CN111778257A

CN111778257A - Genetically modified CD96 protein, monoclonal antibody thereof and application

Info

Publication number: CN111778257A
Application number: CN202010620792.8A
Authority: CN
Inventors: 屠志刚; 刘晗青
Original assignee: Jiangsu Laisen Biotechnology Research Institute Co ltd
Current assignee: Jiangsu Laisen Biotechnology Research Institute Co ltd
Priority date: 2020-07-01
Filing date: 2020-07-01
Publication date: 2020-10-16

Abstract

The invention belongs to the technical field of biology, and particularly relates to a genetically modified CD96 protein, a monoclonal antibody thereof and application thereof. The invention uses FAT10 to modify CD96 to obtain optimized FAT10-CD96 protein, and uses the protein to prepare the mouse monoclonal antibody through immunization, the CD96 resisting monoclonal antibody has high specificity, and can be produced in large quantitiesThe titer of the obtained antibody reaches 1 × 10⁵The above. Can be specifically combined with CD96 protein, can be used for immunological detection of cells, has wide market prospect, and may also have important clinical significance for further developing biological treatment medicaments taking CD96 as targets.

Description

Genetically modified CD96 protein, monoclonal antibody thereof and application

Technical Field

The invention relates to the technical field of biology, in particular to a genetically modified CD96 protein, a monoclonal antibody thereof and application thereof.

Background

Acute Myeloid Leukemia (AML) is a malignant proliferative disease originated from hematopoietic stem cells (LSCs), and has the characteristics of rapid onset, fierce illness, difficult control, easy relapse, poor prognosis, and rising incidence with age, and the like, and the condition is severe. The current treatment methods for AML are mainly combination chemotherapy and hematopoietic stem cell transplantation, with significant side effects and are not effective in improving the 5-year survival rate or complete remission rate of the patient. In recent years, with the ongoing research on AML, targeted therapies directed to cell surface molecular markers of LSCs have become the focus of current research. Phenotypic studies of LSCs in AML showed positive expression of CD96 protein on the cell surface of lcs cells in about 60% of AML patients. Positive expression of CD96 protein was present in different types of AML, and expression of CD96 was up-regulated by about 10-fold in LSCs of AML patients compared to normal human Hematopoietic Stem Cells (HSCs). The positive expression of the CD96 protein is taken as a tumor marker of AML, so that how to efficiently and highly specifically identify the expression of CD96 is particularly important, and the continuous development of a CD96 antibody with high specificity, high affinity and high yield has important significance in the aspects of diagnosis of AML-LSCs, specific immunotherapy drugs taking CD96 as a target spot and the like.

The ubiquitin-like protein is a protein with a similar spatial structure and an enzyme modification system with ubiquitin, plays an important role in various biological functions such as nuclear mass transport, transcription regulation, protein stabilization, stress response, cell cycle regulation and the like, is found in the research of anti-tumor drugs, can change the anti-tumor effect of the drugs by regulating the ubiquitin-like modification, and is a new target of the tumor prevention and treatment drugs. Human leukocyte antigen (FAT 10) is a ubiquitin-like protein and is involved in the development of tumors. It has high expression in various cancers and plays a certain role in immune metabolism regulation. At present, no research report on CD96 protein modification related antibody exists.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a genetically modified CD96 protein, a monoclonal antibody thereof and application thereof. The invention designs a FAT10 modified CD96 fusion expression protein by utilizing a molecular biology technology, develops a CD96 antibody with high specificity, high affinity and high yield, and has important significance in the aspects of specific immunotherapy drugs further taking CD96 as a target spot and the like.

The above object of the present invention is achieved by the following technical solutions:

a CD96 protein gene modified by FAT10 has a nucleotide sequence shown as SEQ ID number 1.

A CD96 protein modified by FAT10 is obtained by expressing the CD96 protein gene modified by FAT10, and the amino acid sequence of the protein is shown as SEQ ID NO. 2.

An expression vector, which contains the CD96 protein gene modified by FAT 10.

In a specific embodiment, the invention also provides an anti-CD 96 monoclonal antibody, wherein the anti-CD 96 monoclonal antibody adopts the expression vector of the FAT10 modified CD96 protein gene as an immunogen; the amino acid sequence of the light chain variable region of the anti-CD 96 monoclonal antibody is shown as SEQ ID number 3, and the amino acid sequence of the heavy chain variable region thereof is shown as SEQ ID No. 4.

In a specific embodiment, the invention also provides a preparation method of the anti-CD 96 monoclonal antibody, which comprises the steps of immunizing a BALB/c mouse with FAT10-CD96 recombinant protein, fusing spleen lymphocytes of the mouse successfully immunized with myeloma cells, screening positive clones to obtain a hybridoma cell line capable of specifically secreting the anti-CD 96 monoclonal antibody, inoculating the cells into abdominal cavities of the BALB/c mouse sensitized with liquid paraffin in advance, producing the anti-CD 96 monoclonal antibody in a large amount, and preparing the high-purity anti-CD 96 monoclonal antibody in a protein purification mode. The preparation process comprises the following steps:

(1) expressing and purifying the recombinant protein;

(2) detecting the immunity and the titer of a BALB/c mouse;

(3) fusing and screening hybridoma cells;

(4) production and purification of anti-CD 96 monoclonal antibody.

In a specific embodiment, the invention also provides a hybridoma cell line capable of producing the anti-CD 96 monoclonal antibody.

The invention also provides application of the anti-CD 96 monoclonal antibody in preparation of an immunological detection tool.

Further, the anti-CD 96 monoclonal antibody is used for preparing a biological detection reagent for detecting CD96 protein. The further application is to prepare a reagent for detecting the CD96 antigen, in particular to detect CD96 protein in cells.

Compared with the prior art, the invention has the beneficial effects that:

the invention utilizes FAT10 to modify CD96 in a covalent connection mode to obtain optimized FAT10-CD96 protein, and is favorable for preparing a mouse-derived monoclonal antibody by the protein immunization, the anti-CD 96 monoclonal antibody has the characteristics of high specificity and mass production, and the titer of the obtained antibody reaches 1 × 10⁵The above. The anti-CD 96 monoclonal antibody obtained by the invention can specifically bind to CD96 protein, antagonize the expression of CD96 on the cell surface of AML-LSCs or provide certain help for immunotherapy targeting CD 96. Can be used for immunological detection of cells, has wide market prospect, and also has important clinical significance for further developing biological treatment medicines taking CD96 as targets.

Drawings

FIG. 1 is an SDS-PAGE electrophoresis of recombinant proteins;

FIG. 2 is a SDS-PAGE electrophoresis of purified fractions of the CD96 monoclonal antibody;

FIG. 3 is a graph showing the results of measurement of the immunotiter of anti-CD 96 monoclonal antibodies at different dilutions;

FIG. 4 shows the specificity and cross-reaction identification of the anti-CD 96 monoclonal antibody;

FIG. 5 is a graph showing the results of Western blot analysis of MOLT-4 and CCRF-CEM cells with the anti-CD 96 monoclonal antibody.

Detailed Description

The invention discloses a genetically modified CD96 protein, a monoclonal antibody thereof and application thereof, and can be realized by appropriately improving process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention. The methods, devices and materials used in the examples which follow, if not specifically indicated, are all conventional and commercially available methods, devices and materials used in the art.

Example 1: preparation of anti-CD 96 monoclonal antibody

(1) Expression and purification of FAT10-CD96 recombinant protein

a. Transformed escherichia coli BL21 expression target recombinant protein

The nucleotide sequences of FAT10 and CD96 genes can be obtained in public databases, wherein the Genbank accession number of FAT10 is 10537, and the Genbank accession number of CD96 is 10225; modifying a CD96 gene by FAT10, wherein the nucleotide sequence of the FAT10 modified CD96 gene is shown as SEQ ID number 1, the sequence is synthesized by Nanjing Kingnshire biotechnology limited and is inserted into a prokaryotic expression vector pET-30a to form a recombinant expression plasmid pET-30a-FAT10-CD96, the recombinant plasmid pET-30a-FAT10-CD96 is transformed into escherichia coli BL21 by adopting a 42 ℃ heat shock 90 s method, kanamycin-resistant bacterial plaques are picked up and cultured in 500 mL LB culture medium until the OD of the bacterial liquid is 0.6-0.8, 0.5 mM IPTG is added to induce the expression of target protein, after the bacterial liquid is cultured at the constant temperature of 25 ℃ for 12 h, the bacterial body is collected, the bacterial body is broken by ultrasound, and the supernatant is collected by centrifugation.

b. Recombinant protein purification

And (b) adding the supernatant of the thallus lysate obtained in the step a into a nickel column chromatography column (purchased from Qiagen), binding at 4 ℃ overnight, discarding the supernatant, washing away unbound protein in the chromatography column by using a Wash Buffer solution, eluting the recombinant protein by using a 500 mM imidazole Buffer solution with 10 times of column volume, collecting and combining the eluted recombinant protein FAT10-CD96, and performing subsequent animal immunization after freeze-drying. The same procedure was used to collect normal CD96 protein (unmodified with FAT 10) as a comparative example. The expression of each protein is separated by SDS-PAGE electrophoresis, FIG. 1 is the SDS-PAGE electrophoresis of the recombinant protein CD96 and FAT10-CD96 elution components, and the elution shown in FIG. 1 obtains CD96 and FAT10-CD96 proteins with higher purity respectively.

(2) BALB/c mouse immunity and potency detection

BALB/c mice used in this example were purchased from the animal model research institute of Nanjing university. Mice were immunized by a conventional method (refer to "modern antibody technology and its applications" published by Beijing university Press and "guidance on antibody preparation and use" from scientific Press).

The serum titer of the immunized mice is detected by an indirect ELISA method. And (2) respectively taking the CD96 protein and the FAT10-CD96 protein in the step (1), diluting the CD96 protein to 1 mu g/mL by using a carbonate buffer solution with the pH of 9.6 and the mol/L of 0.05, adding the diluted protein into a 96-well enzyme label plate, coating each well with 100 mu L at 4 ℃ overnight, taking out the coated enzyme label plate, washing the coated enzyme label plate for 3 times by using TBS-T buffer solution, patting the coated enzyme label plate dry, and storing the coated enzyme label plate at 4 ℃ for later use. After 1 week of the second immunization, a proper amount of blood is collected from the tail vein of the mouse, serum is separated by centrifugation at 5000 g for 15 min, the serum is diluted by a sample diluent (phosphate buffer containing 0.5% bovine serum albumin) according to a gradient of 1:100, 1:1000, 1:10000, 1:100000 and 1:1000000, an ELISA plate to be detected is added to each hole at 100 muL, incubated for 1 h at 37 ℃, washed for 3 times by TBS-T buffer, the ELISA plate is patted dry, HRP-labeled goat anti-mouse secondary antibody (purchased from Jackson Immuno Research) diluted at 1:5000 is added to each hole, and incubated for 30 min at 37 ℃. And (3) taking out the ELISA plate, washing the ELISA plate for 5 times by TBS-T buffer solution, adding 100 mu L of TMB substrate display solution into each hole, developing the ELISA plate for 10-15 min in a dark place at 37 ℃, then adding 50 mu L of stop solution, and reading the light absorption value under the wavelength of 450 nm of an ELISA reader. The selected serum titer reaches 1:10⁵The above mice were immunized for the third time.

(3) Fusion and screening of hybridoma

Preparing feeder layer cells, preparing SP2/0 myeloma cells, and taking mouse spleen cells for cell fusion 3-4 days after the immunization is finished. And (3) paving the fused cells (4 pieces of 96-well plates), screening hybridoma cells for producing the monoclonal antibody by using an indirect ELISA method, and performing two rounds of subclone screening to respectively obtain hybridoma cell strains with the best antibody secretion, wherein the hybridoma cell strains are named as follows: CD96mAb and FAT10-CD96 mAb.

(4) Production and purification of anti-CD 96 monoclonal antibody.

Preparing mouse monoclonal antibody ascites according to a conventional method, purifying the ascites antibody by using an caprylic acid-protein G method to respectively obtain an anti-FAT 10-CD96 monoclonal antibody (FAT 10-CD96 mAb) and a CD96 monoclonal antibody (CD 96 mAb), and verifying the purity of the purified antibodies by SDS-PAGE electrophoresis, wherein the antibodies of the CD96mAb and the FAT10-CD96 mAb can obtain higher purity after elution as shown in figure 2, the molecular weight of the prepared antibody IgG (H + L) is about 160 KD, wherein the heavy chain of the IgG is about 55KD, and the light chain of the IgG is about 25 KD.

Example 2: potency assay and specificity of CD96 monoclonal antibody

(1) Potency assay of monoclonal antibodies:

diluting CD96 protein to 1 μ g/mL with 0.05 mol/L carbonate buffer solution with pH9.6, coating with ELISA plate, 100 μ L/well, and standing overnight at 4 deg.C; the monoclonal antibodies were mixed at a ratio of 1:10 with a sample diluent (phosphate buffer containing 0.5% bovine serum albumin)³、1:10⁴、1：10⁵、1：10⁶Diluting, incubating at 37 ℃ for 1 h at 100 mu L/hole, taking out an ELISA plate, washing for 3 times by TBS-T, beating the ELISA plate, adding 100 mu L of HRP-labeled goat anti-mouse secondary antibody diluted by 1:5000 into each hole, incubating for 30 min at 37 ℃, washing for 5 times by TBS-T, adding 100 mu L of TMB substrate display solution into each hole, developing for 10-15 min at 37 ℃ in a dark place, adding 50 mu L of stop solution to stop the reaction, reading the light absorption value at 450 nm wavelength of an ELISA instrument, and obtaining the result shown in figure 3, wherein the titer of the CD96 monoclonal antibody is 1 × 10⁵The titer of the FAT10-CD96 monoclonal antibody is about 1 × 10⁶About 5 fold higher than the CD96 monoclonal antibody.

(2) Monoclonal antibody specificity recognition and cross reaction identification:

respectively diluting CD96, VEGFA, TIGIT, HB-EGF and HSP90 proteins to 1 mu g/mL by using carbonate buffer solution, coating an enzyme label plate, setting a blank control group to be 1 mu g/mL BSA protein, and carrying out indirect ELISA detection by using FAT10-CD96 monoclonal antibody, wherein a figure 4 shows the identification result of specificity and cross reaction; as shown in FIG. 4, the FAT10-CD96 monoclonal antibody has no cross reaction with VEGFA, TIGIT, HB-EGF and HSP90 proteins, which indicates that the antibody has better specificity.

Example 3: western blot experiment of CD96 monoclonal antibody

Collecting a human lymphoblastic leukemia cell line MOLT-4 (purchased from American ATCC company) and a human T lymphoblastoid cell line CCRF-CEM (purchased from American ACTT company), using RIPA lysate to lyse the cells, centrifuging at 5000 g and 4 ℃ for 20 min, collecting supernatant, and detecting the protein concentration by using a BCA kit (purchased from Biyuntian biotechnology limited); separating cell lysate by 12% SDS-PAGE electrophoresis, then placing PAGE gel (purchased from Biyuntian biotechnology limited) on a PVDF membrane, and transferring 120 mA to the PVDF membrane for 240 min; placing the PVDF membrane into 5% skimmed milk, sealing for 1 h at room temperature, and incubating overnight at 4 ℃ with FAT10-CD96 monoclonal antibody obtained in example 1 of the invention at a ratio of 1:5000 respectively; taking out the PVDF membrane, rinsing with PBS-T for 3 times, 5 min each time, placing in HRP-labeled goat anti-mouse secondary antibody diluted by 1:5000, incubating at room temperature for 1 h, discarding the secondary antibody, washing with PBS-T for 5 times, 5 min each time; the film was added to a developing solution and developed away from light, and the results of the experiment were recorded by photographing, and as shown in fig. 5, the FAT10-CD96 monoclonal antibody prepared in example 1 of the present invention was able to specifically recognize CD96 protein and was able to detect CD96 protein in cells, indicating that the CD96 monoclonal antibody produced in the present invention has high specificity.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

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<120> genetically modified CD96 protein, monoclonal antibody thereof and application

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Claims

1. A CD96 protein gene modified by FAT10 is characterized in that the nucleotide sequence is shown as SEQ ID number 1.

2. A CD96 protein modified by FAT10, which is obtained by expressing the modified CD96 protein gene of claim 1, and the amino acid sequence of the protein is shown as SEQ ID NO. 2.

3. An expression vector comprising the FAT 10-modified CD96 protein gene of claim 1.

4. An anti-CD 96 monoclonal antibody, which is characterized in that the FAT10 modified CD96 protein of claim 2 is used as immunogen; the antibody comprises a heavy chain variable region, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 3.

5. The anti-CD 96 monoclonal antibody of claim 4, wherein the antibody further comprises a light chain variable region having the amino acid sequence shown in SEQ ID No. 4.

6. A hybridoma cell line capable of producing the monoclonal antibody of claim 4.

7. Use of the anti-CD 96 monoclonal antibody according to claim 4 for the preparation of an immunological detection means.

8. The use of the anti-CD 96 monoclonal antibody according to claim 7, in the preparation of a biological detection reagent for detecting CD96 protein.

9. The use of the anti-CD 96 monoclonal antibody according to claim 7, for preparing a reagent for detecting CD96 antigen.