 Induced expression

[0035] The optimized coding sequence obtained in Example 1 was used to construct a recombinant expression vector pColdⅡ-MnMIF, with cloning sites of NdeI and HindIII. The recombinant plasmid was transformed into Escherichia coli TOP10, and resistance screening was performed with ampicillin. The obtained recombinant transformants were sequenced (sequencing was completed by BGI Co., Ltd.). The correctly sequenced transformants were stored in glycerol.

[0036] 2.1 Bacterial Activation

[0037] In a sterile clean bench, use an inoculation loop to dip a small amount of recombinant E. coli TOP10 glycerol bacteria, and inoculate it into LB solid plate culture medium containing Amp with a final concentration of 100 μg / mL by streak method, and culture it at 37°C overnight for no more than 16 hours.

[0038] 2.2 Liquid culture of recombinant strains

[0039] In a sterile clean bench, use a 10 μL sterile white pipette tip to pick up a single colony of the recombinant bacteria grown on the plate, inoculate it into 10 mL of LB liquid culture medium containing Amp at a final concentration of 100 μg / mL, and culture it overnight at 37°C and 180 rpm in a shaker. The culture time should not exceed 16 h.

[0040] In a sterile clean bench, use a sterile pipette tip to inoculate 1% of the overnight culture into 200 mL of LB liquid medium containing Amp at a final concentration of 100 μg / mL, and culture at 37°C, 180 rpm in a shaker until the OD of the culture reaches 600 When the concentration reaches about 0.5-0.6, incubate in an ice water bath for 30 minutes.

[0041] 2.3 Inducible expression of recombinant protein

[0042] In a sterile clean bench, mark the control group 1 and experimental groups 2-6. The control group is a bacterial solution without adding any inducer, and the experimental groups 2-6 are added with IPTG inducer with final concentrations of 0.1mM, 0.25mM, 0.5mM, 0.75mM, and 1mM, respectively, and cultured in a constant temperature shaker at 15°C and 180rpm for 24h.

[0043] 2.4 Total protein extraction

[0044] Collect the induction-completed bacteria into a centrifuge tube and weigh and record. Add non-denaturing lysis buffer (reduction-resistant) at a mass-volume ratio of 1:6, add a protease inhibitor mixture (100×) to the non-denaturing lysis buffer at a volume ratio of 1:100, so that the final concentration of the protease inhibitor mixture (100×) is 1×, and mix it repeatedly by blowing. Then add 100 mg / mL lysozyme solution to the mixed solution of bacterial precipitation and lysis buffer to a final concentration of 1 mg / mL. Use a pipette to absorb the solution and repeatedly blow the bacteria to make the bacterial mud completely dissolved in the lysis buffer and other systems, collect it in a 10mL Ep tube and place it on ice for 30 minutes. Set the parameters of the ultrasonic cell disruptor to 4°C, power 20%, 5min, open 3s, close 6s, put the Ep tube containing the bacterial solution into an ice water bath for ultrasonic disruption, and alternately ultrasonicate the experimental group and the control group for 5 minutes until the solution becomes clear and transparent. The solution was divided into 2 mL centrifuge tubes and balanced, and centrifuged at 4°C, 15,000 g, for 20 min. The supernatant and precipitate were collected and stored in a -20°C refrigerator to avoid repeated freezing and thawing.

[0045] 2.5 SDS-PAGE of recombinant proteins

[0046] Precipitation: Use a 10μL white pipette tip to take a small amount of precipitation and blow it into 20μL of 1×SDS, add 5μL of 5×loding buffer, mix well, and seal with sealing film.

[0047] Supernatant: Take 20 μL of supernatant, add 5 μL of 5× loading buffer, mix well, and seal with sealing film.

[0048] The prepared samples were placed in boiling water and boiled for 10 minutes, and then immediately placed on ice. SDS-PAGE was performed on the non-denatured lysis supernatant and precipitate of the bacteria to confirm that the recombinant protein was expressed in the non-denatured lysis supernatant or precipitate. Figure 2 As shown ( Figure 2 (M: Marker; 1: non-denaturing lysis supernatant of control group; 2: non-denaturing lysis precipitate of control group; 3: non-denaturing lysis supernatant of experimental group; 4: non-denaturing lysis precipitate of experimental group).

[0049] Example 3 Nickel column affinity purification and identification of recombinant proteins using histone tag antibodies

[0050] In this embodiment, nickel column affinity chromatography was used to purify the recombinant protein expressed in the supernatant. After purification, the sample was analyzed by SDS-PAGE, and a weak band was found at a molecular weight of about 30 kDa. Since mammalian studies have found that MIF often appears in the form of aggregates, it is speculated that the band may be a polymer of MIF. The histone tag antibody carried by the recombinant protein was used for protein immunoblotting analysis, and the specific operation steps are as follows:

[0051] 3.1 After sample preparation, place in boiling water bath for 10 min, and load 10 μL of sample into each gel well. Electrophoresis conditions: 60V for stacking gel, 110V for separation gel.

[0052] 3.2 Transfer

[0053] This embodiment adopts the wet transfer method, and clean gloves are used during the transfer process. Put the special instruments for transfer such as sponge, filter paper, glass rod, flat tweezers into the transfer box, and soak them with 1× transfer buffer for standby use. After the electrophoresis is completed, cut the gel according to the pre-stained Marker and cut the PVDF membrane of the same size as the gel. The PVDF membrane is transferred to 1× transfer buffer after soaking and activating in methanol solution for 3min. Fit the protein gel to the membrane tightly to avoid bubbles. Install it in the order of a layer of sponge, three layers of filter paper, gel, PVDF membrane, three layers of filter paper, and a layer of sponge and place it in the electrotransfer tank, and fill the tank with 1× transfer buffer. The transfer condition is a constant current of 170mA and a transfer of 1h 40min.

[0054] 3.3 Antigen-antibody reaction

[0055] Blocking: After transfer, remove the PVDE membrane and incubate it in 5% skimmed milk powder for 1 hour.

[0056] Binding to primary antibody: Take out the PVDF membrane, put it into a hybridization bag containing MIF primary antibody dilution solution and seal it. Be careful not to have bubbles in the hybridization bag, and incubate it at 4℃ overnight.

[0057] Primary antibody washing: Take out the PVDF membrane and wash it in TBST solution for 6 times, 10 min each time.

[0058] Binding secondary antibody: remove the PVDF membrane, place it in HRP-labeled secondary antibody solution, and incubate for 1 hour.

[0059] Secondary antibody washing: Take out the PVDF membrane and wash it in TBST solution for 6 times, 10 min each time.

[0060] ECL development: prepare the developer solution, i.e., solution A: solution B = 1:1, mix thoroughly and then drop it onto the PVDF membrane, incubate at room temperature for 3 minutes, and collect images according to a certain exposure time.

[0061] The results are as follows Figure 3 As shown, it was confirmed that the band at about 30 kDa was not a mixed band, but was also an induced expressed recombinant protein.

[0062] Example 4 Immunization of animals and analysis of antibody titers

[0063] With recombinant protein (recombinant protein sequence:

[0064] MPLAVFVTNVDMSSTMEEFATGLTHILSDRLHREEEKITVHIQPNQ

[0065] FMLRGGSTDPAGYVSLCTSRGFGDVEHRRDTSQKILDFIKEQLKL

[0066] KNPSRFMVYMHTMSADDIGIDGGLVSDRFGCMGQGGPFDVRWHKLTK, as shown in SEQ ID NO.1) solution was used as antigen, and after rapid mixing with antibody adjuvant, 6-8 week old Kunming mice (purchased from Dalian Medical University Experimental Animal Center) were immunized twice. The first immunization dose for each Kunming mouse was 20 μg of recombinant Echinococcus leucophylla MIF protein. The second immunization was performed on the 21st day after the first immunization. The immunization dose for each Kunming mouse was 20 μg of recombinant Echinococcus leucophylla MIF protein. Serum was collected on the 35th day after the first immunization (the adjuvant used in the two immunizations was a five-week standard mouse monoclonal antibody / polyclonal antibody adjuvant, English name SoFastAntibody-Mouse5W, manufacturer: Beijing Solarbio Life Sciences Technology Co., Ltd.), and mouse polyclonal antibodies were obtained. The titer was determined by enzyme-linked immunosorbent assay, and the results are shown in the figure. Figure 4 shown.

[0067] Example 5 Analysis of the Expression of MIF Protein in Sea Urchin Coelomocytes Using the Prepared Polyclonal Antibodies

[0068] Sample preparation: sea urchin coelomocyte lysate

[0069] Methods: Western Blot

[0070] The polyclonal antibody prepared can recognize the native protein MIF in the sea urchin coelomocyte protein sample (see Figure 5 ).

[0071] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical solutions and inventive concepts of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A sea urchin macrophage migration inhibitory factor (MIF) antigen protein, characterized in that: The amino acid sequence of the antigen protein is shown in SEQ ID NO.1; or The amino acid sequence of the antigen protein is formed by substituting, deleting or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO:

1.

2. A gene, characterized in that The gene is the gene encoding the MIF antigen protein of the sea urchin Echinococcus glabrata according to claim 1, and the nucleotide sequence of the gene is shown in SEQ ID NO.

2.

3. A recombinant expression vector of MIF antigen protein of Echinococcus glabrata, characterized in that: It comprises the gene according to claim 2 and an initial vector pColdⅡ, wherein the gene is inserted between the NdeI and HindIII restriction sites of the initial vector.

4. The method for preparing the MIF antigen protein of Echinococcus glabrata according to claim 1, characterized in that: The following steps are included: Transforming the recombinant expression vector into Escherichia coli for screening to obtain a strain containing the recombinant plasmid; The above strains are inoculated into a culture medium for growth and induction of expression of the target protein, the bacteria are collected and the cells are lysed to release the target protein; The protein mixture was purified using a nickel ion affinity purification column, and the concentration of the purified protein was measured using the BSA grayscale analysis method.

5. A polyclonal antibody against MIF of Echinococcus glabrata, characterized in that: The polyclonal antibody is obtained by immunizing mice with the antigen protein according to claim 1.

6. The complete sequence of the MIF gene coding region of the sea urchin Echinococcus glabrata, characterized in that: It has the sequence shown in SEQ ID NO.

3.

7. A primer pair for amplifying the full sequence of the MIF gene coding region of the Echinococcus glabrata according to claim 6, wherein the sequence of the primer pair is shown in SEQ ID NOs. 4 to 5.

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