CN107686519B - Preparation method and application of anti-mouse MXRA7 monoclonal antibody - Google Patents

Abstract

The invention discloses a preparation method and application of an anti-mouse MXRA7 monoclonal antibody, wherein the antibody is generated by a hybridoma cell strain with the preservation number of CCTCC NO: C2017142, and the preparation method comprises the following steps: (1) amplifying MXRA7 protein coding gene by using a primer with a restriction enzyme site, cloning the MXRA7 protein coding gene into a prokaryotic expression vector, and purifying fusion protein by thallus expression; (2) immunizing a mouse by using the purified fusion protein as an antigen, preparing positive hybridoma cells, beating the positive hybridoma cells into the abdominal cavity of the mouse to obtain ascites of the mouse, and purifying the ascites to obtain a monoclonal antibody; (3) the MXRA7 monoclonal antibody was purified and its specificity was verified. The monoclonal antibody of the MXRA7 protein prepared by the method has high titer, good stability and strong specificity, and can be used for immunological detection, thereby providing a reliable and convenient research tool for comprehensively researching the function of the MXRA7 protein.

Description

Preparation method and application of anti-mouse MXRA7 monoclonal antibody

Technical Field

The invention belongs to the technical field of biology, and particularly relates to an anti-mouse gene MXRA7 monoclonal antibody SZ181 and a preparation method thereof; also relates to the application of the monoclonal antibody in Western blot, immunohistochemistry and flow cytometry.

Background

MXRA7 is a single copy gene located on mouse chromosome 11 and encodes an amino acid with a molecular weight of about 20 KDa. The gene is originally discovered that 8 cDNAs are always co-expressed with a group of genes (mainly comprising Mmps, Timps and the like) related to matrix reconstruction when Walker and the like analyze through bioinformatics in 2002, and then the 8 cDNAs are sequentially named as MXRA 1-8. So far, although some members have been successfully cloned and subjected to functional studies, no functional studies on MXRA7 have been reported. The subject group has recently demonstrated that the gene has the following properties: (1) the gene has different expression in various tumor tissues and normal tissues; the over-expression of MXRA7 obviously inhibits the proliferation of prostate cancer cells of mice and the generation and metastasis of tumors, and the MXRA7 can be used as a tumor marker, can inhibit the generation and development of tumors and can become an ideal tumor-targeted therapeutic molecule; (2) the gene influences the tissue repair ability after being knocked out; (3) meanwhile, the gene is closely related to angiogenesis, immune reconstitution and the like. Therefore, the preparation of the monoclonal antibody taking the mouse MXRA7 as a target point is of great significance for further exploring the physiological and pathological functions of MXRA 7.

Disclosure of Invention

The invention aims to provide a preparation method of an anti-mouse MXRA7 monoclonal antibody SZ181, wherein the SZ181 is numbered according to a laboratory antibody library, the antibody is secreted and produced by a hybridoma cell strain SP2/0-MXRA7 of Balb/c mice, and the monoclonal antibody SZ181 can be used for detecting the expression of MXRA7 protein in each tissue of the mice and provides a useful tool for the function research of mouse MXRA 7.

In order to achieve the above object, the present invention provides the following technical solutions:

a preparation method of an anti-mouse MXRA7 monoclonal antibody is secreted and generated by a hybridoma cell strain with the preservation number of CCTCC NO: C2017142;

the preparation method of the MXRA7 monoclonal antibody comprises the following steps:

(1) using a chemically synthesized MXRA7 protein coding gene SEQ ID NO.3 as a template, amplifying the template by using an upstream primer SEQ ID NO.1 and a downstream primer SEQ ID NO.2 to obtain a mouse MXRA7 protein coding gene with enzyme cutting sites at two ends, cloning the mouse MXRA7 protein coding gene into a prokaryotic expression vector pCold-SUMO to construct a prokaryotic recombinant plasmid, and transforming the prokaryotic recombinant plasmid into a competent cell;

(2) culturing and expressing the competent cells to obtain SUMO-MXRA7 fusion protein, and purifying the SUMO-MXRA7 fusion protein;

(3) immunizing a Balb/c mouse with the purified SUMO-MXRA7 fusion protein, measuring the serum titer of the mouse, and taking the spleen to prepare a spleen cell suspension; fusing the homologous SP2/0 cell with a mouse spleen cell to prepare an SP2/0 hybridoma cell, and screening and purifying the positive SP2/0 hybridoma cell;

(4) culturing the positive SP2/0 hybridoma cells in the step (3) by adopting an in vitro cell culture method, beating the positive cells into the abdominal cavity of a mouse to obtain ascites of the mouse, and purifying the ascites to obtain the mouse monoclonal antibody.

The invention relates to a preparation method of a mouse MXRA7 monoclonal antibody, which comprises the steps of (1) carrying out PCR amplification on a plasmid by using an upstream primer containing a fragment GGTACC with an amplified KpnI enzyme cutting site and a downstream primer containing a fragment GGATCC with an amplified BamHI enzyme cutting site, taking a 5 mu LPCR product, carrying out 1.5% agarose gel electrophoresis, purifying the rest PCR product by using a PCR clean kit, carrying out double enzyme digestion on the recovered product by using KpnI and BamHI, carrying out 1.5% agarose gel electrophoresis after enzyme digestion is finished, and recovering a target fragment by using a DNA gel recovery kit; carrying out double enzyme digestion on the expression vector pCold-SUMO by using a KpnI and BamHI double enzyme digestion system to enable the expression vector pCold-SUMO to become a linear fragment, carrying out electrophoresis by using 1.5% agarose gel after the enzyme digestion is finished, and recovering a target fragment by using a DNA gel recovery kit; carrying out ligation reaction on the digested MXRA7 encoding gene and a gel recovery product of pCold-SUMO plasmid at 16 ℃ for 3h by using T4DNA ligase, converting the ligation product into DH5a competence, coating a plate, picking a single colony the next day, carrying out colony PCR, carrying out gel electrophoresis, and screening out a positive colony clone; extracting the recombinant expression plasmid of the positive colony, transferring the recombinant expression plasmid into BL21(DE3) competent cells for protein expression, and purifying to obtain the SUMO-MXRA7 fusion protein.

In the preparation method of the anti-mouse MXRA7 monoclonal antibody, the BL21(DE3) competent cells are inoculated into LB culture solution containing Amp with the concentration of 50 mu g/mL and cultured overnight at 37 ℃; sucking 50 μ L of overnight culture into 5mL LB culture solution containing Amp with concentration of 50 μ g/mL, and shake-culturing at 37 deg.C and 200rpm until OD600 is 0.6-0.7; adding isopropyl thiogalactoside to a final concentration of 0.5mM, inducing at 16 ℃ for 24 hours, centrifuging the bacterial liquid, collecting supernatant, and purifying the collected supernatant by using nickel column NTA resin for column chromatography to obtain the purified SUMO-MXRA7 fusion protein.

The invention discloses an application of a monoclonal antibody SZ181 of an anti-mouse MXRA7 in biological detection, which comprises the following steps:

1. the normal tissues of the mice were collected, paraffin sections were prepared, and the effect of detecting the MXRA7 protein expressed in the normal tissues of the mice with the MXRA7 monoclonal antibody SZ181 was verified.

2. And extracting the total protein of each normal tissue of the mouse, and carrying out Western blot test on the extracted total protein to verify the detection effect of the mouse anti-mouse MXRA7 monoclonal antibody SZ181 on the MXRA7 protein expressed in each tissue of the mouse.

3. Collecting mouse liver cancer cells Hepa1-6-MXRA7 with MXRA7 over-expression and control cells, and verifying the detection effect of the mouse anti-mouse MXRA7 monoclonal antibody SZ181 on HCRCN81 protein expressed in the mouse liver cancer cells by using flow cytometry.

The experimental result shows that the mouse anti-mouse MXRA7 monoclonal antibody SZ181 has a detection effect on MXRA7 natural proteins expressed in normal tissues and cells of a mouse, and can be used as an important tool for further researching the function of the mouse MXRA 7.

Has the advantages that: the invention prepares SP2/0-MXRA7 hybridoma cells for the first time, and provides a novel mouse-resistant MXRA7 monoclonal antibody SZ181 which can detect the presence or absence of MXRA7 protein expression in mouse tissues, and confirms that the antibody can be applied to western blotting, immunohistochemistry and flow cytometry, thereby providing a reliable and convenient research tool for comprehensively exploring MXRA7 protein functions; meanwhile, the antibody can also recognize rat MXRA7 protein, and provides possibility for comparative study of MXRA7 protein among different species.

Biological preservation Instructions

The hybridoma cell strain MXRA7 is preserved in China center for type culture Collection in 2017, 08 and 29 months, and is located in Wuhan university school with eight paths 299 in Wuchang district, Wuhan City, Hubei province, and the preservation number is CCTCC NO: C2017142.

Drawings

FIG. 1 is an agarose gel electrophoresis of a PCR amplified MXRA7 target fragment with corresponding cleavage sites, showing the following lanes: 1. marker, 2, PCR product.

FIG. 2 is a comparison chart of sequencing results. Wherein the sequence A represents a sequencing result sequence, the sequence B represents a sequence SEQ ID NO.3, and the sequence C represents an alignment result.

FIG. 3 is SDS-PAGE patterns of SUMO-MXRA7 fusion protein after induced expression and purification in E.coli BL21(DE3) strain, wherein the lanes are: 1. marker, 2, non-induced expression purification solution, 3, induced expression purification solution, 4, thallus cracking precipitation, 5, thallus cracking supernatant, 6, flow-through solution, 7, Binding Buffer, 8 and Elute Buffer.

The mass spectrometry result of fig. 4 shows that the alignment result of the purified fusion protein is MXRA7 protein. Wherein Top score shows that it is identical to MXRA7 sequence.

FIG. 5 is a graph showing the results of detection of antibody titer and specificity. In the graph A, the ELISA detects the antibody titer of SZ181, the corresponding dilution ratio of the antibody is 1600-102400 times, the dilution ratio is sequentially changed every two times, and the negative control is mouse IgG protein. FIG. B, Western blot to examine the recognition specificity of SZ 181. Wherein lane 1: purified SUMO-MXRA7 fusion protein; lane 2: hepatic tissue protein of WT mice. And in the figure C, the recognition site of the SZ181 is positioned in the MXRA7 first exon by Western blot detection. Wherein lane 1: the control group is GST protein; lane 2: purified GST-MXRA7 exon 1 protein; lane 3: purified GST-MXRA7 exon 2-4 proteins. Panel D, SUMO-MXRA7 fusion protein competition inhibition assay for SZ181 recognition site, wherein lane 1: the control group is the liver tissue protein of WT mice; lane 2: an equal amount of mouse liver tissue protein is incubated with primary antibody and 25 mu g of Sumo-MXRA7 protein; lane 3: an equal amount of mouse liver histones was incubated with primary antibody and 50 μ g of SUMO-MXRA7 fusion protein.

FIG. 6 Western blot plot of MXRA7 expression in rat tissues detected by the antibody.

FIG. 7 Western blot plot of MXRA7 expression in mouse tissues detected by the antibody. Wherein, the graph A is a typical Western blot graph, and the graph B is a gray scale statistical analysis histogram of the two results.

Figure 8 immunohistochemistry graphs for antibody detection of MXRA7 expression in mouse tissues.

Figure 9 immunofluorescence plots of antibody detection of MXRA7 expression in mouse cells.

Figure 10 flow cytometry plots of antibody detection of MXRA7 expression in mouse cells.

Detailed Description

The present invention is further described below with reference to specific examples, which are only exemplary and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.

Example 1 construction of prokaryotic recombinant expression plasmid for mouse MXRA7

In order to obtain a monoclonal antibody with high affinity and good specificity, the invention firstly expresses an immune antigen MXRA7 protein in prokaryotes. The method is characterized in that a chemically synthesized MXRA7 protein coding gene SEQ ID NO.3 is used as a template (synthesized by Nanjing Kingsri Biotechnology Co., Ltd.), a target fragment with KpnI and BamHI enzyme cutting sites is amplified, an upstream primer SEQ ID NO.1 contains KpnI enzyme cutting sites (GGTACC), and a downstream primer SEQ ID NO.2 contains BamHI enzyme cutting sites (GGATCC). (KpnI and BamHI from Takara)

The PCR technology is utilized to amplify the segment of MXRA7 protein coding gene SEQ ID NO.3, and the specific reaction system and reaction conditions are as follows:

10× PCR Buffer 2 μL

dNTP (2.5 mM each) 1.6. mu.L

cDNA template 2. mu.L

Forward primer (10 mM) 0.5. mu.L

Reverse primer (10 mM) 0.5. mu.L

rTaq（5U/μL） 0.2 μL

H₂O 13.2 μL

Total volume (. mu.L) 20. mu.L

Reaction conditions are as follows: (1) pre-denaturation at 94 ℃ for 5 min; (2) denaturation at 94 ℃ for 30 s; (3) annealing at 55 ℃ for 30 s; (4) extension at 72 ℃ for 30s for 30 cycles, extension at 72 ℃ for 5 min.

After the reaction, 5. mu.L of PCR product was subjected to 1.5% agarose gel electrophoresis, and the amplification result was checked; and purifying the residual PCR product by using a PCR clean kit, and carrying out double enzyme digestion on the recovered product by using KpnI and BamHI under the specific reaction conditions as follows. After the reaction, the reaction mixture was electrophoresed through 1.5% agarose gel, and the agarose gel electrophoresis chart is shown in FIG. 1, in which the DNA fragment having a molecular weight of about 500bp was used as the target gene fragment, and the target fragment was recovered by using a DNA gel recovery kit.

The expression vector pCold-SUMO was subjected to double digestion using KpnI and BamHI double digestion systems to obtain a linear fragment, which was subjected to electrophoresis using 1.5% agarose gel after completion of the reaction, and the digestion result was checked and the target fragment was recovered using a DNA gel recovery kit.

The gene encoding MXRA7 was ligated with the gel recovery product of pCold-SUMO plasmid using T4DNA ligase (purchased from Takara), ligation reaction was carried out at 16 ℃ for 3 hours, the ligation product was transformed into DH5a competent cells, after plating, single colony was picked the next day, gel electrophoresis was carried out after colony PCR, and positive clones were selected based on positive amplification.

Colony PCR system:

10× PCR Buffer 2 μL

dNTP (2.5 mM each) 1.6. mu.L

cDNA template 2. mu.L

Forward primer (10 mM) 0.5. mu.L

Reverse primer (10 mM) 0.5. mu.L

rTaq（5U/ μL） 0.2 μL

H₂O 13.2 μL

Total volume 20. mu.L

The clones identified as positive are sent to be sequenced, a sequencing result comparison graph is shown in figure 2, wherein the sequence A represents a sequencing result sequence, the sequence B represents a SEQ ID NO.3 sequence, and the sequence C represents a comparison result, and the comparison result shows that the connection is successful, no mutation exists, and the expression vector is successfully constructed.

Example 2 prokaryotic expression of SUMO-MXRA7 fusion proteins

1) Extracting plasmids of colonies identified as positive clones, and transforming the extracted plasmids into competent cells BL21(DE 3);

2) competent cells BL21(DE3) were inoculated into LB medium containing Amp (50. mu.g/mL) and cultured overnight at 37 ℃;

3) pipetting 50. mu.L of overnight culture into 5mL of LB medium containing Amp (50. mu.g/mL), and shaking at 37 ℃ and 200rpm until OD600= 0.6-0.7; adding IPTG to a final concentration of 0.5mM, inducing at 16 ℃ for 24 hours, centrifuging the bacterial liquid and collecting supernatant; meanwhile, the bacterial liquid induced without IPTG is used as a blank control.

Purification conditions of SUMO-MXRA7 fusion protein:

1) resin filling: taking an empty column, adding 3mL of nickel column NTA resin for pre-packing the column, when the preservation solution descends to the surface of the resin, sequentially washing the column by using 3 times of column volume of sterilized double distilled water, and balancing a column bed by using 3 times of column volume of 1 × Binding Buffer (20mM Tris-HCl, 500mM NaCl, 20mM imidazole, pH 8.4);

2) purifying by column chromatography

1. When the 1 × Binding Buffer is reduced to the surface of the resin, adding an extraction supernatant containing the recombinant protein, repeatedly loading for 2-3 times, and reserving the solution after column passing;

2. adding 1 × Binding Buffer with 10 times of column volume, and reserving the solution after passing through the column;

3. adding 8 times volume of 1 × Washing Buffer (20mM Tris-HCl, 500mM NaCl, 60mM imidazole, pH8.4), eluting the hybrid protein which can not be combined with the resin, and reserving the Washing liquid after passing through the column;

4. adding 1 XElute Buffer (10mM Tris-HCl, 250mM NaCl, 300mM imidazole, pH8.4) in 6 column volumes to Elute the target protein bound to the resin, and collecting the eluate with a 1.5mL clean EP tube to obtain a purified solution for inducing expression;

carrying out column chromatography purification on the non-induced bacterial liquid by the same method to obtain a purified liquid which is not subjected to induced expression;

the purified liquid without induced expression, the purified liquid with induced expression, the thalli lysis sediment, the thalli lysis supernatant, the flow-through liquid, the Binding Buffer and the Elute Buffer are analyzed by 8 percent SDS-PAGE gel electrophoresis, and the recombinant protein with higher purity and the molecular weight of about 50KD is obtained from the purified liquid with induced expression as shown in figure 3.

And detecting the purified recombinant protein by mass spectrometry, comparing and analyzing the results to obtain that the protein sequence corresponding to the highest score is MXRA7 protein (figure 4), and confirming that the recombinant protein is the target protein MXRA7 protein and contains the SUMO tag.

Example 3 production and purification characterization of anti-mouse MXRA7 monoclonal antibody SZ181

The purified SUMO-MXRA7 fusion protein expressed in example 2 was used to immunize 6-8 week-old Balb/c female mice according to the specific immunization scheme shown in Table 1 below.

Table 1 immunization mouse dose and protocol

Number of immunizations

First immunization

Secondary immunization

Triple immunization

Four times of immunization

Five immunizations

Six times of immunity

Date of immunization

0d

14d

28d

42d

56d

73d

Immunization dose

30 mu g/body

30 mu g/body

30 mu g/body

30 mu g/body

40 mu g/body

50 mu g/body

Immunologic adjuvant

Freund's complete adjuvant

Freund's incomplete adjuvant

Freund's incomplete adjuvant

Freund's incomplete adjuvant

Freund's incomplete adjuvant

Is free of

Immunization method

Subcutaneous and intramuscular

Subcutaneous and intramuscular

Subcutaneous and intramuscular

Subcutaneous and intramuscular

Subcutaneous and intramuscular

Abdominal injection

One week after the third booster immunization, i.e., day 35 of immunization, blood was collected from the ocular venous plexus, and the serum titer of the mice was measured by ELISA. The serum titer of the immunized mice is mostly 1:10⁵On the left and right, the titer was low, and the fourth and fifth immunizations were continued. One week after the fifth immunization, namely the 63 th day of immunization, the antiserum titer in the blood collection detection of the ocular venous plexus can reach 1:10⁶In the invention, mice with high titer are selected for the next step of cell fusion. The specific detection steps are as follows:

coating: CBS dilutes SUMO-MXRA7 fusion protein antigen to 10 μ g/mL, 100 μ L/well, incubate for 2h at 37 ℃;

and (3) sealing: 1% gelatin 180. mu.L/well, incubated at 37 ℃ for 2 h;

reaction conditions are as follows: the antiserum containing MXRA7 was diluted in a gradient manner at 100. mu.L/well in a 37 ℃ water bath for 1h → 100. mu.L/well at the working concentration (1:5000 dilution) goat anti-mouse IgG-HRP in a 37 ℃ water bath for 1h → 100. mu.L/well TMB developed for 10min → 50. mu.L/well 1M hydrochloric acid termination reaction → ELISA for OD450 values.

Cell fusion: mouse myeloma cells SP2/0 were recovered 10 days before fusion, and cultured in DMEM medium containing 15% FBS (fetal bovine serum) to ensure good cell growth during fusion. One day before fusion, 4-week-old Balb/c mice are taken, after the eyes are removed and blood is discharged, the mice are killed by cervical dislocation, and macrophages in abdominal cavities of the mice are taken as feeder cells to be paved on a 96-well plate. On the day of fusion, SP2/0 cells in good growth status were collected for use, and then spleens of immunized mice were separated under aseptic conditions, placed in a 200-mesh stainless steel screen, gently ground to obtain a single spleen cell suspension, washed twice with PBS, and the cells were suspended in 20mL DMEM. Mouse myeloma cells SP2/0 were mixed with spleen cells according to a 1: 3, centrifuging at 1000rpm for 5min, discarding the supernatant, and flicking the tube bottom with a finger to mix the two cells into paste. 1mL of 37 ℃ preheated 50% PEG solution is dripped within 1min for mediated fusion at the temperature of 37 ℃, after the fusion is finished, serum-free DMEM medium preheated at the temperature of 37 ℃ is added for termination, finally, the supernatant is removed by centrifugation, HAT medium is added for re-suspension, and the mixture is dripped into a 96-well culture plate containing feeder cells after being mixed uniformly, wherein the concentration is 100 mu L/well, the concentration is 37 ℃ and 5% CO₂And (5) culturing.

Screening monoclonal cell strains: when 1/3-1/4 culture wells are full of fused cells (the culture medium is yellow), culture supernatant can be aspirated, and positive wells are screened by ELISA. The culture medium is replaced timely, and the screening method of the established positive clone is carried out again according to the growth state of the cells. Selecting cells which grow in a single clone, have good shapes and high antibody titer to continue subcloning; enlarging culture and freezing and storing in liquid nitrogen.

Ascites preparation and antibody purification: collecting 8 weeks oldInjecting 0.4mL of liquid paraffin (Pristane) into the abdominal cavity of a female Balb/c mouse for 3-5 days, and then injecting the liquid paraffin (Pristane) into the abdominal cavity according to the proportion of 1 multiplied by 10⁷The hybridoma cells are injected into the abdominal cavity of one side of the mouse, and simultaneously, the other side of the mouse is injected with 0.2mL of the mixture of Pristane and incomplete Freund's adjuvant in the same volume into the abdominal cavity again, so that the hybridoma cells are dispersed in the abdominal cavity by gently massaging the abdomen of the mouse. Observing the abdomen of the mouse after 7-10 days, collecting ascites, removing fibrin by conventional treatment of the ascites, passing through a Protein G affinity chromatography column, eluting with glycine-hydrochloric acid (pH2.8), adjusting the pH to 7.0 with a Tris solution with the pH of 9.0, and subpackaging at-80 ℃ for storage.

The potency of the monoclonal antibody and the subtype thereof are determined: the titer of the purified anti-MXRA 7 monoclonal antibody SZ181 was identified by ELISA. Coating: MXRA7 protein was diluted in PBS to 0.1. mu.g/mL, 50. mu.L/well and incubated at 37 ℃ for 8 h; and (3) sealing conditions: 1% gelatin 200. mu.L/well, incubated at 37 ℃ for 2 h; reaction conditions are as follows: gradient dilution SZ 181100. mu.L/well incubation 1h at 37 ℃ → 100. mu.L/well working concentration goat anti-mouse IgG-HRP (1:5000 dilution), water bath 1h at 37 ℃ → 100. mu.L/well TMB development 10min → 50. mu.L/well 1M hydrochloric acid termination reaction → ELISA instrument to measure OD450 value, as shown in FIG. 5 (A), the titer was determined to be about 1: 102400.

The Ig class and subclass were identified using antibody mouse antibody identification paper (purchased from Roche) showing that the monoclonal antibody is IgG1, kappa type.

Specific identification of anti-MXRA 7 monoclonal antibody: the specificity of the monoclonal antibody was examined using immunoblot (protein blocking) experiments. To 0.5g of mouse liver was added 1mL of RIPA cell lysate containing protease inhibitor, homogenized with a tissue homogenizer, and incubated on ice for 15 min. After centrifugation, the supernatant was collected and quantified for BCA protein. Meanwhile, the protein quantification is carried out on the SUMO-MXRA7 fusion protein which is expressed and purified by pronucleus. After adding SDS loading buffer to boil the sample, 20. mu.g of protein sample was electrophoresed on 10% SDS-PAGE gel and transferred to PVDF membrane. After being blocked by 5% skim milk at room temperature for 2h, the membrane is incubated overnight at room temperature with an anti-MXRA 7 monoclonal antibody SZ 1814 ℃ diluted at a ratio of 1:1000, washed by PBST for 3 times, incubated with a goat anti-mouse secondary antibody labeled by HRP at room temperature for 1h, washed by PBST for 3 times, and then detected by ECL chemiluminescence solution for antibody reaction. As shown in FIG. 5 (B), the sample in lane 1 is SUMO-MXRA7 fusion protein, the sample in lane 2 is liver tissue protein of WT mice, and it can be seen that the antibody can recognize both SUMO-MXRA7 fusion protein and mouse liver tissue protein. Western blot detection is carried out by taking escherichia coli lysates respectively expressing fusion proteins of the MXRA7 exon I and the MXRA7 exon I as detection targets, and the recognition site of the SZ181 in the Western blot detection is located on the MXRA7 exon, which is obtained from a graph (C) in figure 5. In the site competition experiment, different concentrations of SUMO-MXRA7 fusion protein were added to the antibody for blocking while the primary antibody was incubated, and the rest steps were carried out conventionally, and the result is shown in FIG. 5 (D), wherein 50. mu.g/mL of SUMO-MXRA7 fusion protein could completely block the binding of SZ181 and the target protein.

anti-MXRA 7 monoclonal antibody SZ181 cross-reactivity: proteins derived from different tissues (heart, small intestine and skin) of rats were taken, BCA was quantified, 1 xSDS loading buffer was added to the protein for boiling, and 20. mu.g of the protein sample was electrophoresed on 10% SDS-PAGE gel and transferred to an NC membrane. After being blocked with 5% skim milk at room temperature for 2h, the membrane is incubated with anti-MXRA 7 monoclonal antibody SZ 1814 ℃ at 1:1000 dilution overnight, after being washed with PBST for 3 times, the membrane is incubated with goat anti-mouse secondary antibody labeled with HRP at room temperature for 1h, after being washed with PBST for 3 times, the antibody reaction is detected by ECL chemiluminescence solution, and the result is shown in FIG. 6, and the antibody can be used for identifying MXRA7 protein in small intestine and skin of rats. The proteins of human cell lines THP-MXRA7 and SH1-MXRA7 overexpressing MXRA7 and control cells are extracted, and Western blot does not detect a target band, which indicates that the antibody cannot recognize MXRA7 protein in human tissues.

Example 4 detection of the use of MXRA7 monoclonal antibody SZ181

1. Western blot application detection

Proteins from different tissues of mice are taken, BCA is quantified, 1 xSDS loading buffer is added for boiling, 20 mu g of protein sample is taken, 10% SDS-PAGE gel electrophoresis is carried out, and then the protein sample is transferred to an NC membrane. After being blocked by 5% skim milk at room temperature for 2h, the membrane is incubated with anti-MXRA 7 monoclonal antibody SZ 1814 ℃ at 1:1000 dilution overnight, after being washed for 3 times by PBST, the membrane is incubated with goat anti-mouse secondary antibody marked by HRP at room temperature for 1h, after being washed for 3 times by PBST, the antibody reaction is detected by ECL chemiluminescence solution, and the results are shown in FIG. 7, wherein the expression of MXRA7 in different tissues of mice is inconsistent, and the expression of MXRA in the small intestine and the spleen is higher. Wherein, the graph A is a typical Western blot graph in one time, and the graph B is a gray scale statistical analysis of the two results.

2. Immunohistochemical application detection

Paraffin section of mouse tissue prepared by conventional method, antigen retrieval and application of H₂O₂After treatment, the sample was sealed with a normal goat serum working solution for 20min at room temperature. anti-MXRA 7 monoclonal antibody SZ181 was purified using 0.1% BSA 1: diluting with 100 deg.C, adding dropwise onto the tissue slices, incubating at 4 deg.C overnight, incubating overnight at 37 deg.C for 1 hr, washing with PBS for 3 times, and each time for 3 min; adding working liquid drops of goat anti-mouse secondary antibody marked by HRP (horse radish peroxidase) onto the slices, incubating for 30min at 37 ℃, and washing for 3 times with PBS (phosphate buffer solution) for 3min each time;

after DAB color development, conventional method counterstaining, dehydration, transparency, mounting, the result is shown in FIG. 8, antibody SZ181 has positive signal in spleen and lung of mouse, and has no obvious positive signal in liver.

3. Immunofluorescence application detection

Cells of mouse hepatoma cell line Hepa1-6-MXRA7 overexpressing MXRA7 were made into cell slide, treated with 0.5% Triton-X, and blocked with 1% BSA at room temperature for 30 min. anti-MXRA 7 monoclonal antibody was purified with 0.1% BSA 1:100 dilution was added dropwise to the cell slide and incubated overnight at 4 ℃. The next day, the overnight pieces were washed 3 times with PBS, 3min each time; adding working liquid drops of a goat anti-mouse secondary antibody marked by Alexa Fluor 594 to the slices, incubating for 30min at 37 ℃, and washing for 3 times with PBS (phosphate buffer solution) for 3min each time; DAPI was stained at room temperature for 5min and washed 3 times with PBS, 3min each time. After adding the tablet, the tablet is sealed and observed by laser confocal microscope. The immunofluorescence results show (fig. 9) that antibody SZ181 has good binding activity, MXRA7 is able to specifically detect mouse cells, and the red signal is localized in the cytoplasm.

4. Streaming application detection

Digesting mouse liver cancer cell strain Hepa1-6-MXRA7 of over-expression MXRA7 and control cells with 0.25% pancreatin containing EDTA, and reselecting cells to cells containing 1% FBSIn PBS, and adjusted to a concentration of 5X10⁵Adding a purified anti-MXRA 7 monoclonal antibody (or an isotype control antibody) to a final concentration of 1 mu g/mL, incubating for 30min at 4 ℃ in a dark place, washing for 2 times by PBS, adding an FITC-labeled anti-mouse immunoglobulin antibody, incubating for 30min at 4 ℃ in a dark place, washing for 2 times by PBS, suspending cells in 500 mu L of PBS, detecting on a flow cytometer (beckman fc 500) machine, and confirming the transfection of MXRA7 by the detection result so that the high expression of MXRA7 is realized on Hepa1-6 cells; in addition, the MXRA7 monoclonal antibody SZ181 also confirmed that the expression site of MXRA7 in Hepa1-6 cells is located intracellularly, not on the surface of the cell membrane, as shown in FIG. 10.

Sequence listing

<110> Suzhou university affiliated first hospital

Preparation method and application of anti-mouse MXRA7 monoclonal antibody

<141> 2017-09-08

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 33

<212> DNA

<213> biosynthesis (Artificial sequence)

<400> 1

cggggtacca tggaatctcc ggtggagctc tta 33

<210> 2

<211> 33

<212> DNA

<213> biosynthesis (Artificial sequence)

<400> 2

cgcggatcct tacatgtcgt atagacgcag ctg 33

<210> 3

<211> 537

<212> DNA

<213> mouse (Mus musculus)

<400> 3

atggaatctc cggtggagct cttagccgct ctgccggcct tagtgaccgc cctggctctg 60

ctcctggctt ggctgtract gcgtcgcgga gctgcacgcg tgcctgctcc ggagtctaca 120

gctagcgacg aggccccggg cgccccagcg cctccggagc cgcctgaaag ctgtgcacct 180

gaaccggccc ctgagggtcc gagccagtca gaacgcgtgg ctgagcctga agaatcagag 240

gcagaggagc ctgccgcgga gggtcgccaa gacgaagata gcgattcaga gatgggcccg 300

cctacagaag aacctgagga agaagacggc gctgccttca gctttaaata cagcccagga 360

cagttacgtg gtagccagta caaaaagatg atgacgaagg aagagctgga ggaagagcat 420

cgcgttcaga aagagcagct ggccgctatc ttcaagctga tgaaggataa caaggacacc 480

tttggcgaaa tgagcgacgg agatatgcag gagcagctgc gtctatacga catgtaa 537

Claims (2)

1. An anti-mouse MXRA7 monoclonal antibody with the preservation number of CCTCC NO: C2017142.

2. Use of the anti-mouse MXRA7 monoclonal antibody of claim 1 in the biological detection of MXRA7 protein for non-therapeutic and diagnostic purposes.

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