CN108276488B

CN108276488B - Chicken marrow-like cell trigger receptor B2 polyclonal antibody and preparation method and application thereof

Info

Publication number: CN108276488B
Application number: CN201810076437.1A
Authority: CN
Inventors: 白世平; 白洁; 张克英; 丁雪梅; 王建萍; 曾秋凤; 彭焕伟; 玄月; 宿卓薇
Original assignee: Sichuan Agricultural University
Current assignee: Sichuan Agricultural University
Priority date: 2018-01-26
Filing date: 2018-01-26
Publication date: 2020-06-19
Anticipated expiration: 2038-01-26
Also published as: CN108276488A

Abstract

The invention discloses a chicken myeloid cell trigger receptor B2 polyclonal antibody and a preparation method and application thereof, belonging to the field of animal genetic engineering, wherein an antigen polypeptide for inducing the generation of the chicken myeloid cell trigger receptor B2 polyclonal antibody consists of an amino acid sequence shown in SEQ ID NO.1, and an encoding gene of the antigen polypeptide consists of a nucleotide sequence shown in SEQ ID NO. 2. The chicken myeloid cell trigger receptor B2 polyclonal antibody has strong specificity, and provides an important tool for quantitative analysis and related research of the chicken myeloid cell trigger receptor B2 in broiler tissues or cells; secondly, the antigen sequence used by the invention is long, and the antigen sequence is easy to have immunoreaction with target protein, and the detection sensitivity is high.

Description

Chicken marrow-like cell trigger receptor B2 polyclonal antibody and preparation method and application thereof

Technical Field

The invention belongs to the technical field of animal genetic engineering, and particularly relates to a chicken myeloid cell trigger receptor B2 polyclonal antibody, and a preparation method and application thereof.

Background

Myeloid cell Triggering Receptors (TREMs) belong to immunoglobulin superfamily members, and mammals such as human mainly comprise TREM-1, TREM-2 and TREM-3 receptors; on the other hand, poultry mainly comprise TREM-A1, TREM-B1 and TREMB2, and poultry TREM-B2 has the highest protein sequence homology with the mammalian TREM-2, so that the poultry TREM-B2 is supposed to have similar functions of TREM-2. Mammalian studies have shown that TREMs are generally expressed on the membrane surface of myeloid cells such as glial cells, neutrophils, leukocytes, macrophages, and the like, mediate signaling pathways for inflammatory responses and have cascade amplification effects on inflammatory responses. Recent findings indicate that TREM-2 not only regulates the inflammatory response of the body, but also plays an important role in the signal transduction process of some neurological diseases (Alzheimer's disease) and adipogenesis. TREM-2 is also highly expressed on mouse liver endothelial cells and is an important signal molecule in a adipogenesis signal path. A rare variation of TREM-2 is a risk factor for late-onset Alzheimer's disease with a probability of increasing risk of onset similar to apolipoprotein. Furthermore, TREM2 deletion can alter microglial function in a mouse model of alzheimer's disease. No research report on the function of TREM-B2 of poultry is found so far, but the function is supposed to be related to the animal immune function.

Mammalian TREM2 consists of an extracellular region containing a V-Ig superfamily domain, 3N-glycosylation sites presumably containing positively charged transmembrane sequences of lysine residues, and an intracellular short tail structure. Poultry TREM-B2 retains 2V-type Ig superfamily domains, and the other functional regions are identical to mammalian TREM 2. The lysine residues of TREM2 interact electrostatically with the tyrosine residues on the intracellular signal adaptor protein DAP12 at the lipid membrane. DAP12 is structurally a homodimer, comprising a short extracellular region between cysteine residues and a disulfide bond and an immunoreceptor tyrosine activating groupThe intracellular domain of sequence (ITAM). Upon activation of TREM2, the DAP12 tyrosine residue is phosphorylated under the ITAM motif by tyrosine (Src) family kinases, allowing Syk and ZAP70 tyrosine kinases to bind through the C-carboxy terminal Src homology region (SH2) domain, thereby activating downstream signaling pathways including phosphatidylinositol 3-kinase (PI3K), Protein Kinase C (PKC), extracellular regulated protein kinase (ERK), and increasing Ca²⁺And (4) internal flow. Lipids play an important role in regulating TREM2 function. No research report on intracellular signal transduction of the TREM-B2 protein receptor of poultry is found so far.

Currently, only polyclonal or monoclonal antibodies of human and mouse are commercialized TERM antibodies, and no TREM antibodies for poultry are available so far. Because the similarity between the chicken TREM-B2 protein sequence and human TREM-2 is very low, and the similarity of functional regions does not exceed 75%, the TREM-B2 expression effect in broiler tissues and cells cannot be identified by adopting human and mouse TREM-2 antibodies, and the research on the function of broiler TREM-B2 is limited.

Disclosure of Invention

In view of the above, the invention provides a chicken myeloid cell triggering receptor B2(TREM-B2) polyclonal antibody, and a preparation method and application thereof.

In order to solve the technical problem, the invention discloses an antigen polypeptide for inducing chicken myeloid cell trigger receptor B2 polyclonal antibody, which is composed of an amino acid sequence shown in SEQ ID NO. 1.

The invention also discloses a coding gene of the antigen polypeptide.

Furthermore, the coding gene consists of a nucleotide sequence shown as SEQ ID NO. 2.

The invention also discloses an expression vector containing the coding gene.

The invention also discloses a chicken medullary cell trigger receptor B2 polyclonal antibody, wherein the antigen polypeptide of the polyclonal antibody is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO. 1.

Furthermore, the encoding gene of the antigen polypeptide consists of a nucleotide sequence shown in SEQ ID NO. 2.

The invention also discloses a preparation method of the chicken myeloid cell trigger receptor B2 polyclonal antibody, which comprises the following steps: artificially synthesizing a nucleotide sequence of SEQ ID NO. 2; constructing a pET-28a prokaryotic expression vector, cloning a nucleotide sequence of an artificially synthesized chicken TREM-B2 gene fragment SEQ ID NO.2 onto the vector, sequencing, and inducing protein expression; ultrasonic crushing to induce expression bacteria, and purifying with Ni-NTA resin chromatographic column; immunizing a New Zealand white rabbit with the purified recombinant pET-28a-TREM-B2 expression protein, and collecting blood from the heart on the 7 th day after the five-immunization to prepare antiserum; and purifying the antiserum by using a Protein G affinity chromatography column to obtain the chicken TREM-B2 polyclonal antibody.

The invention also discloses a detection kit containing the chicken myeloid cell trigger receptor B2 polyclonal antibody.

The invention also discloses application of the chicken myeloid cell trigger receptor B2 polyclonal antibody in detecting TREM-B2 expression in broiler chicken tissues.

Compared with the prior art, the invention can obtain the following technical effects:

1) the invention prepares chicken TREM-B2 polyclonal antibody for the first time, and provides an important tool for quantitative analysis of TREM-B2 in broiler chicken tissues or cells and related research thereof.

2) The antigen sequence used by the invention is long, different kinds of TREM protein molecules can be distinguished, the generated TREM-B2 polyclonal antibody has strong immunoreaction specificity, high detection sensitivity and high titer.

Of course, it is not necessary for any one product in which the invention is practiced to achieve all of the above-described technical effects simultaneously.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention and not to limit the invention. In the drawings:

FIG. 1 is an electropherogram of a purified antigen after expression of a TREM-B2 recombinant protein according to the present invention; wherein, 1: protein molecular weight standards (Marker); 2: purifying the antigen, wherein the molecular weight (from top to bottom) of the Marker is 116.0KD/66.2KD/45KD/35KD/25KD/18.4KD/14.4 KD;

FIG. 2 is a measurement of the titer of antisera to a rabbit according to the invention;

FIG. 3 is a measurement of the titer of antisera to another rabbit according to the invention;

FIG. 4 is an electropherogram of a purified chicken TREM-B2 antibody of the present invention; wherein, 1: protein molecular weight standards; 2: purifying chicken TREM-B2 antibody (Marker); the molecular weight of the Marker (from top to bottom) is 116.0KD/66.2KD/45KD/35KD/25KD/18.4KD/14.4 KD;

FIG. 5 is a graph of TREM-B2 expression in different tissues of the broiler chicken of the present invention; wherein, 1: duodenum 50 μ g; 2: spleen 50 μ g; 3: liver 25 μ g; 4: liver 50 μ g; 5: abdominal fat 25 μ g; 6: 50 mug of abdominal fat; m: marker; marker molecular weight (from top to bottom): 180KD/130KD/95KD/72KD/55KD/45KD/35KD/25KD/17 KD.

Detailed Description

The following embodiments are described in detail with reference to the accompanying drawings, so that how to implement the technical features of the present invention to solve the technical problems and achieve the technical effects can be fully understood and implemented.

Example 1 Artificial Synthesis of fragment of Chicken myeloid cell triggering receptor B2(TREM-B2) Gene

The specific procedure is as follows:

(1) comparing the amino acid sequence deduced from broiler TREM-B2 sequence (NM-001080868.1) on GenBank with TREM-A1 and TREM-B1, finding out the difference of the amino acid sequences of the functional regions of the amino acid sequences of SEQ ID NO:1 and the corresponding nucleotide sequence SEQ ID NO:2, artificially synthesized by Shanghai Bioengineering Co.

(2) Connecting the artificially synthesized fragment with a pGEM-T vector (Promega, USA) according to a molar ratio of 2:1 for T-A cloning, standing overnight at 4 ℃ to achieve a high connection yield, taking a connection product to convert a DH5 α competent strain (Beijing Tiangen Biotechnology limited company), carrying out shake culture for 2h at 37 ℃ in an LB culture medium, taking 50 mu l of culture solution to inoculate and coat the culture solution on an LB plate, carrying out inversion culture for 16h in a 37 ℃ incubator, carrying out blue-white spot screening, picking a white single colony, carrying out shake culture for overnight at 37 ℃ in an LB ampicillin-containing (100 mu g/mL) selection culture medium, and simultaneously sending the bacterial solution to Shanghai biological engineering company for sequencing, wherein the sequence is shown in SEQ ID No:2 to determine that the artificially synthesized sequence is correct.

Example 2 construction of prokaryotic expression vector for Chicken myeloid cell trigger receptor B2(TREM-B2)

The method comprises the following steps:

(1) constructing a prokaryotic expression vector containing chicken (amino acids 20-125): the expression vector pET-28a was digested simultaneously with EcorI and Hind III (Takara, Inc.). And (4) carrying out 1% agarose gel electrophoresis on the double digestion products, and recovering the expression vector. The artificially synthesized TREM-B2 fragment and the expression vector were ligated at a molar ratio of 3: 1 overnight at 16 ℃. The ligation product is transformed into BL-21 competent strain (Beijing Tiangen Biotechnology Co., Ltd.), the LB culture medium (without kanamycin) is subjected to shaking culture at 37 ℃ for 2h, 50-100 mu L of culture solution is taken to be inoculated to an LB plate containing kanamycin (50 mu g/mL) (Shanghai Biotechnology engineering Co., Ltd.), and the LB plate is subjected to incubator inversion culture at 37 ℃ for more than 16h for screening. The selected single colony was cultured with shaking in LB medium (containing 50. mu.g/mL kanamycin) at 37 ℃ overnight, and PCR was directly performed using the colony solution. Meanwhile, the bacterial liquid is sent to Shanghai bio-engineering company (Shanghai Biotech) to carry out sequencing analysis, and positive bacteria are screened out.

(2) After the expression engineering bacterium BL-21 is constructed, the plasmid pET-28a is transformed into BL21 strain, and the induction expression is carried out according to the plasmid instruction. The induction conditions were: the final concentration of inducer IPTG (Shanghai Biotech engineering Co.) is 0.5 mM; induction was carried out at 30 ℃ for 3 h. And (3) carrying out ultrasonic disruption induced expression bacteria, centrifuging, and carrying out SDS-PAGE protein electrophoresis analysis on the supernatant. The target protein is expressed by supernatant and purified by using a Ni-NTA chromatographic column. The concentration is about 3.0mg/mL, the purity is about 85 percent, and the size is about 27kD (see figure 1).

EXAMPLE 3 preparation of polyclonal antibodies against the broiler myeloid cell trigger receptor B2(TREM-B2)

Two New Zealand white rabbits were immunized with the recombinant PET-28a-GR purified from example 2, the immunization method is shown in Table 1.

TABLE 1 immunization methods

Blood was collected from the heart at day 7 after the five-day immunization to prepare antiserum, and the titer of the antiserum was determined by using TREM-B2 recombinant protein, as shown in FIGS. 2 and 3, and the titer of the sera from two rabbits exceeded 1: 50K.

Purification of the antibody: washing (1mL/min) the ProteinG affinity chromatography column with 10 times the volume of the affinity column in deionized water, and then washing (1mL/min) the ProteinG affinity chromatography column with 10 times the volume of the affinity column in 1% NaAC; then filtering the antiserum sample through a 0.22 mu m filter membrane, adding 1% NaAC with 4 times of volume, and loading at the speed of 0.5 mL/min; washing with 3.5% glacial acetic acid, and collecting the eluted product until no protein flows out; adjusting the pH of the eluted product to be neutral by using saturated sodium carbonate, concentrating the product to be 5mL by using a 10kDa ultrafiltration tube, dialyzing the product by using 1 XPBS overnight, and changing the solution for 1 time the next day; the gel electrophoresis pattern of the purified rabbit antiserum is shown in FIG. 4, the antibody of the rabbit antiserum is 10mg/ml, and the purity is 95%.

EXAMPLE 4 determination of TREM-B2 expression assay in broiler chicken small intestine, liver, spleen and adipose tissue Using TREM-B2 polyclonal antibody

The method comprises the following steps:

injecting pentobarbital solution with the weight of 20mg/kg into AA broiler chickens which are bred for 21 days, collecting the livers and abdominal fat tissues of the broiler chickens, quickly freezing by liquid nitrogen, and storing in a refrigerator at the temperature of minus 80 ℃. And detecting the expression condition of TREM-B2 in duodenum, spleen, liver and adipose tissue of the broiler chicken by using a Western Blot technology.

The specific operation is as follows:

(1) the method comprises the steps of extracting proteins of the liver and abdominal fat weight of the broiler chicken by using a tissue protein extraction kit of Pierce company, and determining the protein concentration by using a BCA trace protein concentration determination kit. Separating gel with concentration of 15% and concentrated gel with concentration of 5% are prepared, and the loading amount is 30 or 50 μ g/hole. Electrophoresis conditions: concentrating the gel at constant pressure of 120V for about 20 min; the gel was separated at 150V and the electrophoresis stop time was determined by prestained protein Marker. And (5) after electrophoresis, performing membrane conversion by adopting a wet conversion method.

(2) Film transfer: cutting the glue into proper size, immersing the glue into a film buffer, and immersing the PVDF film into a nailTransferring the alcohol into a membrane transfer buffer after 1min, and immersing the filter paper into the membrane transfer buffer (the PVDF membrane and the filter paper are both cut into the same size as the glue); leaching the graphite electrode by using a membrane transfer buffer, laying two pieces of filter paper, and dripping a little of the membrane transfer buffer; laying a film, and dripping a little of film buffer; spreading glue, and dripping a little of film buffer (taking care not to generate bubbles); finally, laying two pieces of filter paper, and dripping a little of membrane buffer; covering the electrode, and adjusting the voltage to the maximum value at 1.5mA/cm²The gel volume is transferred to the membrane for 1.5h (the load voltage is not suitable to exceed 1V/cm)²)。

(3) And (3) sealing: after the membrane transfer was completed, the membrane was incubated in a 1% casein-TBST solution at room temperature for 2h to block non-specific binding sites on the membrane.

(4) Primary antibody incubation: the blocked membrane was added to a 1:5000 dilution of the chicken TREM-B2 antibody prepared in example 3, incubated at room temperature for 10min and then at 4 ℃ overnight.

(5) And (3) secondary antibody incubation: after the primary antibody incubation was completed, the membrane was washed three times with TBST (shaking on a horizontal shaker) for 5min each time, and then horseradish peroxidase-labeled goat anti-rabbit IgG antibody (1:10000 dilution) was added and incubated at room temperature for 1 h.

(6) And (3) developing: after the secondary antibody incubation is finished, washing with TBST for three times, 5min each time (shaking on a horizontal shaker); then covering the membrane (single white surface) with ECL (Millipore) for 3-5min, and scanning with film for 1 min; the film is taken out, rinsed by deionized water and dried in the air, a Marker is calibrated, and the experimental result is analyzed, as shown in figure 5, according to the predicted molecular weight (about 30kD) of TREM-B2 protein, the polyclonal antibody can be used for detecting the expression of TREM-B2 in small intestine, liver, spleen and adipose tissue of broiler chicken by a Western blot method.

While the foregoing description shows and describes several preferred embodiments of the invention, it is to be understood, as noted above, that the invention is not limited to the forms disclosed herein, but is not to be construed as excluding other embodiments and is capable of use in various other combinations, modifications, and environments and is capable of changes within the scope of the inventive concept as expressed herein, commensurate with the above teachings, or the skill or knowledge of the relevant art. And that modifications and variations may be effected by those skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.

Sequence listing

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Claims

1. An antigenic polypeptide for inducing chicken myeloid cell trigger receptor B2 polyclonal antibody, which is characterized by consisting of an amino acid sequence shown in SEQ ID NO. 1.

2. A gene encoding the antigenic polypeptide of claim 1.

3. The coding gene of claim 2, consisting of the nucleotide sequence shown in SEQ ID NO. 2.

4. An expression vector comprising the coding gene of claim 2 or 3.

5. A polyclonal antibody of chicken myeloid cell trigger receptor B2, wherein the antigen polypeptide of the polyclonal antibody consists of the amino acid sequence shown in SEQ ID NO. 1.

6. The chicken myeloid cell triggering receptor B2 polyclonal antibody of claim 5, wherein the encoding gene of said antigen polypeptide consists of the nucleotide sequence shown in SEQ ID NO. 2.

7. A preparation method of polyclonal antibody of chicken myeloid cell trigger receptor B2 is characterized by comprising the following steps: artificially synthesizing a nucleotide sequence of SEQ ID NO. 2; constructing a pET-28a prokaryotic expression vector, cloning a nucleotide sequence of an artificially synthesized chicken TREM-B2 gene fragment SEQ ID NO.2 onto the vector, sequencing, and inducing protein expression; ultrasonic crushing to induce expression bacteria, and purifying with Ni-NTA resin chromatographic column; immunizing a New Zealand white rabbit with the purified recombinant pET-28a-TREM-B2 expression protein, and collecting blood from the heart on the 7 th day after the five-immunization to prepare antiserum; and purifying the antiserum by using a Protein G affinity chromatography column to obtain the chicken TREM-B2 polyclonal antibody.

8. A test kit comprising the polyclonal antibody against chicken myeloid cell-triggered receptor B2 according to claim 5 or 6.

9. Use of the chicken myeloid cell triggering receptor B2 polyclonal antibody of claim 5 or 6 for detecting TREM-B2 expression in broiler chicken tissues.