CN110117325B - Pig CD127 polypeptide and encoding gene and application thereof - Google Patents

Abstract

The present invention provides a porcine CD127 polypeptide which is an extracellular portion fragment of porcine CD127 protein. The polypeptide is used as an immunogen, and in the monoclonal antibody screening process, the Elisa and the flow cytometry detection are combined to prepare a monoclonal antibody which is used for pig CD127 genes and can be used for the flow cytometry detection. By using the antibody, the detection and the typing of the lymphocyte subpopulation of the pig, particularly Treg cells, can be performed by a flow cytometer under the condition of maintaining the cell activity. In order to deeply study the functions of the porcine lymphocytes, the research and the application of cell immunity are carried out in pigs, and the further research on the relation between the disease resistance of the pigs and the lymphocytes has important practical significance.

Description

Pig CD127 polypeptide and encoding gene and application thereof

Technical Field

The invention relates to the field of molecular biology, in particular to a monoclonal antibody of a pig CD127 gene and a preparation method thereof.

Background

CD127 (leukocyte differentiation antigen molecule 127) is an important regulator IL-7 for maintaining peripheral mature T cell homeostasis, responds to the alpha chain of receptor on T lymphocytes, and is an important marker gene for lymphocyte subpopulations, especially Treg (regulatory T lymphocytes) detection and typing. anti-CD 127 flow monoclonal antibodies are an important tool antibody for studying lymphocyte subsets using flow cytometry. Currently, only commercial CD127 streaming antibodies against mice and humans are available on the market, and no CD127 streaming monoclonal antibodies against pigs are available.

Regulatory T cells (tregs) are a subpopulation of T cells with immunoregulatory function among T lymphocytes, and play an important role in maintaining autoimmune tolerance and regulating adaptive immune responses. Treg cells generally have the properties of cd4+cd25+foxp3+, in particular FoxP3 protein, one of the fork-head transcription factor family members, is considered as the marker of the most specific Treg cells. However, foxp3 is an intracellular protein, and is expressed in cells, and it is necessary to fix and perforate the cells during labelling, so that detection and sorting of Treg cells by Foxp3 cannot be performed while maintaining cell activity. It was found that intracellular FoxP3 was inversely correlated with extracellular CD127 expression in Treg cells. Further studies have shown that the promoter of CD127 has a binding site for FoxP3, and that FoxP3, upon expression, binds to the promoter region of CD127, resulting in down-regulation of the expression level of CD127, and thus Treg cells also have the characteristics of CD127 low. By taking advantage of this property, researchers often use the Treg cell surface marker CD127 for detection and analysis when detecting and isolating Treg cells. T cells are defined as Treg cells when they have the characteristics cd4+cd25+cd127 low. The Treg cells obtained by detection and separation by utilizing the characteristics can keep the integrity and the activity of the cells, and can be subjected to further culture and biological function detection. Thus CD127 antibodies are critical for T lymphocyte typing, especially Treg cell typing.

Flow cytometry detection and typing have become an important tool in immunological research. The specificity requirement for the antibody is high when the flow cytometry is used for detecting and parting lymphocyte subpopulations. The CD127 antibodies currently on the market against human and mouse are not universal between species, nor are there currently streaming monoclonal antibodies against the porcine CD127 gene. Due to the lack of antibodies, the typing and detection of lymphocyte subpopulations such as tregs of pigs is not possible.

The invention aims to provide a monoclonal antibody for pigs, which has high specificity and high sensitivity, is suitable for flow cytometry detection, and can be used for Elisa detection and immunoblotting detection.

Disclosure of Invention

In order to achieve the aim, the invention obtains extracellular fragment information of the pig CD127 gene protein through analysis, clones to obtain cDNA fragments corresponding to the genes, prokaryotic expresses corresponding gene protein peptide fragments, further purifies to obtain recombinant protein of pig CD127, then utilizes the obtained recombinant protein to immunize Balb/c mice, fuses spleen cells of the immunized mice with SP2/0 myeloma cells, combines Elisa detection and screens by a flow cytometer to obtain subcloned antibodies of flow detection.

In order to realize the technical scheme, the invention adopts the following technical means.

The invention provides a polypeptide coded by a pig CD127 gene, wherein the sequence of the polypeptide comprises a sequence shown as SEQ ID NO. 1.

Preferably, the polypeptide sequence is shown as SEQ ID NO. 1.

The invention further provides a pig CD127 gene fragment, which codes for the polypeptide.

Preferably, the sequence of the gene fragment is shown as SEQ ID NO. 2.

The present invention provides a vector comprising the above gene fragment.

The present invention provides primer pairs for amplifying the above gene fragments.

Preferably, the sequences of the primer pairs are shown in SEQ ID NO. 3-4.

The invention further provides application of the polypeptide, the gene fragment, the vector and the primer pair in preparation of the pig CD127 antibody.

The invention further discloses an antibody, which binds to the polypeptide. Specifically, it is composed of

The polypeptide is prepared by immunizing animals as an immunogen.

Preferably, the antibody is a monoclonal antibody, a humanized antibody, a polyclonal antibody. The invention screens out a hybridoma cell strain with excellent comprehensive properties and obvious secreted antibody grouping to resist pig CD127-2F8 in the preparation process of monoclonal antibodies, and the hybridoma cells are preserved in China Center for Type Culture Collection (CCTCC) NO: C2018162.

the invention discloses application of the antibody in a flow cytometer.

The invention selects extracellular fragments of pig CD127 protein as antigens, combines Elisa with flow cytometry detection in the process of monoclonal antibody screening, and prepares monoclonal antibodies which are used for pig CD127 genes and can be used for flow cytometry detection. By using the antibody, the detection and the typing of the lymphocyte subpopulation of the pig, particularly Treg cells, can be performed by a flow cytometer under the condition of maintaining the cell activity. The method can be used for analyzing the lymphocyte subpopulation of the pig by a flow cytometer, and has important practical significance for further researching the relationship between the disease resistance of the pig and the lymphocyte in order to deeply research the function of the lymphocyte of the pig and develop the research and application of cell immunity in the pig.

Drawings

Fig. 1 bioinformatics analysis of pig CD 127.

FIG. 1A is a signal peptide prediction.

FIG. 1B is a transmembrane region analysis.

FIG. 2 prokaryotic expression and purification of porcine CD127 protein.

FIG. 2A is a prokaryotic expression gel profile in which M-way protein MARKER, 1-way Pet28a induction, 2-way Pet28a no induction control, 3-way Pet32A induction bacteria, and 4-way Pet32A no induction control.

FIG. 2B shows a gel profile of protein purification, wherein M lanes of protein MARKER,1, 2 lanes of pre-protein sample loading, and 3, 4, 5, 6 lanes of imidazole elution samples are 10mM, 50mM, 100mM, and 500mM, respectively.

FIG. 3 six mice (M-M6) serum, CD3+ co-labeled flow assay (C is normal mouse serum control)

FIG. 4 mix pool M2, M4 media supernatant with CD3+ cell co-labeling assay.

FIG. 5 Co-labelling detection of secreted antibodies with porcine CD3+ in the supernatant of each subclone medium (C as control).

FIG. 6 subclone 1D8,2F3,2F8,3C6 secreted antibody Western Blot detection (T thymus-like, M-muscle protein-like).

Detailed Description

Hybridoma cell strain anti-pig CD127-2F8, this hybridoma cell has been preserved in China center for type culture collection (address: university of Wuhan, china) in 7 months 10, collection number CCTCC NO: C2018162.

the following description of the present invention refers to the accompanying drawings and examples, but is not limited to the same, and modifications and equivalents of the present invention can be made without departing from the spirit and scope of the present invention.

1) The invention firstly refers to cDNA and corresponding protein sequence of pig CD127 gene on GenBank. And then analyzing and obtaining information such as a signal peptide transmembrane domain of the gene protein by using an analysis platform such as SignalP 4.1 server,TMHMM Server and the like. Finally obtaining the extracellular fragment of the pig CD127 gene protein.

2) Based on the extracellular fragment of the gene sequence, a primer is designed and synthesized to amplify cDNA sequence corresponding to the extracellular sequence of the gene protein by PCR. And then connecting the amplified DNA sequence into a T vector, converting, enzyme cutting, then, adding the DNA sequence into an expression vector, and obtaining the recombinant protein of the pig CD127 through converting and IPTG induction expression. And then purifying by using an NI affinity chromatographic column to obtain the purified recombinant protein.

3) Mice were immunized intraperitoneally with purified protein. Mouse antisera were taken. Antibody titers of antisera were measured using Elisa. And simultaneously, the co-labeling condition of the flow cytometry antiserum and the porcine CD3 antibody on lymphocytes in the porcine blood is utilized. The method comprises the steps of selecting mouse spleen cells with higher co-labeling ratio for preparing monoclonal antibodies to carry out cell PEG fusion with SP2/0 myeloma cells, and taking cell culture supernatant to continuously detect the secretory antibody titer and the public labeling condition of the monoclonal antibodies with CD3 antibodies.

4) And selecting the subcloned cell strains from the fusion cell strains with high co-labeling ratio. And simultaneously, continuously combining Elisa with a flow cytometer for detection until the content of the antibody is stable, and obtaining a stable subclone cell strain. Finally, the monoclonal antibody of the pig CD127 gene which is applicable to the detection of the flow cytometry is selected.

Example 1 acquisition of porcine CD127 extracellular fragment recombinant protein

The cDNA sequence (NM-001146128.2) and the protein sequence (NP-001139600.1) of the pig CD127 gene on GenBank are used. The sequence was analyzed with a SignalP 4.1 server and protein signal peptides were predicted. The transmembrane domain was predicted using TMHMM Server v.2.0 (http:// www.cbs.dtu.dk/services/TMHMM /). And obtaining the extracellular fragments of the porcine CD127 gene protein 22-240. Analysis showed that the full length of the pig CD127cDNA was 1999bp, wherein the coding region (ORF) was from 49bp to 1428bp, encoding a 459aa protein. The signal peptide was predicted to show that the first 21aa of CD127 protein is the signal peptide (fig. 1A). The mature CD127 protein is 438aa, has a molecular weight of 49kD, and has an isoelectric point (pI) of 6.52. The transmembrane region showed that 220-242 of the mature peptide was the transmembrane region, N-terminal 1-219 was the extracellular region, and C-terminal 242-459 was the intracellular region (FIG. 1B).

Primers P1:5'-GGATCCAGTGGCTATGCAGAGAATGGAG-3', P2:5'-CTCGAGTCAAGGATCCACCTCCCCTGTG-3' were designed and synthesized based on the extracellular fragment of the gene sequence. The pig thymus cRNA is used as a template, and cDNA sequences corresponding to 219 amino acids in the 22 th to 240 th (namely 1 st to 219 th mature peptides) of the gene protein sequences are amplified by PCR. PCR reaction 50.0. Mu.L: 25 mu L F and Fast long PCR Mater Mix, 1 mu l of each primer, 22 mu l of ddH2O,1 mu l of cDNA template. The reaction procedure: pre-denaturation at 94℃for 2min; 15s at 94 ℃, 15s at 55 ℃ and 1min at 72 ℃ for 35 cycles; extending at 72℃for 10min. After the size of the PCR product is verified to be correct by agarose gel electrophoresis, cutting the gel, and recycling the gel by a gel recycling kit for standby.

And (3) connecting the amplified cDNA sequence corresponding to 219aa total peptide fragments of the porcine CD127 protein 22-240 into a T-Vector pMDTM 19 Vector through TA cloning, and transforming DH5 alpha competence. The positive clones were identified by PCR, inoculated with the extracted plasmid, cut with BamHI and XhoI, ligated into the double digested pET28a and pET32a vectors and transferred into the competence of the expression strain DL21 (DE 3). After the sequence of the prepared monoclonal is correct through sequencing identification, inoculating the monoclonal strain into LB liquid medium (100 mug/mL AMP), and shake culturing at 37 ℃ overnight; the next day 5mL of the culture broth was transferred to 500mL of LB liquid medium (100. Mu.g/mL AMP), cultured with shaking at 37℃for 3 hours, and induced to express by adding IPTG at a final concentration of 0.5 mM. And collecting thalli for ultrasonic crushing, centrifuging, collecting thalli supernatant and precipitate respectively, and performing SDS-PAGE analysis to obtain fusion expression proteins with the sizes between 43kD and 34kD, wherein recombinant proteins of two clone bacteria are mainly expressed in the form of inclusion bodies, and the expression quantity of pET28a vectors is large (figure 2A). The inclusion body of the protein expressed by pET28a is dissolved by 8M urea solution and then purified by an NI affinity chromatography column, and the pig CD127 recombinant protein with higher purity is obtained after imidazole elution (figure 2B).

EXAMPLE 2 mouse immunization with recombinant CD127 protein

After the recombinant protein of the CD127 extracellular fragment obtained by purification was completely emulsified with complete fries adjuvant, intraperitoneal immunization was performed with 100 μg of CD127 protein per mouse. After primary immunization, CD127 proteins emulsified with incomplete freund's adjuvant at 7, 14 and 28d, respectively, were each continued for 1 time; after 3 days of final 1 immunization, mouse serum was taken, and after prokaryotic expression of CD127 protein was coated, the antibody titer of mouse serum was detected by Elisa. The results showed that the titers of antibodies to recombinant proteins in the sera of 6 mice were all greater than 1:8000 (Table 1). And simultaneously, detecting the co-labeling condition of the mouse antiserum and the pig CD3 antibody on lymphocytes in pig blood by using a flow cytometer.

Table 1: antibody titre of 6 mice serum

The flow cytometer detection flow is as follows: pig EDTA anticoagulation 100 μl was taken, lysed by adding erythrocyte lysate, centrifuged at 800g for 5min, the supernatant was discarded, and the cell pellet was resuspended in 100 μl PBS and added with 10 μl of self-made primary antibody. After incubation at 37 ℃ for 30min, washing 3 times with PBS, AF 488-labeled goat anti-mouse IgG was added. After incubation at 37℃for 30min, the cells were resuspended in 100. Mu.L of PBS and then 1. Mu.L of CD3 antibody (BD, USA) was added, after incubation at 37℃for 10min, after 3 washes with PBS, the co-labelling of CD3 with the self-made CD127 antibody was detected by flow cytometry. The results showed that polyclonal antibodies in the serum of 6 mice were able to co-label CD3 positive cells in porcine lymphocytes. Wherein the sera of both M2 and M4 mice were able to cluster more clearly in the co-labelling of CD3 positive cells (FIG. 3).

EXAMPLE 3 monoclonal antibody screening

Mixing spleen cells of mice M2 and M4 with SP2/0 myeloma cells according to the proportion of 1:10, centrifuging 1000g for 3min, discarding supernatant, vibrating the cells to be completely and uniformly mixed, adding 1mL of preheated PEG into a water bath kettle at 37 ℃, standing for 45s, adding 10mL of RPMI1640 culture medium to stop reaction, centrifuging 1000g for 10min, discarding supernatant, adding HAT culture medium containing 20% serum, culturing for 48h at 37 ℃ in a cell culture bottle, and taking culture medium supernatant Elisa of the mixed culture cells to detect, wherein the result shows that the two mixed pool cells can secrete antibodies against pig CD 127. The primary Elisa detection OD of the M2, M4 mixed pool culture medium supernatant was 2.813,2.713 (negative control 0.088,0.079). Simultaneously, culture medium supernatant 1:100 dilution, as anti-CD 127 antibody, using flow cytometry to detect cell cd3+ cell co-labeling, showed that 2.3% of cd3+ positive cells were able to co-label with secreted antibody in M2 mix pool medium supernatant, while 67% of cd3+ cells were able to co-label with secreted antibody in M4 mix pool medium supernatant, and the clustering was clear (fig. 4).

And (3) taking M4 mixed pool cells, uniformly mixing, performing dispersion culture in a 96-well plate, screening and culturing for 10d, coating by using purified CD127, and detecting the content of secreted antibodies in the supernatant in a culture medium by using Elisa. And taking the cell strain with higher secretion of the antibody for subsequent screening until the content of the antibody is stable, and obtaining a stable subclone cell strain. A total of 6 subcloned cell lines with stable Elisa titers were obtained as 1E2,1D8,2F3,2F8,3C6,4E1. Using these subclone culture supernatants as CD127 antibodies to detect co-labelling of CD3+ cells in porcine lymphocytes, the results showed that: each subclone can label a portion of the cd3+ cells. Wherein the marked cd3+ cells of the four 1D8,2F3,2F8,3C6 subclones are more, the subclones 2F8 are more significantly clustered (fig. 5), and the hybridoma cells are preserved in the China center for type culture collection (cctccc) NO: C2018162.

example 4 validation of porcine CD127 antibodies

To verify the effectiveness of the subcloned cell lines in secreting antibodies, long white pig thymus tissue samples and muscle tissue samples were taken, homogenized after adding RIPA Buffer, 12000g, centrifuged at 4℃to take the supernatant, added protein Loading Buffer, incubated at 96℃for 10min, and separated by electrophoresis on a 12% SDS-PAGE gel. After electrophoresis, transferring the membrane, taking subcloned strain cells (1D8,2F3,2F8,3C6) with better flow detection and grouping, using culture supernatant as self-made CD127 primary antibody, using HRP-marked goat anti-mouse IgG as secondary antibody, and detecting antibody marking condition by western Blot. Western blotting procedure was as follows: the PVDF film treated by the methanol is stuck to SDS-PAGE gel, and then the two sides are soaked with electrotransfer liquid filter paper to form a sandwich structure, and 100V electrotransfer is carried out for 1.5h. The transferred film was blocked with 5% nonfat dry milk for 3min, washed 3 times with TBST wash for 10min each, and diluted CD127 antibody was added overnight at 4 ℃. TBST was washed 3 times and secondary antibody (1:500 dilution) was incubated for 1h. After washing 3 times with TBST, the color was developed by ECL color development. Scanning imaging with an LI-Cor chemiluminescent imager and analyzing the electrophoretic band size. The antibodies secreted by each subclone were detected as a single protein band of approximately 50kDa in both thymus and muscle samples, and the protein was expressed in the thymus samples in a higher amount than in the muscle samples (FIG. 6). This experiment further demonstrates the effectiveness of this antibody against porcine CD 127.

The above-described embodiment is only a preferred embodiment of the present invention, and is not limited in any way, and other variations and modifications may be made without departing from the technical aspects set forth in the claims.

Sequence listing

<110> Chongqing city, stock raising academy of sciences; chongqing market pig industry technology institute of technology

<120> a pig CD127 polypeptide, its coding gene and application

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Claims (5)

1. The polypeptide coded by the porcine CD127 with immunogenicity is characterized in that the sequence of the polypeptide is shown as SEQ ID NO. 1.

2. A pig CD127 gene fragment, which encodes the polypeptide of claim 1, has a sequence shown in SEQ ID NO. 2.

3. A vector comprising the gene fragment of claim 2.

4. A primer pair for amplifying the gene fragment of claim 2;

the primer pair is characterized in that the sequences of the primer pair are shown in SEQ ID NO. 3-4.

5. The use of the polypeptide of claim 1, the gene fragment of claim 2, the vector of claim 3, and the primer pair of claim 4 for the preparation of a porcine CD127 antibody.

Images