CN114409791B

CN114409791B - Fully human anti-human erythrocyte RhD full molecular IgG and preparation method and application thereof

Info

Publication number: CN114409791B
Application number: CN202210095345.4A
Authority: CN
Inventors: 杨婷婷; 王哲越; 王布强; 常新霞; 陈芳芳; 唐奇; 杨永林; 王根银; 冯振卿; 傅强
Original assignee: Jiangsu Libo Medicine Biotechnology Co ltd; Nanjing Medical University
Current assignee: Jiangsu Libo Medicine Biotechnology Co ltd; Nanjing Medical University
Priority date: 2022-01-26
Filing date: 2022-01-26
Publication date: 2023-01-24
Anticipated expiration: 2042-01-26
Also published as: WO2023143629A1; CN114409791A

Abstract

The invention relates to a fully human anti-human erythrocyte RhD full molecular IgG and a preparation method and application thereof. The fully human anti-human erythrocyte RhD full molecular IgG comprises a heavy chain variable region and a light chain variable region, and is characterized in that: the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO.3, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 4. The invention utilizes the phenotype-differentiated red blood cells to screen and enrich B cells which can specifically express the anti-human red blood cell RhD antigen, obtains the paired light chain variable region and heavy chain variable region gene sequences of the fully-humanized anti-RhD antibody through single B cell BCR region library-building PCR sequencing analysis, and then adopts the gene engineering antibody technology to express the full-molecule antibody in eukaryotic cells. The recombinant expression fully human anti-human erythrocyte RhD full molecular antibody can effectively identify the RhD antigen on the surface of human erythrocytes, and leads to agglutination reaction of RhD positive erythrocytes.

Description

Fully human anti-human erythrocyte RhD full molecular IgG and preparation method and application thereof

Technical Field

The invention belongs to the field of immunology, and particularly relates to a fully human anti-human erythrocyte RhD full molecular IgG, and a preparation method and application thereof.

Background

The molecule with antigenicity on the surface of the erythrocyte is called erythrocyte blood group antigen, the erythrocyte blood group antigen and the corresponding in vivo antibody thereof are important indexes for clinical treatment and detection such as clinical blood transfusion, organ transplantation, newborn hemolysis and the like, and are also the basis of blood group related disease research. At present, according to the nomenclature principles of the International blood transfusion society for human erythrocyte surface antigens, 368 erythrocyte blood group antigens are found and confirmed, and can be classified into 39 blood group systems, 5 blood group sets and 2 blood group series (low frequency antigen 700 series: 17 antigens; high frequency antigen 90-series: 7 antigens). Of the 39 blood group systems, the most important is the ABO blood group system, the coding gene of which is located on chromosome 9, and other blood group systems include MNS, P1PK, rh, LU, KEL, LE, FY, JK, DI, etc. Among them, rh blood group system is the most complex, and the coding gene is located on chromosome 1, because recombination and mutation of the gene generate a plurality of different antigens, besides common D, C, C, E and E antigens, a plurality of rare antigens such as HrB, bea, evans and other antigens are also expressed, and among them, rhD has the strongest antigenicity.

Since the Rh blood group system antigens were first found on Rhesus monkey (Rhesuus monkey) erythrocytes, they are named with their initials Rh. The main antigens D, C, C, E and E are expressed by the RhD gene and the RhCE gene. RhD gene expression on chromosome 1P ^34.3-36.1 And the total length is 57295bp. The RhD gene and RhCE gene are arranged in series in opposite directions, the two genes are separated by 30kb only, the RhD protein has 417 amino acids, the type IV crosses overThe membrane protein has 12 times of total transmembrane and has 6 extracellular region structures.

Antibodies against RhD are rarely produced in the natural state, and are generally produced when RhD-positive blood infants are pregnant with RhD-negative individuals, or when exposure is made clearly, for example, by miscarriage of RhD-positive blood. After the RhD antibody is produced by the individual, it causes intravascular hemolysis and extravascular hemolysis, and as a result of the destruction of red blood cells, hemolytic disease of the fetuses and newborn (HDFN) occurs in the newborn, which may lead to hemolysis, anemia, hyperbilirubinemia and even death of the fetus. The prophylactic use of RhD antibodies to avoid HDFN and delayed hemolytic transfusion reactions is a clinically essential treatment. Meanwhile, in idiopathic thrombocytopenic purpura diseases, the RhD antibody also has a therapeutic effect and is one of combined medicines for treating the diseases.

Most of the anti-human RhD protein antibodies currently used for clinical examination are rhesus hybridoma or polyclonal antibodies. Meanwhile, anti-human RhD protein antibodies are often used for the treatment of hemolytic diseases of newborn, idiopathic Thrombocytopenic Purpura (ITP), and transfusion errors of RhD (-) individuals. In order to avoid rejection caused by species differences, related therapeutic antibodies are required to be humanized in the clinical application process. At present, most of therapeutic antibodies used at home and abroad for the RhD protein are separated and purified from peripheral blood of donors with positive expression of the RhD protein, and the quantity is limited, so that the antibodies cannot be industrially produced.

The existing antibody development techniques for preparing anti-RhD antibodies suffer from a number of problems and drawbacks. For example, the high-titer serum produced by repeatedly immunizing RhD antigen negative erythrocytes is used for preparing the polyclonal antibody of the murine source and the rabbit source, so that the risk of spreading viruses or other pathogens from blood sources among different individuals exists, the titer among different batches is unstable, and the quality control cost is high. The classical hybridoma monoclonal antibody preparation method requires an antigen with higher immunogenicity to immunize a mouse. The RhD protein not only has large molecular weight and complex spatial structure, but also has a large amount of glycosylation site modification in the natural RhD protein, so that the preparation of the recombinant human RhD protein by adopting a gene synthesis and artificial expression mode is very difficult. Therefore, it is difficult for mice to generate an effective immune response. If a phage surface display antibody library technology is adopted, a large number of RhD positive B cell samples are needed to be used for phage library construction, meanwhile, enrichment, screening and amplification are needed repeatedly for many times, so that a proper antibody can be screened, the process is complicated, the cost is high, then light chain variable regions and heavy chain variable regions of the antibody obtained by phage screening are randomly paired, the affinity of the antibody is poor, and the antibody engineering technology is needed to be utilized for affinity maturation modification in the later period, so that the antibody is easy to have strong antigenicity, and the clinical treatment application is influenced. Single cell transcriptome sequencing technology is also applied to monoclonal antibody development, and high-throughput B cell transcriptome genetic information including paired antibody variable region gene sequences can be rapidly obtained. However, the application of the protein is limited aiming at the surface antigen RhD protein of human erythrocyte. The erythrocyte surface antigen system is complex, and the common ABO antigen system and the RhD antigen system are co-expressed, so that B cells capable of secreting RhD antibodies cannot be obtained through cell sorting, and erythrocyte positive to the RhD antigens cannot be sequenced (the erythrocyte does not contain transcriptome genetic information).

Disclosure of Invention

The invention discloses a fully human source full molecular antibody with the specificity of recognizing human red blood cell RhD antigen, a preparation method and application thereof, belonging to the field of biological pharmacy.

The invention discloses a fully human antibody with specificity recognition of human red blood cell RhD antigen, wherein the nucleic acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO.1, and the nucleic acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO. 2; the amino acid sequence of the antibody light chain variable region is shown in SEQ ID NO.3, and the amino acid sequence of the antibody heavy chain variable region is shown in SEQ ID NO.4; the full-length nucleic acid sequence (containing a constant region) of the antibody light chain is shown as SEQ ID NO.5, and the amino acid sequence is shown as SEQ ID NO. 7; the full-length nucleic acid sequence (containing a constant region) of the heavy chain of the antibody is shown as SEQ ID NO.6, and the amino acid sequence is shown as SEQ ID NO.8; the nucleic acid sequence of the antibody light chain antigen complementary region CDR is shown in SEQ ID NO.9, SEQ ID NO.10 and SEQ ID NO.11 in sequence; the amino acid sequence is shown in SEQ ID NO.15, SEQ ID NO.16 and SEQ ID NO.17 in sequence; the nucleic acid sequence of the antibody heavy chain antigen complementary region CDR is shown in SEQ ID NO.12, SEQ ID NO.13 and SEQ ID NO.14 in sequence; the amino acid sequence is shown in SEQ ID NO.18, SEQ ID NO.19 and SEQ ID NO.20 in sequence.

The invention discloses a preparation method of a fully human antibody with the specificity of recognizing a human erythrocyte RhD antigen. The method comprises the following steps: recruiting donors who have RhD antibody detection as positive to obtain peripheral blood and isolating PBMCs; sorting to obtain specific B cells; screening and enriching B cells capable of specifically expressing anti-human erythrocyte RhD antigen by using phenotypically different erythrocytes; obtaining paired gene sequences of light and heavy chain variable regions of the fully human RhD antibody through single B cell BCR library construction PCR sequencing and biological information analysis; a genetic engineering antibody technology is adopted to express a fully human anti-RhD full molecular antibody in eukaryotic cells.

The invention also discloses an expression vector containing the DNA molecule for encoding the fully human anti-human erythrocyte RhD full molecular IgG and a cell containing the expression vector.

A method for preparing the fully human anti-human erythrocyte RhD whole IgG comprises the steps of culturing the cell containing the expression vector, and recovering the fully human anti-human erythrocyte RhD whole IgG from a cell culture.

A composition comprising the fully human anti-human erythrocyte RhD whole IgG or antigen-binding fragment of the invention and a pharmaceutically acceptable carrier.

A method of treating a pathological condition characterized by exposure to red blood cell RhD antigen in a subject, comprising administering to the subject an effective amount of the composition.

The fully human anti-human erythrocyte RhD full molecular antibody or the antigen binding fragment thereof is conjugated with a therapeutic agent.

The fully human anti-human erythrocyte RhD full molecular antibody or the antigen binding fragment thereof is conjugated with a label. Wherein the label is selected from the group consisting of: radioisotopes, fluorescent dyes, and enzymes.

The invention relates to application of fully human anti-human erythrocyte RhD whole molecular IgG or an antigen binding fragment thereof in detecting erythrocyte RhD protein in a sample from human peripheral blood.

A method of detecting a red blood cell RhD protein in a sample from human peripheral blood by contacting said fully human anti-human red blood cell RhD whole molecule antibody or antigen binding fragment thereof with said sample and detecting said fully human anti-human red blood cell RhD whole molecule antibody bound to said red blood cell RhD protein. Wherein the fully human anti-human erythrocyte RhD whole molecule antibody or antigen binding fragment thereof is used in an erythrocyte agglutination assay or an ELISA assay.

The invention relates to application of a fully human anti-human erythrocyte RhD whole molecular IgG in preparing a medicament for preventing hemolytic diseases of newborn, treating Idiopathic Thrombocytopenic Purpura (ITP) or preventing sensitization to a rhesus D antigen after mistakenly inputting RhD (+) blood into a RhD (-) individual.

The method has the advantages and innovativeness that the prepared anti-RhD antibody is fully humanized, rejection caused by species specificity cannot be generated in clinical treatment and use, the requirement of clinical medication safety is met, and the traditional technological development process of humanization of the murine antibody is avoided. Meanwhile, in the gene sequence of the antibody obtained by the method through preparation and screening, the heavy chain and the light chain of the variable region of the antibody belong to natural pairing, and have higher affinity and stability.

The invention discloses an application of a fully human source full molecular antibody with the specificity of recognizing a human erythrocyte RhD antigen in clinical diagnosis and clinical treatment. The fully human anti-human erythrocyte RhD full molecular antibody can effectively identify the RhD antigen on the surface of the human erythrocyte, and can be used for hemagglutination analysis or ELISA analysis related to the RhD antigen of the human erythrocyte; can be used for preventing hemolytic disease of newborn, treating Idiopathic Thrombocytopenic Purpura (ITP), or preventing sensitization to rhesus D antigen after mistaken transfusion of RhD (+) blood into RhD (-) individuals.

Drawings

Fig. 1 is a B-cell magnetic bead sorting enrichment flow assay. Wherein: a is PBMC, B is 9.57% of CD19+ B cell population in lymphocyte population; c is the B cell group after enrichment of the magnetic beads, and D is 83.3 percent of the CD19+ B cell group.

FIG. 2 is a flow-through map of RhD antibody-positive B cells. Wherein: a is lymphocyte group, B is CD19+/IgG + B cell group, the percentage is 3.64%, C is CD19+/IgG + B cell group combined with erythrocyte, and IgG +/CD19+/CD235a/B + RBC/B cell group is 0, 83%.

FIG. 3 is a graph of a cluster analysis screen. And performing cluster analysis on the antibody sequences, wherein the antibody sequences are divided into five cluster, and one example of the candidate antibody gene sequences is selected from each cluster and is respectively G9, G11, D7, D3 and E.

Figure 4 is a UV map of antibody expression purification. The Protein purification UV picture can be seen, the early antibody Protein is adsorbed on a Protein A column, and the later eluted antibody has a high-tip absorption peak.

FIG. 5 is an electrophoretic staining pattern of antibody expressed purified supernatant protein wherein: a is coomassie staining and B is silver staining). The heavy and light antibody chain bands and other unrelated protein bands are shown in an Input lane, only unrelated protein bands are shown in a Flow through lane, obvious heavy and light antibody chain bands and no other protein bands are shown in an elusion lane, and the antibody protein is purified and enriched.

FIG. 6 is the specific detection of antibodies against red blood cell antigens. The antibody with clone number D7 can specifically agglutinate RhD positive erythrocyte, and RhD negative erythrocyte can not agglutinate.

FIG. 7 is a graph showing the measurement of antibody hemagglutination titer. The results suggest that the potency of the agglutination RhD positive erythrocytes obtained by preparing the fully human anti-human erythrocyte RhD antibody (D7) (0.1 mg/ml) as described above, and the strong positive results reached 1: above 512, weak positivity reaches 1:2048.

FIG. 8 is the silver staining pattern of antibody co-immunoprecipitated protein electrophoresis. The expression of the target protein is detected by adopting a silver staining method, a specific protein band can be seen at 40kDa, and the protein can be confirmed to be human RhD protein by mass spectrum detection.

FIG. 9 antibody-mediated erythrocyte lysis assay. The OD value of the RhD positive erythrocyte group is obviously increased (P is less than 0.05) relative to the negative control group, which indicates that after the fully human full-molecular antibody (D7) identifies and binds erythrocytes, the damage of monocytes to the erythrocytes is promoted, the hemoglobin content in the supernatant is high, and the OD value is increased.

FIG. 10 is a flow assay of antibody-mediated erythrocyte lysis. A is a result graph of incubation of unsensitized RhD positive erythrocytes and monocytes; b is a result graph of re-incubation of mononuclear cells after the fully human full molecular antibody (D7) and RhD negative red blood cells are incubated; and the C picture is a result picture of re-incubation of mononuclear cells after the incubation of the fully human antibody (D7) and the RhD positive erythrocytes, and the result indicates that the proportion of erythrocytes is obviously increased compared with the control group after the incubation of the RhD positive erythrocytes combined by the mononuclear cells and the fully human antibody (D7).

FIG. 11 is a full human anti-RhD antibody protection assay. It is demonstrated that after 9h of erythrocyte infusion, it can be seen that the human erythrocytes in the experimental mice are lower than those in the control group (p < 0.05), and the fully human anti-human erythrocyte RhD antibody (D7) can mediate the elimination of O-type RhD + erythrocytes.

Detailed Description

Example 1: enrichment of B cells secreting RhD antibodies

Example 2: differential screening of erythrocyte phenotypes

Example 3: single B cell BCR region library-building PCR sequencing

Example 4: screening and analysis of fully human RhD antibody gene

Example 5: expression and purification of fully human RhD antibody

Example 6: characteristic analysis of fully human anti-RhD antibody

Example 7: detection of ADCC (antibody-mediated isothermal amplification) effect of fully-human anti-RhD antibody

Example 8: detection of protective experiment of fully human anti-RhD antibody

Example 1: enrichment of B cells secreting RhD antibodies

Donors who tested positive for RhD antibody (serum antibody titer >1 128) were recruited, peripheral blood was obtained and blood typing was performed.

Extracting PBMC:

preheating 1640 culture medium and lymphocyte separation liquid at 1.37 ℃;

2. taking 15ml of lymphocyte separation liquid into a 50ml centrifuge tube;

3. extracting 15ml of RhD antibody positive peripheral blood into a new 50ml centrifuge tube, diluting with 1640 culture medium with the same volume, and reversing and uniformly mixing;

4. adding the diluted peripheral blood (1 dilution) into a centrifuge tube containing the lymphocyte separation solution (diluted peripheral blood: lymphocyte separation solution = 2;

centrifuging at 5.800 g/min for 20min, and setting the starting and braking acceleration of the centrifuge to 1;

6. absorbing liquid on the upper layer of the centrifugal tube at a constant speed, and stopping absorbing when the distance is 2-3cm from the tunica albuginea layer;

7. sucking the tunica albuginea cells, transferring the tunica albuginea cells into a new 50ml centrifuge tube, and supplementing the volume to 45ml by using 1640 culture medium;

8. turning upside down, mixing uniformly, cleaning cells, and centrifuging at 300g/min for 5min;

9. after discarding the supernatant, resuspending the suspension by using 1640 culture medium, and centrifuging the suspension at 500g/min for 5min; centrifuging for 5min;

10. resuspend the cell pellet using RPMI1640+10% FBS +1% P/S complete medium and calculate the number of cells, subpackaged and frozen in liquid nitrogen.

B cell enrichment:

1. taking PBMC cells obtained by separation (at least 10) ⁷ Cells), resuspended in 40ul PBS + BSA buffer;

2. according to the operation procedure of B cell isolation Kit (Miltenyi), incubating 10ul of each B cell and biotin-labeled mixed antibody at 2-8 ℃ for 10min;

3. adding 30ul buffer solution and 20ul avidin micro magnetic beads into the mixture, and incubating for 10min at the temperature of 2-8 ℃;

4. placing the magnetic column in a suitable MACS separator magnetic field, and pre-washing the magnetic column with a buffer;

5. adding the PBMC cell suspension into a magnetic column, and collecting the cell flow-through liquid after sample loading;

6. counting cells by using a cell counter;

7. after incubation with anti-human CD 19-APC/cysteine 7 (Biogend), detection was performed by flow cytometry (FIG. 1).

FIG. 1 shows the flow-type results, wherein A is PBMC and B is CD19 in lymphocyte population ⁺ The proportion of B cell population is 9.57%; c is magnetic bead enrichmentThe later B cell population, D is CD19 ⁺ The proportion of B cell population is 83.3%.

Example 2: differential screening of erythrocyte phenotypes

1. 1ml of cell culture solution is added into the total B cells enriched by the magnetic beads for resuspension;

2. mixing three parts of A, B and O type RhD negative washing erythrocytes in equal volume, washing with normal saline for three times, and washing with a cell culture medium for one time;

3. adding 1ml of mixed red blood cells into the total B cell suspension, fully and uniformly mixing, incubating at 37 ℃ for 1h, and gently shaking and uniformly mixing every 15 min;

4. taking 5ml of lymphocyte separation liquid in a 15ml centrifuge tube; the incubated cell mixture was slowly added to the upper layer of the lymphocyte separation medium (2 ml sample, an equal volume of medium was added);

5. centrifuging for 20min at a speed of 800g/min after the transferring is finished;

6. collecting the upper layer B cells which are not combined with the red blood cells into a new centrifugal tube, and adding cell culture solution to wash for one time;

7. collecting 3 parts of O-type RhD positive washing red blood cells, washing the red blood cells with physiological saline for three times, and then washing the red blood cells with a cell culture medium for one time;

8. resuspending the B cells obtained in the last step by using 1ml of cell culture medium, adding 1ml of O-type RhD positive washing red blood cells, fully and uniformly mixing, incubating for 1 hour at 37 ℃, and gently shaking and uniformly mixing every 5min;

9. adding flow antibodies of anti-human CD19-APC/Cyanine7 (Biogend), anti-human IgG-Brilliant Violent 421 (Biogend) and anti-human CD235a/b-PE (Biogend) into the cell mixture for incubation, washing and detecting by a flow cytometer;

10. first screening for CD19 Using flow cytometry ⁺ /IgG ⁺ Screening for CD235 in the B cell population ⁺ B cells of (a), i.e., B cells that bind red blood cells (fig. 2);

11. IgG to be screened ⁺ /CD19 ⁺ /CD235a/b ⁺ The cells of (2) were sorted into 96-well cell culture plates at 1/well.

FIG. 2A shows lymphocyte population and B shows CD19 ⁺ /IgG ⁺ B cell population (3.64%), C is erythrocyte-binding B cell population, igG ⁺ /CD19 ⁺ /CD235a/b ⁺ The RBC/B cell population was 0, 83%.

Example 3: single B cell BCR region library-building PCR sequencing and screening

1. Adding cell lysate into a 96-well plate, and capturing mRNA by oligo-dT;

2. one strand of cDNA full length was obtained by reverse transcription of mRNA, and an Index was introduced on a Template-Switch Oligo, one Index sequence for each row on a 96-well plate.

3. PCR Master Mix was prepared and added to 96-well plates and cDNA enrichment was performed by Oligo-dT and Universal PCR primer on Template Switch Oligo.

And 4, carrying out posing and purifying on each column of cDNA enrichment products on a 96-well plate by cDNA quality detection. The cDNA concentration was determined by Qubit and the fragment distribution of the cDNA was determined by capillary electrophoresis.

And 5, enriching the BCR, and respectively designing two PCR primers in the constant regions (C regions) of the heavy chain and the light chain of the BCR through semi-nested PCR.

6. The specificity of the BCR-enriched product was increased by two rounds of specific enrichment (semi-nested PCR).

7. And (3) amplifying and building a library, and designing a Primer for PCR amplification and library building according to a PCR Handle sequence added on the BCR second round specific enrichment Primer.

8. After each library is split according to Index, BCR is independently assembled, variable region clustering analysis is carried out, and the effective heavy-light chain variable region nucleic acid sequence of the antibody is obtained by screening and eliminating redundant data.

Example 4: screening and analysis of fully human RhD antibody gene

1. Sequencing to obtain 78 pairs of effective paired antibody heavy and light chain variable region nucleic acid sequences, eliminating defect and duplication, similar sequence and translating into amino acid sequence;

2. removing the defective sequences, and splicing high-similarity sequences to obtain 42-pair heavy and light chain sequences;

3. simulating the 3D structures of RhD antigen and 42 antibody variable regions by using a Discovery studio molecule, and evaluating the energy of antigen-antibody docking;

4. the antibody sequence was subjected to cluster analysis and divided into five clusters, one example of the candidate antibody gene sequences, which are G9, G11, D7, D3 and E5 (FIG. 3), were selected from each cluster, and the amino acid sequences of the candidate antibodies G9, G11, D7, D3 and E5 are as follows:

example 5: expression and purification of fully human RhD antibody

1. The 5 pairs of antibody (G9, G11, D7, D3, E5) variable region sequences were nucleic acid synthesized and recombined into eukaryotic expression vectors pFUSE-CHIg-hG1, pFUSE-CLIg-hl and pFUSE-CLIg-hk containing human antibody constant region sequences, respectively (vector plasmids were purchased from Invitrogen, USA);

2. transforming the expression vector into competent bacteria, shake bacteria and amplify, and extracting the plasmid of the expression vector;

3. after the concentration of the plasmid is measured, subpackaging and freezing;

4. transfection of the antibody expression plasmid into 293F cell expression System:

1) At 8% of CO ₂ Culturing 293Freestyle cells in a shaking incubator at 37 ℃ and 120g/min, wherein the cell activity reaches more than 98%;

2) 293Freestyle cells were replaced by fresh medium in a 30ml culture at a cell concentration of 1X 10 ⁶ /ml；

3) Taking out 20ug of heavy chain plasmid and 20ug of light chain plasmid, and diluting to 600ul with Opti-MEM medium to obtain solution A;

4) 60ul of the transfection reagent EZ Trans Plus (Liji organism) was removed and diluted to 600ul with Opti-MEM medium as solution B;

5) Mixing the solutions A and B, and standing at room temperature for 20min;

6) Adding the mixed solution into the 293F cells, and slowly shaking the culture bottle to mix uniformly;

5. collecting cell culture supernatant 7 days after transfection;

6. filtering the collected cell culture supernatant with 0.45um and 0.22um filter membranes;

7. purification of antibody proteins was performed by an AKTA purifier100 instrument using a Protein a purification column (fig. 4);

8. passing the eluted protein through a 30kDa ultrafiltration column, and centrifuging at 4000g/min for 20min to obtain a concentrated antibody solution;

9. measuring the concentration of the antibody, marking, subpackaging and freezing;

10. SDS-page was performed on a portion of the antibody eluate (Elution) and the above cell culture supernatant (Input), a purified Flow-through (Flow through) and an ultrafiltration eluate (hyperfiltraction), and then subjected to Cookie staining and silver staining (FIG. 5, A is Cookie staining, B is silver staining).

FIG. 4 shows the UV pattern of Protein purification, the early antibody Protein is adsorbed on Protein A column, and the later eluted antibody shows a high-peak absorption peak. In FIG. 5, heavy and light antibody bands and other unrelated protein bands are shown in the input lane, only unrelated protein bands are shown in the Flow through lane, obvious heavy and light antibody bands are shown in the resolution lane, no other protein band is shown, and the antibody protein is purified and enriched.

Example 6: characterization of fully human RhD antibodies

Agglutination test of specific antigen red blood cell

1. Collecting and identifying red blood cells with Dcee, dccEE, DCCee and DCcEe antigen phenotypes, and washing 3-4 times with physiological saline respectively;

2. collecting and identifying 5 kinds of red blood cells with RhD antigen negative of different individual sources, mixing, and washing with normal saline for 3-4 times;

3. washing red blood cells, and diluting with normal saline to 2-5% by volume;

4. respectively sucking 100ul of the washed red blood cells and adding the red blood cells into a U-shaped 96-well plate;

5. adding 100ul of the prepared antibody (10 ug/ml) into a U-shaped plate, mixing, incubating at 37 deg.C for 30min;

6. mixing, adding 50ul of the mixture into anti-human immunoglobulin antibody gel blood type card, incubating at 37 deg.C for 10min;

7. placing the blood type card in a card type centrifuge, centrifuging for 900g/min, 2min,1200g/min and 3min;

8. observing the agglutination condition of the blood type card, and obtaining the result shown in figure 6;

the above 5 different fully human antibodies, wherein the antibody with clone number D7 can specifically agglutinate RhD positive erythrocytes, and RhD negative erythrocytes do not agglutinate (see FIG. 6), indicate that D7 is a fully human antibody with antigen of specific recognition of human erythrocyte RhD.

The full-length of the light chain amino acid sequence of the fully human anti-human erythrocyte RhD antibody (D7) is SEQ ID NO.7, and the full-length of the heavy chain amino acid sequence is SEQ ID NO.8; wherein the sequence of the light chain amino acid variable region is SEQ ID NO.3, and the sequence of the heavy chain amino acid variable region is SEQ ID NO.4; CDR1 in the light chain amino acid variable region sequence is SEQ ID NO.15, CDR2 is SEQ ID NO.16, CDR3 is SEQ ID NO.17, CDR1 in the heavy chain amino acid variable region sequence is SEQ ID NO.18, CDR2 is SEQ ID NO.19, and CDR3 is SEQ ID NO.20. The full length of a corresponding light chain nucleic acid sequence in an expression plasmid of the antibody is SEQ ID NO.5, and the full length of a heavy chain nucleic acid sequence is SEQ ID NO.6; the light chain variable region is SEQ ID NO.1, the heavy chain variable region is SEQ ID NO.2, wherein CDR1 in the light chain variable region is SEQ ID NO.9, CDR2 is SEQ ID NO.10, and CDR3 is SEQ ID NO.11; in the heavy chain variable region, CDR1 is SEQ ID NO.12, CDR2 is SEQ ID NO.13, and CDR3 is SEQ ID NO.14.

Antibody titer detection

1. Sucking 100ul PBS and adding into a U-shaped 96-well plate, and adding into 24 wells in total;

2. sucking 200ul (0.1 mg/ml) of fully human anti-human erythrocyte RhD antibody (D7) and adding the antibody into the blank hole in the U-shaped plate,

3. pipette 100ul of this solution into PBS containing wells;

4. pipetting 100ul of the antibody diluent from the first well, adding the antibody diluent to the second well containing PBS, and mixing;

5. by analogy, antibody was diluted in 24 wells at double ratio;

6. adding 100ul of standard RhD positive erythrocyte (Sanquin) into the U-shaped plate hole, and incubating for 30min at 37 ℃;

9. sucking 50ul of the above erythrocyte antibody mixture, adding into anti-human immunoglobulin antibody gel blood type card, incubating at 37 deg.C for 10min;

10. placing the blood type card in a card type centrifuge, centrifuging for 900g/min, 2min,1200g/min and 3min;

11. observing agglutination of blood type cards (fig. 7B);

12.50 ul of anti-human secondary antibody (1 diluted in 5000) was added to the U-shaped plate, and incubated at 37 ℃ for 30min

The agglutination of the red blood cells was observed under a lamp in the U-shaped plate (FIG. 7).

FIG. 7A shows the agglutination result of U-plate erythrocytes, and FIG. 7B shows the agglutination result of anti-human globulin microcolumn, which suggests that the titer of agglutinated RhD-positive erythrocytes obtained by preparing fully human anti-human erythrocyte RhD antibody (D7) (0.1 mg/ml) as described above, and the strong positive is 1: above 512, weak positivity reaches 1:2048.

antibody binding protein assay

1. Incubating the prepared antibody and Protein A magnetic beads at 4 ℃ overnight;

2. washing RhD positive erythrocytes and RhD negative erythrocytes with physiological saline;

3, cracking and washing red blood cells by using the ACK lysis solution to obtain a cracked red blood cell membrane;

4. protein lysate RIPA (Beyotime) lyses erythrocyte membrane proteins;

5. mixing the Protein A magnetic bead-antibody complex with RhD positive erythrocyte and RhD negative erythrocyte membrane Protein respectively, and incubating for 1 hour at 4 ℃;

6. adsorbing a Protein A magnetic bead-antibody-Protein compound on a magnetic frame, and washing with PBS (phosphate buffer solution) for three times;

heating the compound at 7.98 ℃, and removing magnetic beads on a magnetic frame to obtain an antibody-protein compound;

8. carrying out protein electrophoresis on the compound;

9. detecting the expression of the target protein by silver staining (fig. 8);

10. in the experimental group lane, a protein band is visible at 40kDa relative to the control group, and gel is cut and recovered and subjected to protein mass spectrometry;

the results of mass spectrometry showed that the extracellular domain polypeptide fragment of protein of specific RhD antigen (YLPGCNR, FPHLAVGF, YFDDDQVFWK) bound by fully human anti-human red blood cell RhD antibody (D7) could be detected in the experimental group, and the related sequence was consistent with the extracellular domain amino acid sequence of NCBI's gene database, gene ID:6007, RHD Rh blood group D antigen (human) ].

1. Monocyte THP-1 with 2X 10 ⁶ Cell concentration per ml, 100ul per well added to 96 well plate, cultured overnight in incubator, to make cell semi-adherent and activated;

2.200ul of 5% RhD positive RBCs and 200ul of the above prepared antibody (0.1 mg/ml) were pre-sensitized, i.e., incubated at 37 ℃ for 1h,

3. the control group is incubated with RhD negative erythrocytes and fully human whole-molecule antibody (D7);

4. adding 50ul of the sensitized erythrocyte with the concentration of 5% or the control erythrocyte into a THP-1 culture plate for culture;

and 5.24 hours later, sucking cell supernatant, and adding a luminescent color development solution to detect the hemoglobin content in the supernatant.

6. The OD value of the cell supernatant was measured by a microplate reader (FIG. 9);

7. culturing with monocyte THP-1 in 10 wells ⁶ A cell;

8. culturing overnight to ensure that the growth of the mononuclear cells is stable, and partial cells are semi-adherent and activated;

9. sensitized and non-sensitized erythrocytes were added to the mononuclear cell culture wells, respectively, (0.5 ul of packed volume of erythrocytes per well) for culture;

after 10.24 hours, excess red blood cells in the culture supernatant were washed;

11. separating mononuclear cells, phagocyte red blood cell mononuclear cells and combined red blood cell mononuclear cells by lymphocyte separating medium to remove unbound red blood cells;

12. washing the upper mononuclear cells, adding a red blood cell marker CD235a/b-PE antibody and a human IgG-BV421 antibody, and incubating for 20min at normal temperature in a dark place;

13. monocytes were washed with PBS and detected by flow cytometry (fig. 10).

Fig. 9 indicates that OD value of RhD positive erythrocyte group is obviously increased compared with negative control group, which indicates that after the fully human anti-human erythrocyte RhD antibody (D7) recognizes and binds erythrocytes, damage of monocytes to erythrocytes is promoted, hemoglobin content in supernatant is high, and OD value is increased; FIG. 10A is a graph showing the results of incubation of unsensitized RhD-positive erythrocytes with monocytes; b is a result graph of the incubation of the fully human full molecular antibody (D7) and RhD negative red blood cells and the re-incubation of mononuclear cells; and the graph C is a result graph of re-incubation of the fully human full molecular antibody (D7) and the RhD positive erythrocytes with the monocytes, and shows that the proportion of the erythrocytes is obviously increased compared with that of a control group after the incubation of the RhD positive erythrocytes combined with the monocytes and the fully human full molecular antibody (D7), so that the antibody in the research has a promotion effect on the recognition of the monocytes to phagocytize erythrocytes.

Example 8: detection of protective experiment of fully human anti-RhD antibody

1. Feeding NOD-SCID mouse, collecting tail vein blood, centrifuging at 12000g/min for 10min;

2. centrifuging, layering, and sucking upper layer serum;

3. detecting the IgG concentration in the serum by an ELISA detection kit,

4. mice with knock-out concentrations >10 ng/ul;

5. the control group was injected with only 10mg human recombinant IgG by intraperitoneal injection of 10mg IgG and 5ug of fully human anti-human erythrocyte RhD antibody (D7);

injecting 5ul O-type RhD + erythrocytes and 35ul O-type RhD-erythrocytes through tail vein after 6.24 hours;

7. taking blood through tail vein 4h and 9h after red blood cell injection;

8. obtaining red blood cells in blood, repeatedly washing with normal saline for three times to obtain washed red blood cells,

9. erythrocyte incubation anti-human CD235-PE (Biogend) flow antibody;

10. the ratio of human erythrocytes remaining in the mice was measured by flow cytometry, and the results are shown in FIG. 11.

FIG. 11 illustrates that after 9h of erythrocyte infusion, it can be seen that human erythrocytes in experimental mice are lower than those in control mice (p < 0.05), and that fully human anti-human erythrocyte RhD antibody (D7) can mediate the clearance of O-type RhD + erythrocytes.

SEQUENCE LISTING

<110> Nanjing university of medical science

Jiangsu Libo biomedical technology Co., ltd

<120> fully human anti-human erythrocyte RhD full molecular IgG and preparation method and application thereof

<130>

<160> 28

<170> PatentIn version 3.3

<210> 1

<211> 318

<212> DNA

<213> Artificial sequence

<400> 1

tcctatgagc tgactcagcc accctcagtg tccgtgtccc caggacagac agccagcatc 60

acctgctctg gagataaatt gggggataaa tatgcttgct ggtatcagca gaagccaggc 120

cagtcccctg tgctggtcat ctatcaagat agcaagcggc cctcagggat ccctgagcga 180

ttctctggct ccaactctgg gaacacagcc actctgacca tcagcgggac ccaggctatg 240

gatgaggctg actattactg tcaggcgtgg gacagcagca ctgtggtatt cggcggaggg 300

accaagctga ccgtccta 318

<210> 2

<211> 384

<212> DNA

<213> Artificial sequence

<400> 2

gaggtgcaac tggtgcagtc tggagcagag gtgaaaaagc ccggggagtc tctgaagatc 60

tcctgtaagg gttctggata cagttttacc acctactgga tcgcctgggt gcgccagatg 120

cccgggaaag gcctggagtg gatggggatc atctatcctg gtgactctga taccagatac 180

agcccgtcct tccaaggcca ggtcaccatc tcagccgaca agtccatcag caccgcctac 240

ctgcagtgga gcagcctgaa ggcctcggac accgccattt attactgtgc gagagtcccg 300

aaaagtggct ggtaccccta cccctactac tactactacg gtatggacgt ctggggccaa 360

gggacctcgg tcaccgtctc ctca 384

<210> 3

<211> 106

<212> PRT

<213> Artificial sequence

<400> 3

Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln

1 5 10 15

Thr Ala Ser Ile Thr Cys Ser Gly Asp Lys Leu Gly Asp Lys Tyr Ala

20 25 30

Cys Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Val Leu Val Ile Tyr

35 40 45

Gln Asp Ser Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Ala Trp Asp Ser Ser Thr Val Val

85 90 95

Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210> 4

<211> 128

<212> PRT

<213> Artificial sequence

<400> 4

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu

1 5 10 15

Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Thr Tyr

20 25 30

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

35 40 45

Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe

50 55 60

Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Ile Tyr Tyr Cys

85 90 95

Ala Arg Val Pro Lys Ser Gly Trp Tyr Pro Tyr Pro Tyr Tyr Tyr Tyr

100 105 110

Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser

115 120 125

<210> 5

<211> 636

<212> DNA

<213> Artificial sequence

<400> 5

tcctatgagc tgactcagcc accctcagtg tccgtgtccc caggacagac agccagcatc 60

acctgctctg gagataaatt gggggataaa tatgcttgct ggtatcagca gaagccaggc 120

cagtcccctg tgctggtcat ctatcaagat agcaagcggc cctcagggat ccctgagcga 180

ttctctggct ccaactctgg gaacacagcc actctgacca tcagcgggac ccaggctatg 240

gatgaggctg actattactg tcaggcgtgg gacagcagca ctgtggtatt cggcggaggg 300

accaagctga ccgtcctaca gcccaaggcc aaccccaccg tgaccctgtt ccccccatct 360

tctgaggagc tgcaagccaa caaggccacc ctggtgtgcc tgatctctga cttctaccct 420

ggcgctgtga cagtggcctg gaaggctgat ggctctcctg tgaaggctgg cgtggagacc 480

accaagccat ctaagcagtc taacaacaag tatgctgcct cttcttacct gtctctgacc 540

cctgagcagt ggaagagcca ccggtcttac tcttgccagg tgacccatga gggctctaca 600

gtggagaaga cagtggcccc cacagagtgc tcttga 636

<210> 6

<211> 1377

<212> DNA

<213> Artificial sequence

<400> 6

gaggtgcaac tggtgcagtc tggagcagag gtgaaaaagc ccggggagtc tctgaagatc 60

tcctgtaagg gttctggata cagttttacc acctactgga tcgcctgggt gcgccagatg 120

cccgggaaag gcctggagtg gatggggatc atctatcctg gtgactctga taccagatac 180

agcccgtcct tccaaggcca ggtcaccatc tcagccgaca agtccatcag caccgcctac 240

ctgcagtgga gcagcctgaa ggcctcggac accgccattt attactgtgc gagagtcccg 300

aaaagtggct ggtaccccta cccctactac tactactacg gtatggacgt ctggggccaa 360

gggacctcgg tcaccgtctc ctcagcatcc accaagggcc catctgtctt ccccctggcc 420

ccatcctcca agagcacctc tggcggcaca gctgccctgg gctgcctggt gaaggactac 480

ttccctgagc ctgtgacagt gtcctggaac tctggcgccc tgaccagcgg cgtgcacacc 540

ttccctgctg tgctccagtc ctctggcctg tactccctga gcagcgtggt gacagtgcca 600

tccagcagcc tgggcaccca gacctacatc tgcaatgtga accacaagcc cagcaacacc 660

aaggtggaca agcgggtgga gcccaagtcc tgtgacaaga cccacacctg ccccccatgc 720

cccgcccctg agctgctggg cggcccatct gtcttcctgt tcccccccaa gcccaaggac 780

accctgatga tctcccggac ccccgaggtg acctgtgtgg tggtggatgt gagccatgag 840

gaccccgagg tgaagttcaa ctggtatgtg gatggcgtgg aggtgcacaa cgccaagacc 900

aagccccggg aggagcagta caacagcacc taccgggtgg tgagcgtgct gacagtgctg 960

catcaggact ggctgaatgg caaggagtac aagtgcaagg tgtccaacaa ggccctgcct 1020

gcccccattg agaagaccat ctccaaggcc aagggccagc cccgggagcc ccaggtctac 1080

accctgcccc cctcccggga ggagatgacc aagaaccagg tgagcctgac ctgcctggtg 1140

aagggcttct accccagcga cattgctgtg gagtgggaga gcaacggcca gcctgagaac 1200

aactacaaga ccaccccccc tgtgctggac tctgatggct ccttcttcct gtacagcaag 1260

ctgacagtgg acaagagccg gtggcagcag ggcaatgtct tctcctgctc tgtgatgcat 1320

gaggccctgc acaaccacta cacccagaag agcctgtccc tgtcccccgg caagtga 1377

<210> 7

<211> 211

<212> PRT

<213> Artificial sequence

<400> 7

Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln

1 5 10 15

Thr Ala Ser Ile Thr Cys Ser Gly Asp Lys Leu Gly Asp Lys Tyr Ala

20 25 30

Cys Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Val Leu Val Ile Tyr

35 40 45

Gln Asp Ser Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Ala Trp Asp Ser Ser Thr Val Val

85 90 95

Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gln Pro Lys Ala Asn Pro

100 105 110

Thr Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala Asn Lys

115 120 125

Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly Ala Val Thr

130 135 140

Val Ala Trp Lys Ala Asp Gly Ser Pro Val Lys Ala Gly Val Glu Thr

145 150 155 160

Thr Lys Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser Ser Tyr

165 170 175

Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser Tyr Ser Cys

180 185 190

Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val Ala Pro Thr

195 200 205

Glu Cys Ser

210

<210> 8

<211> 458

<212> PRT

<213> Artificial sequence

<400> 8

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu

1 5 10 15

Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Thr Tyr

20 25 30

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

35 40 45

Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe

50 55 60

Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Ile Tyr Tyr Cys

85 90 95

Ala Arg Val Pro Lys Ser Gly Trp Tyr Pro Tyr Pro Tyr Tyr Tyr Tyr

100 105 110

Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser

115 120 125

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

130 135 140

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

145 150 155 160

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

165 170 175

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

180 185 190

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

195 200 205

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

210 215 220

Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

225 230 235 240

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

245 250 255

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

260 265 270

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

275 280 285

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

290 295 300

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

305 310 315 320

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

325 330 335

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

340 345 350

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

355 360 365

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

370 375 380

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

385 390 395 400

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

405 410 415

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

420 425 430

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

435 440 445

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

450 455

<210> 9

<211> 18

<212> DNA

<213> Artificial sequence

<400> 9

aaattggggg ataaatat 18

<210> 10

<211> 9

<212> DNA

<213> Artificial sequence

<400> 10

caagatagc 9

<210> 11

<211> 27

<212> DNA

<213> Artificial sequence

<400> 11

caggcgtggg acagcagcac tgtggta 27

<210> 12

<211> 24

<212> DNA

<213> Artificial sequence

<400> 12

ggatacagtt ttaccaccta ctgg 24

<210> 13

<211> 24

<212> DNA

<213> Artificial sequence

<400> 13

atctatcctg gtgactctga tacc 24

<210> 14

<211> 63

<212> DNA

<213> Artificial sequence

<400> 14

gcgagagtcc cgaaaagtgg ctggtacccc tacccctact actactacta cggtatggac 60

gtc 63

<210> 15

<211> 6

<212> PRT

<213> Artificial sequence

<400> 15

Lys Leu Gly Asp Lys Tyr

1 5

<210> 16

<211> 3

<212> PRT

<213> Artificial sequence

<400> 16

Gln Asp Ser

1

<210> 17

<211> 9

<212> PRT

<213> Artificial sequence

<400> 17

Gln Ala Trp Asp Ser Ser Thr Val Val

1 5

<210> 18

<211> 8

<212> PRT

<213> Artificial sequence

<400> 18

Gly Tyr Ser Phe Thr Thr Tyr Trp

1 5

<210> 19

<211> 8

<212> PRT

<213> Artificial sequence

<400> 19

Ile Tyr Pro Gly Asp Ser Asp Thr

1 5

<210> 20

<211> 21

<212> PRT

<213> Artificial sequence

<400> 20

Ala Arg Val Pro Lys Ser Gly Trp Tyr Pro Tyr Pro Tyr Tyr Tyr Tyr

1 5 10 15

Tyr Gly Met Asp Val

20

<210> 21

<211> 104

<212> PRT

<213> Artificial sequence

<400> 21

Gly Phe Thr Phe Ser Ser Tyr Gly Met His Trp Val Arg Gln Ala Pro

1 5 10 15

Gly Lys Gly Leu Glu Trp Val Ala Val Ile Trp Tyr Asp Gly Ser Asn

20 25 30

Lys Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp

35 40 45

Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu

50 55 60

Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp Gln Val Thr Met Val Arg

65 70 75 80

Gly Val Tyr Lys Thr Tyr Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln

85 90 95

Gly Thr Thr Val Thr Val Ser Ser

100

<210> 22

<211> 112

<212> PRT

<213> Artificial sequence

<400> 22

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

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser

20 25 30

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

35 40 45

Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala

85 90 95

Leu Gln Met Pro Val Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 23

<211> 99

<212> PRT

<213> Artificial sequence

<400> 23

Gly Gly Ser Ile Ser Ser Ser Ser Tyr Tyr Trp Gly Trp Ile Arg Gln

1 5 10 15

Pro Pro Gly Lys Gly Lys Arg Val Ile Gly Ser Ile Tyr Tyr Ser Gly

20 25 30

Ser Thr Tyr Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr Ile Ser Val

35 40 45

Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala

50 55 60

Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Arg Tyr Cys Ser Ser Thr

65 70 75 80

Ser Cys Tyr Tyr Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr

85 90 95

Val Ser Ser

<210> 24

<211> 106

<212> PRT

<213> Artificial sequence

<400> 24

Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln

1 5 10 15

Thr Ala Ser Ile Thr Cys Ser Gly Asp Lys Leu Gly Asp Lys Tyr Ala

20 25 30

Cys Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Val Leu Val Ile Tyr

35 40 45

Gln Asp Ser Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Ala Trp Asp Ser Ser Thr Val Val

85 90 95

Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210> 25

<211> 113

<212> PRT

<213> Artificial sequence

<400> 25

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Val Trp Val

35 40 45

Ser Arg Ile Asn Ser Asp Gly Ser Ser Thr Ser Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Ile Ala Arg Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Val Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Glu Val Asp Gly His Trp Gly Gln Gly Thr Leu Val Thr Val Ser

100 105 110

Ser

<210> 26

<211> 110

<212> PRT

<213> Artificial sequence

<400> 26

Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Glu Ala Pro Arg Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn

20 25 30

Ala Val Asn Trp Tyr Gln Gln Leu Pro Gly Lys Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Tyr Asp Asp Leu Leu Pro Ser Gly Val Ser Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Thr Trp Asp Asp Ser Leu

85 90 95

Asn Ala Gly Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 27

<211> 97

<212> PRT

<213> Artificial sequence

<400> 27

Gly Phe Thr Phe Gly Ser Tyr Ala Phe Tyr Trp Val Arg Gln Ala Pro

1 5 10 15

Gly Lys Gly Leu Glu Trp Val Ser Tyr Ile Ser Ser Ser Ser Ser Thr

20 25 30

Ile Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp

35 40 45

Asn Ala Lys Asn Ser Leu Tyr Leu Asp Met Thr Ser Leu Arg Lys Gly

50 55 60

Asp Thr Ala Thr Tyr Tyr Cys Ala Arg Asp Tyr Thr Thr Trp Arg Gly

65 70 75 80

Ala Asp Ala Leu Glu Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser

85 90 95

Ser

<210> 28

<211> 108

<212> PRT

<213> Artificial sequence

<400> 28

Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln

1 5 10 15

Thr Ala Arg Ile Thr Gly Gly Gly Asp Asn Ile Gly Ser Lys Ser Val

20 25 30

His Trp Tyr Gln Gln Arg Pro Gly Arg Ala Pro Val Leu Ala Ile Tyr

35 40 45

Tyr Asp Ser Asp Arg Ser Ser Ala Ile Pro Glu Arg Phe Ser Ala Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Arg Pro Ser Asp His

85 90 95

Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

Claims

1. A fully human anti-human erythrocyte RhD whole molecular IgG comprises a heavy chain variable region and a light chain variable region, and is characterized in that: the amino acid sequence of the light chain variable region is shown as SEQ ID NO.3, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 4.

2. A DNA molecule encoding the fully human anti-human red blood cell RhD whole IgG of claim 1, comprising a heavy chain variable region and a light chain variable region, wherein: the light chain variable region nucleic acid sequence is shown in SEQ ID NO.1, and the heavy chain variable region nucleic acid sequence is shown in SEQ ID NO. 2.

3. The fully human anti-human erythrocyte RhD whole molecule IgG of claim 1, which is characterized in that: the full-length nucleic acid sequence of the light chain of the full-human anti-human erythrocyte RhD full-molecular IgG is encoded is SEQ ID No.5, and the full-length nucleic acid sequence of the heavy chain is SEQ ID No.6.

4. The antigen-binding fragment of a fully human anti-human erythrocyte RhD whole IgG of claim 1, wherein: the nucleic acid sequences of CDR1, CDR2 and CDR3 of the light chain antigen complementarity determining region are respectively shown as SEQ ID NO.9, SEQ ID NO.10 and SEQ ID NO.11, and the amino acid sequences are respectively shown as SEQ ID NO.15, SEQ ID NO.16 and SEQ ID NO. 17; the nucleic acid sequences of the heavy chain antigen complementarity determining regions CDR1, CDR2 and CDR3 are respectively shown as SEQ ID NO.12, SEQ ID NO.13 and SEQ ID NO.14, and the amino acid sequences are respectively shown as SEQ ID NO.18, SEQ ID NO.19 and SEQ ID NO.20.

5. An expression vector comprising a DNA molecule encoding the fully human anti-human erythrocyte RhD full IgG of claim 1.

6. A cell comprising the expression vector of claim 5.

7. A method of producing the fully human anti-human red blood cell RhD whole IgG of claim 1, comprising the steps of culturing the cell of claim 6 and recovering the fully human anti-human red blood cell RhD whole IgG from the cell culture.

8. A composition comprising the fully human anti-human red blood cell RhD whole IgG of claim 1 or 3 or the antigen-binding fragment of claim 4 and a pharmaceutically acceptable carrier.

9. Use of the fully human anti-human red blood cell RhD whole IgG of claim 1 or 3 in the preparation of a formulation for detecting red blood cell RhD protein in a sample from human peripheral blood.

10. Use of the antigen-binding fragment of claim 4 in the preparation of a formulation for detecting red blood cell RhD protein in a sample from human peripheral blood.

11. Use of the fully human anti-human red blood cell RhD whole IgG of claim 1, in the manufacture of a medicament for preventing hemolytic disease of newborn, treating Idiopathic Thrombocytopenic Purpura (ITP), or preventing sensitization to rhesus D antigen following erroneous input of RhD (+) blood into RhD (-) individuals.