CN113151186B

CN113151186B - Monoclonal antibody of anti-human CD271 and application

Info

Publication number: CN113151186B
Application number: CN202110156939.7A
Authority: CN
Inventors: 路慧丽; 杨慧; 王紫嫣; 朱建伟
Original assignee: Shanghai Jiaotong University
Current assignee: Shanghai Jiaotong University
Priority date: 2021-02-04
Filing date: 2021-02-04
Publication date: 2022-02-18
Anticipated expiration: 2041-02-04
Also published as: CN113151186A; WO2022166802A1

Abstract

The invention belongs to the technical field of biology, and particularly relates to a hybridoma cell strain secreting an anti-human CD271 monoclonal antibody, an antibody thereof and application thereof. The anti-human CD271 antibody has high specificity and affinity, can be used for preparing a detection reagent related to CD271 molecules, or preparing a biotechnology medicament related to CD271, or coupling the anti-human CD271 antibody with a magnetic nano material to prepare immunomagnetic beads to sort tumor stem cells, mesenchymal stem cells, nerve cells and the like, or combining the immunomagnetic beads with other targets to design antibody coupling molecules such as bispecific antibodies and the like, and is used for detecting or treating certain tissue repair diseases and nervous systems or tumor related diseases.

Description

Monoclonal antibody of anti-human CD271 and application

Technical Field

The invention belongs to the technical field of biology, and particularly relates to an anti-human CD271 monoclonal antibody and application thereof.

Background

CD271 is a low affinity nerve growth factor receptor (LNGFR), a member of the tumor necrosis factor receptor superfamily, with a relative molecular mass of 75kD, also known as p75NTR, and it contains 3 regions: a cysteine-rich extracellular domain, a transmembrane domain and an intracellular domain of 155 amino acid residues. CD271 is not only expressed in the nervous system, closely related to development, differentiation and survival of nerve cells, but also is a relatively suitable marker molecule for enriching mesenchymal stem cells, and has the advantages of high specificity, high purity, strong colony forming capability and the like compared with the traditional sorting method.

Mesenchymal Stem Cells (MSCs) are a class of adult stem cells with self-renewal and multi-differentiation potential, and have a wide application prospect in regenerative medicine research. Clinical trials with mesenchymal stem cells have mainly involved tissue injury repair, such as repair of bone, cartilage, and joint injuries, treatment of cardiac, hepatic, and spinal cord injuries, and neurological diseases. However, the use of MSCs for targeted tissue repair has been limited by the engraftment and proliferation of stem cells at the damaged site. Therefore, in the treatment of tissue injury sites or diseases related to nervous system, the elimination or enrichment of specific cells in grafts is the most critical issue in the current field of transplantation. The CD271, CD105, CD133 and the like are used as surface markers of the mesenchymal stem cells, can effectively separate or enrich the mesenchymal stem cells, can be differentiated into cartilage, muscle, tendon and the like under in-vivo or in-vitro specific induction conditions, and achieves the purpose of tissue repair. On the other hand, CD271 is also one of the markers of various tumor stem cells (CSCs). CD271 is used as a surface marker molecule of MSC and CSC, has better specificity to stem cells compared with molecules such as CD44, CD105 and the like, and the monoclonal antibody not only can be used for screening and identifying MSC, but also can be used for anti-tumor research, and has important research and application values. However, at present, there is still a lack of anti-CD 271 monoclonal antibodies having a broad recognition ability for mesenchymal stem cells, tumor stem cells, and the like.

Disclosure of Invention

In view of the above-mentioned disadvantages of the prior art, the present invention aims to provide a monoclonal antibody against human CD271 and its use.

The extracellular structure of native CD271 comprises 4 cysteine-rich CRD structures (CRD1-4) and a linking region (talk region). After CD271 expression, CRD regions are cleaved by proteolytic enzymes, and thus binding of antibodies targeting CRD regions to CD 271-positive cells is affected, and CD271 expression is not detected even at some stage of cellular metabolism. And the talk area close to the cell membrane has no glycosylation, phosphorylation modification or high-level spatial structure, the structure is stable, and the antibody for identifying the area theoretically has better universality. Therefore, in order to obtain a monoclonal antibody capable of stably recognizing human CD271, we select the talk region as a target to develop a novel anti-CD 271 monoclonal antibody, and provide a new tool for the research in the field.

In order to achieve the above objects and other related objects, the present invention adopts the following technical solutions:

the invention provides a hybridoma cell strain CD271T-2 capable of resisting and secreting an anti-human CD271 monoclonal antibody, wherein the preservation number of the hybridoma cell strain is CGMCC 21494. Is preserved in the China general microorganism strain preservation management center, and the preservation address is No.3 of Xilu No.1 of Beijing, Chaoyang, North Chen. The preservation date is 2021 year, 1 month and 25 days. The obtained cell line is classified and named as hybridoma cell line CD 271T-2.

The second aspect of the invention provides an anti-human CD271 monoclonal antibody, wherein the anti-human CD271 monoclonal antibody is secreted by a CD271T-2 hybridoma cell strain.

The anti-human CD271 monoclonal antibody has good binding affinity with human mesenchymal stem cells.

Optionally, the antibody is a murine monoclonal antibody, and belongs to an IgM subtype.

The third aspect of the invention provides an engineered antibody against human CD271, which comprises a light chain and a heavy chain, wherein the light chain comprises LCDR1, LCDR2 and LCDR3, the amino acid sequence of the LCDR1 is shown in SEQ ID No.1, the amino acid sequence of the LCDR2 is shown in SEQ ID No.2, and the amino acid sequence of the LCDR3 is shown in SEQ ID No. 3; the heavy chain comprises HCDR1, HCDR2 and HCDR3, wherein the amino acid sequence of the HCDR1 is shown as SEQ ID NO.4, the amino acid sequence of the HCDR2 is shown as SEQ ID NO.5, and the amino acid sequence of the HCDR3 is shown as SEQ ID NO. 6.

Specifically, the light chain complementarity determining region LCDR1, length: 10aa, the amino acid sequence is shown as SEQ ID NO.1, and specifically comprises: QSVDYDGDSY are provided.

Light chain complementarity determining region LCDR2, length: 3aa, the amino acid sequence is shown as SEQ ID NO.2, and specifically comprises: and (5) AAS.

Light chain complementarity determining region LCDR3, length: 9aa, the amino acid sequence is shown as SEQ ID NO.3, and specifically comprises: QQSNEDPFT are provided.

Heavy chain complementarity determining region HCDR1, length: 10aa, the amino acid sequence is shown as SEQ ID NO.4, and specifically comprises: GFSLSTSGMG are provided.

Heavy chain complementarity determining region HCDR2, length: 7aa, the amino acid sequence is shown as SEQ ID NO.5, and specifically comprises: IWWDDDK.

Heavy chain complementarity determining region HCDR3, length: 13aa, the amino acid sequence is shown as SEQ ID NO.6, and specifically comprises: ARRDYGNYYAMDY are provided.

Further, the amino acid sequence of the light chain variable region of the anti-human CD271 engineering antibody is shown in SEQ ID NO. 7; or certain substitution, addition and/or deletion of one or more amino acids are carried out on the SEQ ID NO.7 to obtain an amino acid sequence with the same function. The heavy chain variable region amino acid sequence is shown as SEQ ID NO.8, or the amino acid sequence with the same function is obtained by carrying out certain substitution, addition and/or deletion on one or more amino acids of SEQ ID NO. 8.

Specifically, the sequence shown in SEQ ID NO.7 specifically comprises:

DIVLTQSPASLAVSLGQRATISCKASQSVDYDGDSYMNWYQQKPGQPPKLLIYAASN LESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSNEDPFTFGSGTKLEIK。

the sequence shown in SEQ ID NO.8 specifically comprises:

QVTLKESGPGILKPSQTLSLTCSFSGFSLSTSGMGVGWIRQPSGKGLEWLAHIWWDDDKYYNPSLKSQLTISKDTSRNQVFLKITSVDTADTATYYCARRDYGNYYAMDYWGQGTSVTVSS

alternatively, the engineered antibody may be a monoclonal antibody.

Alternatively, the monoclonal antibody is of murine origin.

In one embodiment, the monoclonal antibody subclass is IgM.

Alternatively, the antibody is prepared from a CD271T-2 hybridoma cell line by a reverse transcription-polymerase chain reaction.

In a fourth aspect, the present invention provides a polynucleotide encoding the heavy chain variable region and/or the light chain variable region or the full-length amino acids of the anti-human CD271 antibody.

The heavy chain variable region and/or the light chain variable region of the anti-human CD271 antibody is shown as SEQ ID NO.9-SEQ ID NO. 14.

Specifically, light chain complementarity determining region LCDR1, DNA, length: 30bp, the nucleotide sequence is shown as SEQ ID NO.9, and the nucleotide sequence specifically comprises the following components: CAAAGTGTTGATTATGATGGTGATAGTTAT are provided.

Light chain complementarity determining region LCDR2, DNA, length: 9bp, the nucleotide sequence is shown as SEQ ID NO.10, and the nucleotide sequence specifically comprises: GCTGCATCC are provided.

Light chain complementarity determining region LCDR3, DNA, length: 27bp, the nucleotide sequence is shown as SEQ ID NO.11, and the nucleotide sequence specifically comprises: CAGCAAAGTAATGAGGATCCATTCACG are provided.

Heavy chain complementarity determining region HCDR1, DNA, length: 30bp, the nucleotide sequence is shown as SEQ ID NO.12, and the nucleotide sequence specifically comprises: GGGTTTTCACTGAGCACTTCTGGTATGGGT are provided.

Heavy chain complementarity determining region HCDR2, DNA, length: 21bp, the nucleotide sequence is shown as SEQ ID NO.13, and the nucleotide sequence specifically comprises: ATTTGGTGGGATGATGATAAG are provided.

Heavy chain complementarity determining region HCDR3, DNA, length: 39bp, the nucleotide sequence is shown as SEQ ID NO.14, and the nucleotide sequence specifically comprises: GCTCGAAGGGACTATGGTAACTACTATGCTATGGACTAC are provided.

Furthermore, the nucleotide sequence of the light chain variable region of the anti-human CD271 antibody is shown in SEQ ID NO.15, and the nucleotide sequence of the heavy chain variable region of the anti-human CD271 antibody is shown in SEQ ID NO. 16.

Specifically, the sequence shown in SEQ ID NO. 15:

GACATTGTGCTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGGG CCACCATCTCCTGCAAGGCCAGCCAAAGTGTTGATTATGATGGTGATAGTTATATGAAC TGGTACCAACAGAAACCAGGACAGCCACCCAAACTCCTCATCTATGCTGCATCCAATC TAGAATCTGGGATCCCAGCCAGGTTTAGTGGCAGTGGGTCTGGGACAGACTTCACCCT CAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCAAAGTAAT GAGGATCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAA。

the sequence shown in SEQ ID NO. 16:

CAAGTTACTCTAAAAGAGTCTGGCCCTGGGATATTGAAGCCCTCACAGACCCTC AGTCTGACTTGTTCTTTCTCTGGGTTTTCACTGAGCACTTCTGGTATGGGTGTAGGCTG GATTCGTCAGCCTTCAGGGAAGGGTCTGGAGTGGCTGGCACACATTTGGTGGGATGAT GATAAGTACTATAACCCATCCCTGAAGAGCCAGCTCACAATCTCCAAGGATACCTCCAG AAACCAGGTATTCCTCAAGATCACCAGTGTGGACACTGCAGATACTGCCACTTACTAC TGTGCTCGAAGGGACTATGGTAACTACTATGCTATGGACTACTGGGGTCAAGGAACCT CAGTCACCGTCTCCTCAG。

in a fifth aspect, the present invention provides a polynucleotide encoding the recombinant sequences of the light chain variable region and the heavy chain variable region of the Fab fragment of the anti-CD 271 antibody, which enables to obtain a single chain antibody having a smaller molecular weight.

In a fifth aspect, the invention provides a construct comprising a polynucleotide as described above.

The construct may be constructed by inserting the isolated polynucleotide into a multiple cloning site of an expression vector.

The expression vector of the present invention is generally referred to various commercially available expression vectors well known in the art, and may be, for example, a bacterial plasmid, a bacteriophage, a yeast plasmid, a plant cell virus, a mammalian cell virus such as an adenovirus, a retrovirus, or other vectors. Specific expression vectors that may be used include, but are not limited to: pET series expression vector, pGEX series expression vector, pcDNA series expression vector, etc.

The host cell of the invention, which comprises the aforementioned construct or a polynucleotide having an exogenous sequence integrated into its genome. Any cell suitable for expression of an expression vector may be used as the host cell, for example, the host cell may be a prokaryotic cell, such as a bacterial cell or the like; or lower eukaryotic cells such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Specifically, cells of, for example, yeast, insect, plant, etc. may be mentioned. Preferably, the host cell is a eukaryotic cell, and mammalian host cell lines that do not produce antibodies can be used, including but not limited to: ovary cells of Chinese Hamster (CHO), kidney cells of baby hamster (BHK, ATCC CCL 10), Sertoli cells of baby mouse (Sertoli cells), kidney cells of monkey (COS cells), kidney CVI cells of monkey transformed by SV40(COS-7, ATCC CRL 1651), human embryonic kidney cells (HEK-293), kidney cells of monkey (CVI, ATCC CCL-70), kidney cells of African green monkey (VERO-76, ATCC CRL-1587), human cervical cancer cells (HELA, ATCC CCL-2), and the like.

Suitable conditions for expression of the antibody will be known to those skilled in the art and one skilled in the art can empirically select a suitable medium for culturing under conditions suitable for growth of the host cell. After the host cells have been grown to an appropriate cell density, the selected promoter is induced by suitable means (e.g., temperature shift or chemical induction) and the cells are cultured for an additional period of time. The recombinant polypeptide in the above method may be expressed intracellularly or on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (such as salting-out), centrifugation, cell disruption by osmosis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, High Performance Liquid Chromatography (HPLC), and various other liquid chromatography techniques, and combinations thereof.

In a sixth aspect, the invention provides a host cell comprising the aforementioned construct or having exogenous polynucleotide integrated into its genome.

In a seventh aspect, the present invention provides the use of the host cell of the above cell strain, or the above monoclonal antibody against human CD271, or the above engineered antibody against human CD271, or the above polynucleotide, or the above construct, or the above antibody, wherein the host cell is selected from any one of the following:

a) cells expressing CD271, including but not limited to mesenchymal stem cells, tumor stem cells, cells of the nervous system, for their in vitro detection, identification, isolation, enrichment, targeting;

b) mediating mesenchymal stem cells to repair tissues in a targeted way;

c) detecting and tracing stem cells, tumor stem cells and nervous system cells;

d) preparing a nervous system targeted drug;

e) preparing a tumor treatment medicament;

f) coupled with other chemical micromolecules, biological macromolecules, radioactive molecules, nano materials and the like, and is used for various purposes of a) to e).

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

the monoclonal antibody secreted by the hybridoma cell strain has the advantages of high titer, good specificity and strong affinity with natural antigen. The anti-human CD271 engineering antibody is prepared from CD271T-2 hybridoma through reverse transcription-polymerase chain reaction, can be specifically combined with a human CD271 molecule, and has certain affinity for human mesenchymal stem cells in vitro. CD271 is expressed on the surfaces of mesenchymal stem cells, tumor stem cells and cells of nervous system related diseases, and is a potential target for mediating the mesenchymal stem cells or targeting the nervous system related cells and tumors. Therefore, the anti-human CD271 monoclonal antibody is a medical antibody with great potential.

The anti-human CD271 antibody has high specificity and affinity, can be coupled with magnetic nano materials to prepare immunomagnetic beads for sorting mesenchymal stem cells, can be used together with other targets to design a bispecific antibody, or can be used together with other chemical molecules, radioactive molecules, polypeptides or proteins, nano materials and the like for treating certain tissue repair diseases and nervous system or tumor related diseases.

The preservation information of the strain of the invention is as follows:

cell line name: hybridoma cell line CD 271T-2;

the preservation number is as follows: CGMCC 21494;

the preservation date is as follows: 1 month, 25 days 2021;

the name of the depository: china general microbiological culture Collection center;

the preservation unit is abbreviated as: CGMCC;

the address of the depository: xilu No.1 Hospital No.3, Beijing, Chaoyang, North.

Drawings

FIG. 1 is a graph of serum titers of mice after ELISA detection of anti-human CD271 immunization: the coating antigen is full-length CD271 protein antigen, the antigen concentration is 4 mug/mL, the sample serum is diluted from 1:2000 to 1:256000, and the serum titers of No.1, No.2, No.3, No.4 and No.5 mice are all 1: 128000 above, 5 mice all had higher reactivity to antigen;

FIG. 2 is a SDS-PAGE image of mouse CD271T-2 ascites purified antibody: in the non-reduction case, the ascites antibody band is single, and in the reduction case, the ascites antibody band is respectively the light chain and the heavy chain of the antibody at 26kDa and 53 kDa;

FIG. 3 shows ELISA identification of mouse ascites purified antibodies: the coating antigen is full-length CD271 protein antigen, the antigen concentration is 2 mug/mL, each hole is 100 mug L, the concentration gradient of the loading antibody is 0.625 mug/mL, 1.25 mug/mL, 2.5 mug/mL, 5 mug/mL, 10 mug/mL, 20 mug/mL and 40 mug/mL, each hole is 100 mug L, and the result shows that the CD271T-2 antibody can recognize and bind to the human CD271 antigen;

FIG. 4 is a ForteBio validation of the antibody affinity profile after purification;

FIG. 5 shows the results of PCR cloning of heavy and light chain variable region gene of the anti-human CD271 antibody of the present invention: FIG. 5A shows the results of PCR cloning of the heavy and light chain variable region of the antibody of the CD271T-2 hybridoma, and FIG. 5B shows the results of PCR cloning of the chimeric ScFv gene of the heavy and light chain variable region of the antibody of the CD271T-2 hybridoma;

FIG. 6 shows the detection of the expression of CD271 on the surface of mesenchymal stem cells by using anti-human CD271 monoclonal antibody: FIG. 6A shows a positive control antibody derived from Biolegend APC, coat anti human CD271, the immunogen is from melanoma cells, the cell surface of which is highly expressing CD271 molecules, and the antibody is slightly migratory compared to the negative control; FIG. 6B shows a Rabbit anti human CD271 negative control antibody derived from Abcam, the immunogen is from the 350-450 amino acid region within the human CD271 cell membrane, and there is no deviation compared to the positive control; FIG. 6C shows that the CD271T-2 ascites purified antibody has a higher degree of deviation compared with the positive and negative controls, which indicates that the prepared anti-human CD271 monoclonal antibody has very high affinity and specificity, and is superior to the commercial APC mouse anti human CD271 control antibody in the proportion of positive cells.

Detailed Description

The term "antibody" as used herein refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains, each pair having one "light" (L) chain and one "heavy" (H) chain. In a general sense, a heavy chain is understood to mean the polypeptide chain of the antibody having the larger molecular weight, and a light chain is understood to mean the polypeptide chain of the antibody having the smaller molecular weight. Light chains can be classified as kappa and lambda light chains. Heavy chains can be generally classified as μ, δ, γ, α or ε, and the antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of 3 domains (CH1, CH2, and CH 3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain CL. The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1 q). The VH and VL regions can also be subdivided into regions of high denaturation, called Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, called Framework Regions (FRs). Each VH and VL are composed of, in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 are composed of 3 CDRs and 4 FRs arranged from amino terminus to carboxy terminus. The variable regions (VH and VL) of each heavy/light chain pair form the antibody binding sites, respectively. The assignment of amino acids to the various regions or domains follows Kabat Sequences of Proteins of immunological interest (National Institutes of Health, Bethesda, Md. (1987and 1991)), or Chothia & Lesk (1987) J.mol.biol.196: 901-; chothia et al (1989) Nature 342: 878-883. In particular, the heavy chain may also comprise more than 3 CDRs, for example 6, 9, or 12. For example, in the bifunctional antibody of the invention, the heavy chain may be an ScFv in which the C-terminus of the heavy chain of an IgG antibody is linked to another antibody, in which case the heavy chain contains 9 CDRs. The term "antibody" is not limited by any particular method of producing an antibody. For example, it includes, in particular, recombinant antibodies, monoclonal antibodies and polyclonal antibodies. The antibody may be of different isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM antibodies.

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments, and other advantages and effects of the present invention will be apparent to those skilled in the art from the disclosure of the present specification.

Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention. Test methods in which specific conditions are not specified in the following examples are generally carried out under conventional conditions or under conditions recommended by the respective manufacturers.

When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.

Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed herein all employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related arts.

"CD 271-T2" used in the present invention represents a cell line when used together with a hybridoma cell line; when used alone or together with monoclonal antibodies, etc., are designated monoclonal antibodies or engineered antibodies of the invention.

Example 1 preparation of anti-human CD271 hybridoma cell line and monoclonal antibody

1. Immunization of mice

The amino acid sequence of the immune antigen is shown as SEQ ID NO.17, and specifically comprises the following steps:

EEIPGRWITRSTPPEGSDSTAPSTQEPEAPPEQDLIASTVAGVVTTVMGSSQPVVTRGT TDN。

1) preparation of a priming antigen: taking a 1mL sterile EP tube, diluting 100 mu g of immunogen with PBS to 100 mu L, and placing the diluted immunogen in the EP tube; fully shaking the Freund's complete adjuvant to uniformly mix the deposited bifidobacteria; 100 μ L of freund's complete adjuvant was added to a 5mL sterile EP tube, and the diluted immunogen was added so that the ratio of the volume of antigen to the volume of adjuvant was 1:1, placing a centrifugal tube in an ice box; preparing an ultrasonic crusher, and placing the centrifugal tube in an ice box for ultrasonic treatment. The ultrasonic conditions are total power of 200W, total time of 10min, working time of 5 seconds and interval time of 6 seconds. Observing the emulsification condition of the solution in real time; the emulsified antigen was aspirated with a 1ml disposable syringe and placed in an ice box for future use.

2) Preparing a secondary immune antigen and a tertiary immune antigen: taking a 1mL sterile EP tube, diluting 100 mu g of immunogen with PBS to 100 mu L, and placing the diluted immunogen in the EP tube; fully shaking up Freund's incomplete adjuvant to uniformly mix the deposited Bifidobacterium; 100 μ L of Freund's incomplete adjuvant was added to a 5mL sterile EP tube, and the diluted immunogen was added to make the ratio of the volume of antigen to the volume of adjuvant 1:1, placing a centrifugal tube in an ice box; preparing an ultrasonic crusher, and placing the centrifugal tube in an ice box for ultrasonic treatment. The ultrasonic conditions are total power 200W, total time 10min, working time 5s and interval time 6 s. Observing the emulsification condition of the solution in real time; the emulsified antigen was aspirated with a 1ml disposable syringe and placed in an ice box for future use.

3) Preparing a booster immune antigen: a1 mL sterile EP tube was taken, 200. mu.g of immunogen was diluted to 400. mu.L with PBS, aspirated with a 1mL disposable syringe, and placed in an ice box for use.

4) Mouse immunization method

Sucking immune antigen by a 1mL syringe, dividing into 2-3 points, and injecting the immune antigen into the back (or abdomen) of a mouse by subcutaneous injection at about 0.1mL per point; blood is collected on the third day after the third immunization, the antibody titer ELISA detection is carried out, the specific result is shown in figure 1, and the immunity can be strengthened when the titer reaches 1: 100000.

2. Preparation of feeder cells

Feeder cells promote the growth and propagation of individual or a small number of discrete cells.

1) Sterile surgical instruments and glass plates were prepared, 96-well plates, 5mL disposable syringes, 300mL 70% alcohol, Balb/c mice, serum-free DMEM medium, and sterile PBS.

2) Balb/c mice were sacrificed by pulling their necks and immersed in 70% alcohol for 10 min.

3) The mice were removed and the excess alcohol was drained and placed on a sterilized glass plate.

4) The skin of the mouse abdomen was cut open and separated (without cutting the muscle layer). 5mL of DMEM serum-free culture solution was aspirated by a 5mL syringe, injected into the abdomen of the mouse, and the body of the mouse was gently massaged and shaken to thoroughly mix macrophages into the culture solution. The culture was carefully aspirated back through a syringe and placed into a 15mL centrifuge tube.

5) Repeating the above method for 1-2 times to obtain macrophage as much as possible.

6) Centrifuging at 1500rpm for 10min, and discarding the supernatant. Prepared by suspending the culture solution, about 10 can be taken out of 1 adult Balb/c mouse⁶3-4 pieces of 96-well plates can be paved on each abdominal cavity cell.

PEG-mediated fusion of rat spleen B cells and mouse myeloma cells

1) Cell: sp2/0-Ag14, medium: DMEM and 10% FBS, passage is carried out for 48 hours before fusion, and the bottle bottom is covered by about 80% after 48 hours according to the density.

2) The Balb/c mice were sacrificed by tail-cutting and neck-pulling, and immersed in 70% alcohol for 10 min.

3) The left upper abdomen was cut to the left back of the mouse, the spleen was isolated, connective tissue was removed from the spleen, placed in a clean dish, and a small amount of DMEM basal medium (without serum) was added.

4) The mouse spleen was cut into 4-5 pieces, ground with a 5mL syringe, stoppered at the top, and a small amount of DMEM was added while grinding the edges, and the ground spleen cells were washed into a plate.

5) And (3) transferring all the cells in the plate into a 15mL centrifuge tube, adding a proper amount of DMEM basic culture medium, uniformly mixing, centrifuging for 200g, and carrying out 5 min.

6) Washed twice with PBS, centrifuged, 200g, 5 min.

7) All Sp2/0-Ag14 cells were harvested and washed once with PBS and counted.

8) Will 10⁸Spleen cells and 10⁷The individual myeloma cells were mixed in a 50mL centrifuge tube (the number of myeloma cells was increased depending on the number of harvested cells), 30-40mL serum-free DMEM was added thereto, the mixture was thoroughly mixed, centrifuged at 1500rpm for 3min, and the supernatant was removed.

9) The bottom of the centrifuge tube was tapped to break up the pellet sufficiently.

10) The cells were placed in a 37 ℃ water bath, and PEG was slowly dropped in the following manner.

11) Slowly dripping 1mL of PEG solution along the tube wall with a dropper, lasting for 1min, gently oscillating while adding, centrifuging for 2min at 100 g;

12) dripping 4.5mL of DMEM basal medium (preheated and serum-free) by the same method for 3min while gently oscillating;

13) dripping 5mL of DMEM basal medium (preheated and serum-free) by the same method for 2min, and slightly oscillating for 30s after finishing the dripping;

14) slowly adding 45mL of DMEM basal medium (preheating and serum-free),

15) centrifuge at 100g for 5min, remove supernatant, tap the bottom.

16) HAT-containing DMEM complete medium (20% FBS) was added, resuspended gently, cell counted using trypan blue, 100. mu.L/well, and plated into 5 96-well plates.

17)37℃，5％CO₂The incubator is used for culturing without shaking the culture plate during culturing.

18) ELISA was performed 11-13 days after the fusion, and the cell growth was measured, and the amount of antibody in the supernatant was sufficiently accumulated without changing the solution 2-3 days before the measurement.

4. Limiting dilution screening of monoclonal

1) Taking out the antibody positive hole cells, and preparing the cell suspension by using HT culture solution. Samples were taken for trypan blue staining and counted.

2) Dispersing 320 cells in 6.4mL of HT culture solution, fully and uniformly mixing, adding into the 1 st to 4 th rows of a 96-well plate by using a discharging gun, wherein each hole is 100 mu L, and enabling 5 cells to be in each hole; adding 3.2mL of HT culture solution into the rest cell solution, mixing well, adding 5-8 rows per well of 100 μ L to make 2.5 cells per well; the remaining cell fluid (1 mL) was added with 2mL of the culture medium, and after mixing well, 9-12 rows were added to 100. mu.L of each well so that 0.625 cells per well were obtained.

3) Placing 96-well plates in CO₂The culture was carried out in an incubator at 37 ℃.

4) On the fifth day, the growth of the cells was observed under a microscope, and the wells in which the cells grew were recorded.

5) After the clone is propagated in a large quantity, the antibody of the culture solution is detected when 1/3-1/2 of the bottom of the hole is covered.

6) Selecting the cells in the wells with highest Elisa positivity and good growth state from the wells with 0.625 cell per well, and performing the next round of limiting dilution; simultaneously, another well of the same condition is selected and transferred to one well of a 24-well plate containing feeder cells, and then frozen and preserved after amplification.

7) After three rounds of limiting dilution, cells which are screened and meet the strain building conditions are subjected to amplification culture and frozen storage.

5. Preparation method of mouse ascites generated by hybridoma cells

1) 7-10 days before cell injection, 0.5mL of autoclaved pristane (paraffin oil) was injected intraperitoneally into each nude mouse.

2) Recovering the cell strain of the strain, culturing, subculturing and collecting supernatantDetecting ELISA, when positive, centrifugally collecting cells, and carrying out trypan blue staining technology; according to each Balb/c 5X 10⁵Cells were resuspended in 0.2mL PBS.

3) 8 weeks female Balb/c mice were injected intraperitoneally.

4) Ascites began to appear about 1-2 weeks. Ascites extraction can be initiated when the animal body is imaged as a pregnant female mouse. Ascites was withdrawn from the periphery of the abdominal cavity with 5mL syringes, each approximately 2-3 mL.

5) Ascites fluid was centrifuged at 3000rpm (1500g) for 10 minutes, and the supernatant was aliquoted and stored at-70 ℃.

6) Under the condition of keeping the health of the nude mice, the nude mice can be repeatedly extracted at intervals of 1-3 days according to the generation condition of ascites.

Example 2 mouse ascites purification and identification and ForteBio validation of purified antibody affinity

1. Mouse ascites Mab Select Sure^TMPurification of

1) Ascites from mice were immediately centrifuged after collection and the supernatant was frozen at-80 ℃. Unfreezing at room temperature or 4 ℃ before purification, centrifuging to obtain a supernatant, adding 1/10 volumes of 1M Tris-HCl and 0.5M NaCl, adjusting the pH value to 8.0, filtering by 0.22 mu M to obtain a sample to be loaded on a column, and sampling S;

2) the column was equilibrated with 10CV of 1M Tris-HCl, 0.5M NaCl, pH 8.0;

3) sampling, collecting all flow-through and sampling FL;

4)Mab Select Sure^TMwashing with 10CV of 100mM Tris-HCl,50mM NaCl, pH 8.0;

5)Mab Select Sure^TMwashing with 10CV of 10mM Tris-HCl,5mM NaCl, pH 8.0;

6) 0.1CV of 1M Tris-HCl pH 9.0 was added to the collection tube, and 0.5CV of 100mM NaCt/HCt pH3.0 was added each time during elution, and mixed immediately; elute the Elute of the

Elute

1, 2, 3 … tube and sample in half.

2. Purified antibody SDS-PAGE and ELISA identification

1) SDS-PAGE identification

The ascites antibody CD271T-2 obtained by purification was subjected to SDS-PAGE protein electrophoresis analysis, and the specific results are shown in FIG. 2.

2) ELISA identification

Collecting all the collected tubes, measuring the protein concentration after ultrafiltration and centrifugation, and performing ELISA identification on CD271T-1 and CD271T-2, wherein the substrate is CD271 full-length protein, the primary antibody is a Mouse ascites purified antibody, the secondary antibody is Goat Anti Mouse IgG (H + L) -HRP, and the specific result is shown in figure 3.

Validation of antibody affinity after purification by ForteBio

Full-length antigen CD271 was immobilized with a proA sensor and 30, 50, 70, 90, 110, 130nM CD271T-1, CD271T-2 antibody affinities were detected. Curing for 5 minutes (curing height around 1 nm) for 5 minutes, and dissociation for 5 minutes (dissociation fit first 1 minute). Regeneration for 5s, neutralization for 15s, and circulation for three times. All dilutions of antigen antibody were PBS, regeneration buffer was pH1.510mM glycine buffer, equilibration eluent was 0.02% Tween PBS, and neutralization was 0.05% Tween PBS. The concentration of the cured product (i.e., antigen) was 10. mu.g/mL. The curing height is about 1 nm. The specific parameters are shown in Table 1, and the graph is shown in FIG. 4.

TABLE 1

	KD(M)	Kon(1/Ms)	Kdis(1/s)	Full X²	Full R²
						CD271T-2	3.37×10^-9	6.69×10⁴	2.25×10^-4	4.4542	0.98420

Example 3 cloning of heavy and light chain variable region Gene of anti-human CD271 and construction of chimeric antibody 2

RNA extraction: adopting Trizol one-step method to obtain hybridoma cell CD271T-2 about 10⁶Adding Trizol, extracting RNA according to an Ultrapure RNA Kit CW0581S Kit in the Kangji century, and identifying the integrity of the RNA by nucleic acid electrophoresis; the preservation number of the hybridoma CD271T-2 is CGMCC 21494. Is preserved in the China general microorganism strain preservation management center, and the preservation address is No.3 of Xilu No.1 of Beijing, Chaoyang, North Chen. The preservation date is 2021 year, 1 month and 25 days. Classified and named as hybridoma cell strain CD 271T-2;

2. reverse transcription to cDNA (20 μ L): taking Oligo dT Primer (50 muM) 1 muL, dNTP mix (10nM)1 muL and template RNA 5 mug, adding water to 10 muL, keeping the temperature at 65 ℃ for 5min, and rapidly cooling on ice; adding 10 μ L of the above denatured reaction solution, 4 μ L of 5 × primescript II Buffer, 0.5 μ L of RNase Inhibitor (40U/μ L), 1 μ L of PrimeScirpt II RTase (200U/μ L), adding water to 20 μ L, reacting at 42 deg.C for 60min, and inactivating at 70 deg.C for 15 min;

PCR amplification of heavy and light chain variable region genes of anti-CD 271 antibody: the light and heavy chain variable region gene PCR amplification is operated in Hongxin biotechnology company of Suzhou, and the PCR result is shown in FIGS. 5A and 5B;

4. construction of sequencing vector: PMD19-T vector was purchased from takara, heavy and light chain variable region gene pcr products were recovered, connected to T vector, DH5 α transformed, positive clones were selected at 100 μ g/mL ampicillin concentration, sequenced and aligned to several conserved framework amino acids in the protein database. The obtained monoclonal antibody comprises a light chain and a heavy chain, wherein the light chain comprises LCDR1, LCDR2 and LCDR3, the amino acid sequence of the LCDR1 is shown as SEQ ID NO.1, the amino acid sequence of the LCDR2 is shown as SEQ ID NO.2, and the amino acid sequence of the LCDR3 is shown as SEQ ID NO. 3; the heavy chain comprises HCDR1, HCDR2 and HCDR3, the amino acid sequence of the HCDR1 is shown as SEQ ID NO.4, the amino acid sequence of the HCDR2 is shown as SEQ ID NO.5, and the amino acid sequence of the HCDR3 is shown as SEQ ID NO. 6. The amino acid sequence of the light chain variable region is shown in SEQ ID NO. 7. The amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 8. The nucleotide sequence of the code light chain complementarity determining region LCDR1 is shown in SEQ ID NO. 9; the nucleotide sequence of the light chain complementation determining region LCDR2 is shown as SEQ ID NO. 10; the nucleotide sequence of the code light chain complementarity determining region LCDR3 is shown in SEQ ID NO. 11; the nucleotide sequence of the code heavy chain complementarity determining region HCDR1 is shown in SEQ ID NO. 12; the nucleotide sequence of the code heavy chain complementarity determining region HCDR2 is shown in SEQ ID NO. 13; the nucleotide sequence encoding the heavy chain complementarity determining region HCDR3 is shown in SEQ ID NO. 14. The nucleotide sequence of the light chain variable region is shown in SEQ ID NO.15, and the nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO. 16.

[ example 4 ] flow-compatible expression and identification of anti-human CD271 ascites antibody and Hu-MSC

Collecting appropriate well-grown Hu-MSC cells by trypsinization and centrifugation, re-suspending and washing by using FACS (PBS + 2% FBS), centrifuging at 1000rpm for 3min, and discarding supernatant; add primary antibody according to table 3, incubate in dark at room temperature for 30min, resuspend wash with FACS (PBS + 2% FBS), centrifuge 1000rpm, 3min, repeat 3 times, discard supernatant, add secondary antibody according to table 1, photophobic at room temperature for 30min, resuspend wash with FACS (PBS + 2% FBS), centrifuge 1000rpm, 3min, repeat 3 times, add 300 μ L FACS to resuspend cells, detect with flow cytometer, see figure 6 for specific results. It is shown that positive antibodies derived from APC mouse anti human CD271 from Biolegend, the immunogen being from melanoma cells, with high expression of CD271 molecules on the cell surface, are slightly migratory compared to the negative control (FIG. 6A); the positive control antibody of Rabbit anti human CD271 derived from Abcam, the immunogen from the 350-450 amino acid region in the human CD271 cell membrane, did not have any shift compared with the negative control (FIG. 6B); the ascites purified antibody to CD271T-2 was more shifted than the positive and negative controls, indicating that the prepared anti-human CD271 monoclonal antibody had very high affinity and specificity (fig. 6C).

TABLE 2

While the invention has been described with respect to a preferred embodiment, it will be understood by those skilled in the art that the foregoing and other changes, omissions and deviations in the form and detail thereof may be made without departing from the scope of this invention. Those skilled in the art can make various changes, modifications and equivalent arrangements, which are equivalent to the embodiments of the present invention, without departing from the spirit and scope of the present invention, and all changes, modifications and equivalents of the technical disclosure can be made; meanwhile, any changes, modifications and variations of the above-described embodiments, which are equivalent to those of the essential techniques of the present invention, are within the scope of the technical solution of the present invention.

Sequence listing

<110> Shanghai university of transportation

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Ala Ala Ser

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<213> Artificial Sequence (Artificial Sequence)

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Gln Gln Ser Asn Glu Asp Pro Phe Thr

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Gly Phe Ser Leu Ser Thr Ser Gly Met Gly

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Ile Trp Trp Asp Asp Asp Lys

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<213> Artificial Sequence (Artificial Sequence)

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Ala Arg Arg Asp Tyr Gly Asn Tyr Tyr Ala Met Asp Tyr

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<213> Artificial Sequence (Artificial Sequence)

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Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly

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Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp

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Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

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Lys Leu Leu Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala

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Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His

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Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Asn

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Glu Asp Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

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Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Lys Pro Ser Gln

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Gly Met Gly Val Gly Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu

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Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Tyr Tyr Asn Pro Ser

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Leu Lys Ser Gln Leu Thr Ile Ser Lys Asp Thr Ser Arg Asn Gln Val

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Phe Leu Lys Ile Thr Ser Val Asp Thr Ala Asp Thr Ala Thr Tyr Tyr

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Cys Ala Arg Arg Asp Tyr Gly Asn Tyr Tyr Ala Met Asp Tyr Trp Gly

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Gln Gly Thr Ser Val Thr Val Ser Ser

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<213> Artificial Sequence (Artificial Sequence)

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caacagaaac caggacagcc acccaaactc ctcatctatg ctgcatccaa tctagaatct 180

gggatcccag ccaggtttag tggcagtggg tctgggacag acttcaccct caacatccat 240

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tataacccat ccctgaagag ccagctcaca atctccaagg atacctccag aaaccaggta 240

ttcctcaaga tcaccagtgt ggacactgca gatactgcca cttactactg tgctcgaagg 300

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tcag 364

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Ser Asp Ser Thr Ala Pro Ser Thr Gln Glu Pro Glu Ala Pro Pro Glu

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Gln Asp Leu Ile Ala Ser Thr Val Ala Gly Val Val Thr Thr Val Met

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Gly Ser Ser Gln Pro Val Val Thr Arg Gly Thr Thr Asp Asn

50 55 60

Claims

1. A hybridoma cell strain CD271T-2 secreting anti-human CD271 monoclonal antibody, wherein the preservation number of the hybridoma cell strain is CGMCC 21494.

2. An anti-human CD271 monoclonal antibody secreted by the CD271T-2 hybridoma cell line of claim 1.

3. An engineered antibody against human CD271, wherein the engineered antibody against human CD271 comprises a light chain and a heavy chain, the complementarity determining regions of the light chain comprise LCDR1, LCDR2 and LCDR3, the amino acid sequence of the LCDR1 is shown in SEQ ID No.1, the amino acid sequence of the LCDR2 is shown in SEQ ID No.2, and the amino acid sequence of the LCDR3 is shown in SEQ ID No. 3; the complementarity determining region of the heavy chain comprises HCDR1, HCDR2 and HCDR3, the amino acid sequence of the HCDR1 is shown as SEQ ID NO.4, the amino acid sequence of the HCDR2 is shown as SEQ ID NO.5, and the amino acid sequence of the HCDR3 is shown as SEQ ID NO. 6.

4. The engineered anti-human CD271 antibody of claim 3, wherein the amino acid sequence of the light chain variable region of the engineered anti-human CD271 antibody is represented by SEQ ID No. 7and the amino acid sequence of the heavy chain variable region is represented by SEQ ID No. 8.

5. A polynucleotide encoding the variable region of the heavy chain and the variable region of the light chain of the engineered anti-human CD271 antibody of any one of claims 3-4.

6. The polynucleotide of claim 5, wherein the nucleotide sequence encoding the light chain complementarity determining region LCDR1 is set forth in SEQ ID No. 9; the nucleotide sequence of the code light chain complementarity determining region LCDR2 is shown in SEQ ID NO. 10; the nucleotide sequence of the code light chain complementarity determining region LCDR3 is shown in SEQ ID NO. 11; and the nucleotide sequence encoding the heavy chain complementarity determining region HCDR1 is shown in SEQ ID NO. 12; the nucleotide sequence of the code heavy chain complementarity determining region HCDR2 is shown in SEQ ID NO. 13; the nucleotide sequence encoding the heavy chain complementarity determining region HCDR3 is shown in SEQ ID NO. 14.

7. A construct comprising the polynucleotide of any one of claims 5-6.

8. A host cell comprising the construct or genome of claim 7 having integrated therein an exogenous polynucleotide according to any one of claims 5-6.

9. Use of the anti-human CD271 monoclonal antibody of claim 2, or the anti-human CD271 engineered antibody of any one of claims 3-4 in the preparation of a reagent for in vitro detection and sorting of CD 271-expressing cells.