CN107760690B - A kind of preparation method and application of high throughput human antibody - Google Patents
A kind of preparation method and application of high throughput human antibody Download PDFInfo
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- C07—ORGANIC CHEMISTRY
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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- C07—ORGANIC CHEMISTRY
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/52—Constant or Fc region; Isotype
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
Abstract
The present invention relates to genetic engineering and Antibody preparations, specifically disclose a kind of preparation method and application of high-throughput full human monoclonal antibody.The preparation method includes separating periphery blood monocytic cell, sort the unicellular of thick liquid cell or antigen specific memory bone-marrow-derived lymphocyte, the heavy chain and light chain gene variable region of the single B cell antibody of primer pair provided using the present inventor are expanded, the expression system containing heavy chain of antibody, light chain gene is built using the linear expression systemic vectors containing heavy chain fragment or light chain segments constant region, finally realizes isolating and purifying for full human monoclonal antibody.Wherein, amplification heavy chain of antibody and chain variable region gene are combined using primer provided by the present invention, the natural pairing of light chain, heavy chain variable region can be retained, have gene diversity is good, potency is high, Quan Renyuan, affinity of antibody are good, high specificity, without advantages such as xenogenesis seroreaction, the danger for not propagating other communicable diseases.
Description
Technical field
The present invention relates to genetic engineering and Antibody preparations, in particular to the system of high-throughput full human monoclonal antibody
Preparation Method and application.
Background technology
Monoclonal antibody (Monoclonal antibodies, MAbs) drug has compared with traditional small-molecule chemical drug
There are higher targeting and specificity, is a kind of biotech drug that compound growth rate is most fast in recent years, has rapidly become
Treat various communicable diseases, the important tool of the human diseases such as autoimmunity disease and tumour.The side of production human antibody at present
Method mainly has various antibody phage display libraries technology, EBV conversion B cell clone technologies and transgcnic mouse techniques.Antibody is bitten
The development of phage display library technology makes it possible in vitro without immune acquisition antibody, but the antibody thus obtained is not
Primary pairing, efficiency is low, is selected without human immunity monitoring/tolerance, is not human antibody truly, has latent
Autoimmune reaction and immunogenicity, often affine force difference；The full people source that EBV converts the production of B cell clone technology is anti-
Body, it is difficult to generate stable cell line, antibody producing efficiency is low；The human antibody produced using transgenic mice is by the anti-of people
Weight chain and light chain gene strike people mouse, are resisted by being immunized to obtain to clone using traditional mouse monoclonal antibody technology after mouse
Body.But containing being in Mice Body by the interior generation of human antibody heavy chain and B cell/antibody of light chain gene and ripe process
Occur, is selected by immunosurveillance/tolerance of mouse, thus nor human antibody truly, has potential
Autoimmune reaction and immunogenicity.
CN102464717A discloses a kind of preparation method of human antinuclear antibody, is substantially a kind of phage display
All obtained mixing with cells are detached heavy chain light chain gene by library technology jointly together, and then random pair combines, can not be true
Guarantor obtains and the identical antibody of human body natural antibody.
CN101451134A discloses the side of amplification human antibody heavy chain and light chain gene segment in a kind of trace B cells from people
Method is to obtain B cell with the method for magnetic bead sorting, has only used a cell marking, is used without flow sorter several thin
The cell accuracy that born of the same parents mark compounding separation to obtain is good；In addition this method carries out collaborating genes amplification with 10 B cells, will expand
The several heavy chains and several light chain gene random pairs gone out are still unable to ensure to obtain identical anti-with human body natural antibody
Body.
Invention content
In order to solve the problems in the existing technology, the object of the present invention is to provide a kind of high-throughput human antibodies
Preparation method.
In order to realize the object of the invention, technical scheme is as follows：
A kind of preparation method of high throughput human antibody, the method includes：
1, separating periphery blood monocytic cell (PBMC).
The blood sample of communicable disease self-healing patient is either suffered from through communicable disease, autoimmune disease or tumour
The blood sample of the autoantibody generated in person's body detaches PBMC.
Preferably, the time point that antibody titer is high in sample and thick liquid cell ratio is high is selected to acquire blood sample.
Preferably, PBMC is detached using Ficoll density gradient centrifugations or HES centrifugals or blood cell separator.
It is directed to the selection of each communicable disease respectively to be commercialized on the market and verified effective vaccine for man, note
Normal human volunteers are penetrated, and are drawn blood in different time points, volunteer's internal antibody production and the variation of thick liquid cell ratio are detected
Situation；Another approach can also cooperate with hospital, be signed with patient《Informed consent form》In the case of, obtain symbol
The normal person of conjunction condition either autoimmune disease or tumour patient self-healing patient or generate autoantibody patient whole blood
Sample.Antibody titer highest and thick liquid cell ratio highest time point in above-mentioned sample are selected, people volunteer blood is extracted, is utilized
The method of density gradient centrifugation detaches PBMC.
Preferably PBMC separation methods are：The blood sample of fresh acquisition, room temperature 2400rpm centrifuge 7min, take another centrifuge tube,
It is spare that isometric Ficoll is added；After centrifugation, upper plasma is transferred to new -80 DEG C of preservations of centrifuge tube, middle level leucocyte
It draws into another centrifuge tube, the PBS dilutions containing 5%FBS in equal volume is added, piping and druming is uniform, is carefully slowly added into Ficoll,
Liquid level is kept to understand；20 DEG C of 2200rpm of room temperature centrifuge 30min, draw middle layer, i.e. PBMC cells.
2, it sorts unicellular
2.1 thick liquid cells of the sorting containing full human monoclonal antibody gene.
In view of human body, antigen-specific plasma cell dramatically increases after receiving immunity inoculation, therefore, to there is clearly immune connect
The sample in kind and appropriate sampling time, will use the single thick liquid cell of selected by flow cytometry apoptosis.
Preferably, the thick liquid cell fluorescent marker that thick liquid cell of the sorting containing full human monoclonal antibody gene uses is selected from following
Three combinations：
Group unification：AqVD-Amycan-/CD19-BV605+/CD3/14/16/235a-PE-Cy5-/CD20-PerCP-/
IgD-PE-/CD138-FITC-/CD38-AF647+/CD27-BV421+；
Combination two：AqVD-Amycan-/CD19-PE+/CD3/14/16/235a-PE-Cy5-/CD20-PerCP-/IgD-
BB515-/CD138-PE-CF594-/CD38-AF647+/CD27-BV421+；
Combination three：AqVD-Amycan-/CD19-PerCP+/CD3/14/16/235a-PE-Cy5-/CD20-PE-Cy7-/
IgD-PE-/CD138-FITC-/CD38-BV605+/CD27-BV421+。
2.2 antigen specific memory bone-marrow-derived lymphocytes of the sorting containing full human monoclonal antibody gene.
Since thick liquid cell is restored to foundation level after immunity inoculation several weeks, it is single to detach the full people source of specificity using thick liquid cell
The efficiency of Cloning Human Immunoglobulin Genes will substantially reduce.Also some cases we can not to analyze internal thick liquid cell horizontal, just utilize note
Recalling bone-marrow-derived lymphocyte has the characteristics of cell surface antibodies, using the specific antigen of fluorescent marker as probe bait, utilizes streaming
Cell instrument sorts the single memory B lymphocytes of antigentic specificity.
Preferably, the fluorescence that antigen specific memory bone-marrow-derived lymphocyte of the sorting containing full human monoclonal antibody gene uses
Label is selected from following three combinations：
Group unification：AqVD-AmCyan-/CD16-PE-Cy7-/CD14-BV570-/CD3-PerCP5.5-/CD20-
FITC-/IgD-PE-/CD27+/specific antigen-AF647+/specific antigen-BV421+；
Combination two：AqVD-AmCyan-/CD3/CD16/CD235a-PE-Cy5-/CD14-FITC-/IgD-PE-/IgM-
APC-/CD20-BV605+/CD27-APC-H7+/specific antigen-PE-Cy7+/specific antigen-BV421+；
Combination three：AqVD-AmCyan-/CD3/CD16/CD235a-PE-Cy5-/CD14-PerCP-/IgD-PE-/IgM-
FITC-/CD20-PE-CF594+/CD27-APC-H7+/specific antigen-AF647+/specific antigen-BV421+.
3, single B cell gene magnification and analysis
Reverse transcription is carried out to single B cell first and synthesizes cDNA, primer is as shown in table 1；Then amplification heavy chain of antibody and light
Chain gene；And the variable region gene of separation Cloning of Immunoglobulin heavy chain and light chain is analyzed.
Preferably, by nested PCR amplification heavy chain of antibody and light chain gene, preferred first round nested PCR amplification heavy chain, light
Chain λ, light chain κ full-length genes, the second wheel nest-type PRC expand heavy chain, light chain λ, light chain κ variable region genes respectively.
Preferably, one terminal sequence of heavy chain variable region amplified production and part Ig guiding region sequence overlap, another terminal sequence and
The constant-region sequences of Partial heavy overlap；Amplified production one terminal sequence in the variable regions light chain λ is overlapped with part Ig guiding region sequences, separately
One terminal sequence is overlapped with the constant-region sequences of part light chain λ；One terminal sequence of the variable regions light chain κ amplified production and the part boot sections Ig
Sequence overlaps, and another terminal sequence is overlapped with the constant-region sequences of part light chain κ.
Further, the sequence overlapped with part Ig guiding region sequences is CTGGGTTCCAGGTTCCACTGGTGAC.
Further, the sequence overlapped with the constant-region sequences of Partial heavy is GCGGCCCTGGGCTGCCTGGTCAAG.
Further, the sequence overlapped with the constant-region sequences of part light chain λ is AGGAGAGTGTCACAGAGCAGGACAG.
Further, the sequence overlapped with the constant-region sequences of part light chain κ is
CTTCAAGCCAACAAGGCCACACTGGTG。
First round nested PCR amplification the primer is as shown in table 2,3 and 4；Second wheel nested PCR amplification the primer such as table
5, shown in 6,7.
Table 1RT-PCR primers
| Direction | PRIMER ID | 5’-3’SEQUENCE | Tm(℃) | GC% |
| Reverse | IgG114RT | GCGCCTGAGTTCCACGACACC | 67.2 | 66.7 |
| Reverse | IgA99RT | TGTCCGCTTTCGCTCCAGGTC | 65.2 | 61.9 |
| Reverse | IgE93_RT | GGCTCCGGGAAGTAGCCCGTG | 69.1 | 71.4 |
| Reverse | IgM93_RT | GGCTCCGGGAAGTAGCCCGTG | 69.1 | 71.4 |
| Reverse | IgD92_RT | ACGGACGTTGGGTGGTACCCAG | 67.7 | 63.6 |
| Reverse | KC93RT | CTTTGGCCTCTCTGGGATAGAAG | 64.6 | 52.2 |
| Reverse | LC92RT | ACGGCTCCCGGGTAGAAGTCAC | 67.7 | 63.6 |
2 first round of table nest-type PRC heavy chain PCR primer
3 first round of table nest-type PRC light chain κ PCR primers
| Direction | PRIMER ID | 5’-3’SEQUENCE | Tm(℃) | GC% |
| Forward | VK0.1EXT | GTCCCCGCTCAGCTCCTGGGGCT | 73.6 | 73.9 |
| Forward | VK0.2EXT | CAGCTCCTGGGGCTGCTAATGC | 67.7 | 63.6 |
| Forward | VK0.3EXT | CAGCTTCTCTTCCTCCTGCTAC | 64 | 54.5 |
| Forward | VK0.4EXT | GTCCCTGCTCAGCTCCTGGGGCT | 71.8 | 69.6 |
| Forward | VK0.5EXT | ATGAGGSTCCCYGCTCAGCTGCTGG | 70 | 65.2 |
| Reverse | KC59.EXT | CACACAACAGAGGCAGTTCCAG | 64 | 54.5 |
4 first round of table nest-type PRC light chain λ PCR primers
| Direction | PRIMER ID | 5’-3’SEQUENCE | Tm(℃) | GC% |
| Forward | VL1.1EXT | TCCTCACCCTCATCACTCACTG | 64 | 54.5 |
| Forward | VLL1.2EXT | TCCTCACCCTCCTCACTCACTG | 65.9 | 59.1 |
| Forward | VLL2.EXT | CTCCTCACTCAGGGCACAG | 61.6 | 63.2 |
| Forward | VL3.EXT | GCCTCCTCACTCTCTGCACAG | 65.2 | 61.9 |
| Forward | VL4.1EXT | CTGCCCTTCATTTTCTCCACAG | 62.1 | 50 |
| Forward | VL4.2EXT | CCTCTCCTCCTCCACTGGACAG | 67.7 | 63.6 |
| Forward | VL5.EXT | TGCTCCTCTCTCACTGCACAG | 63.3 | 57.1 |
| Forward | VL6.EXT | CCCTCCTCGCTCACTGCACAG | 67.2 | 66.7 |
| Forward | VL7.EXT | TCCTCCTCACTTGCTGCCCAG | 65.2 | 61.9 |
| Forward | VL8.EXT | GACTCCTTGCTTATGGATCAG | 59.4 | 47.6 |
| Forward | VL9.EXT | CCCTCCTCAGTCTCCTCACAG | 65.2 | 61.9 |
| Forward | VL10.EXT | CCCTCCTCACTCACTCTGCAG | 65.2 | 61.9 |
| Reverse | LC70.EXT | CTTATGAGACACACCAGTGTGGC | 64.6 | 52.2 |
Table 5 second takes turns nest-type PRC heavy chain variable region PCR primer
Table 6 second takes turns nest-type PRC light chain κ variable region PCR primers
| Direction | PRIMER ID | 5’-3’SEQUENCE | Tm(℃) | GC% |
| Forward | VK1_int | GACATCCAGATGACCCAGTCTCC | 66.4 | 56.5 |
| Forward | VK2.1_int | GATATTGTGATGACCCAGACTCC | 62.9 | 47.8 |
| Forward | VK2.2_int | GATGTTGTGATGACTCAGTCTCC | 62.9 | 47.8 |
| Forward | VK3.1_int | GAAATTGTGTTGACGCAGTCTCC | 62.9 | 47.8 |
| Forward | VK3.2_int | GAAATTGTAATGACACAGTCTCC | 59.3 | 39.1 |
| Forward | VK4_int | GACATCGTGATGACCCAGTCTCC | 66.4 | 56.5 |
| Forward | VK5_int | GAAACGACACTCACGCAGTCTCC | 66.4 | 56.5 |
| Forward | VK6_int | GAAATTGTGCTGACTCAGTCTCC | 62.9 | 47.8 |
| Reverse | KC30int | CTGCTCATCAGATGGCGGGAAG | 65.9 | 59.1 |
Table 7 second takes turns nest-type PRC light chain λ variable region PCR primers
It should be noted that IgL64Ftag will be added in 5 ' ends of 2~table of table, 7 all primers.
Reverse transcription synthesizes cDNA, preferred method：In the PCR plate of the holes 96- cDNA, 50ng/ μ l are synthesized with 20 μ l reaction systems
Random hexamer Primers, 1.5 μ l 25mMdNTPs and 50U Superscript III reverse transcriptase, 65 DEG C
It is reacted 1 hour in PCR instrument；
Nested PCR amplification antibody I gH, Ig λ and Ig κ variable region genes, preferred method：First round PCR：Contain in 50 μ l systems
There are the RT reaction products of 5 μ l, the HotStarTaq Plus enzymes of 5 units, 0.2mM dNTPs and 0.5 μM of IgH (VH1-
VH6), Ig κ (VK1-VK7) or Ig λ (VL1-VL10) variable region primers, IgM, IgG, IgD, IgA1 and IgA2 or Ig κ or
Ig λ antibody constant region primers.Reaction condition：95 DEG C of 5min of pre-degeneration, then carry out 35 PCR cycles, and each cycle is：95℃
× 30sec, (V λ × 60sec, finally use 72 DEG C and extend 7min by 72 DEG C × 90sec for 55 DEG C (VH with V κ) or 50 DEG C.Second wheel
PCR：Contain the first round PCR reaction product of 3 μ l in 50 μ l systems as masterplate, the HotStarTaq Plus enzymes of 5 units,
0.2mM dNTPs and 0.5 μM of IgH (VH1-VH6), Ig κ (VK1-VK7) or Ig λ (VL1-VL10) variable region primers, IgM,
IgG, IgD, IgA1 and IgA2 either Ig κ or the antibody variable regions Ig λ primer, reaction condition：95 DEG C of 5min of pre-degeneration, then
35 PCR cycles are carried out, each cycle is：95 DEG C × 30sec, 58 DEG C (VH), 60 DEG C (V κ) or 64 DEG C of (V λ) × 60sec,
72 DEG C × 90sec, finally with 72 DEG C of extension 7min.
4, structure is containing heavy chain of antibody, the linear expression system of light chain gene
Isolated heavy chain of antibody, chain variable region gene are connected into respectively by the method for overlapping PCR
In linear carrier expression vector.
Isolated heavy chain of antibody and chain variable region gene, carrier construction and for express antibody when, such as using pass
The plasmid vector of system, then time-consuming, efficiency is low, is not suitable for high-throughput application.
Linear expression systemic vectors consist of two parts, and first part is made of promoter and the boot sections Ig, second part
It is made of heavy chain fragment or light chain segments constant region and BGH polyA signal peptide fragments.
The preferably linear Ig expression vectors of linear carrier, it is permanent by CMV promoter, the boot sections Ig, heavy chain fragment or light chain segments
Determine area and BGH poly (A) signal peptide fragments (B-H, B-K, B-L) composition.
Preferably, heavy chain linear expression vector consists of two parts, and first part is made of CMV promoter, the boot sections Ig,
Second part is made of heavy chain constant region and BGH poly (A) signal peptide fragment.Overlapping PCR resist isolated
Heavy chain variable region gene is connected among first part and second part, to form CMV promoter, the boot sections Ig, heavy chain can
Become the linear expression system of heavy chain of antibody that area, heavy chain constant region and BGH poly (A) signal peptide fragment form.
Preferably, light chain λ linear expression vectors consist of two parts, and first part guides district's groups by CMV promoter, Ig
At second part is made of light chain λ constant regions and BGH poly (A) signal peptide fragment.Overlapping PCR will be isolated
Antibody light chain λ variable region genes be connected among first part and second part, to form the CMV promoter, boot sections Ig, light
The linear expression systems of antibody light chain λ that the variable regions chain λ, light chain λ constant regions and BGH poly (A) signal peptide fragment form.
Preferably, light chain κ linear expression vectors consist of two parts, and first part guides district's groups by CMV promoter, Ig
At second part is made of light chain κ constant regions and BGH poly (A) signal peptide fragment.Overlapping PCR will be isolated
Antibody light chain κ variable region genes be connected among first part and second part, to form the CMV promoter, boot sections Ig, light
The linear expression systems of antibody light chain κ that the variable regions chain κ, light chain κ constant regions and BGH poly (A) signal peptide fragment form.
Further, the boot sections Ig (Ig L) sequence is：
MKVRGIQRNYPQWWIWSMLGLWMLMICNG。
CMV promoter sequence is as shown in SEQ ID NO.95.
The present invention using overlapping PCR method by isolated heavy chain of antibody, chain variable region gene with
Linear carrier expression system combines, and forms overall length linear antibodies gene using the method for One_step PCR, expressing recombinant antibody is for resisting
The screening and identification of body.We are directed to several strain antibodies of HCV, and the expression quantity for comparing glm gene and plasmid gene compares figure, show
Linear expression system is more efficient than traditional plasmid cloning gene expression recombinant antibodies.
5, antibody producing and purifying
Expression vector after recombination is imported host cell to express, collects the supernatant of expression, antibody purification.
Expression vector after recombination can be imported into host cell by any of method；The heavy chain and light chain of pairing
Expression vector can express in eukaryocyte or prokaryotic cell；Expression supernatant is collected, centrifugation discards cell fragment, using normal
Antibody purification Technology antibody purification.
By heterologous polynucleotide import mammalian cell method be it is known in the art, including dextran mediate
Transfection, calcium phosphate precipitation, polybrene mediate transfection, protoplast fusion method, electroporation, polynucleotides lipid
Body encapsulating, the injection of biological rifle and the direct microinjections of DNA into intracellular.
Host cell as expression is mammalian cell known in the art, including many can be from U.S.'s strain allusion quotation
The immortal cell line that Tibetan center (ATCC) obtains.These cell lines include mainly Chinese hamster ovary (CHO) cell, NSO, SP2
Cell, HeLa cells, baby hamster kidney (BHK) cell, MK cells (COS), human liver cancer cell, A549 cells, 3T3 cells, 293
Cell and many other cell lines.By measuring which plant cell line particularly preferred cell is selected with high expression level
System.Other available cell lines include insect cell line, amphibian cell, bacterial cell, plant cell and fungal cell.
It is led when the recombinant expression carrier of encoding heavy chain or its antigen-binding site, light chain and/or its antigen-binding site
When entering mammalian host cell, it can persistently be enough antibody is enable to express in host cell by cultivating this host cell
Generate this antibody for a period of time.Preferably, make in antibody-secreting to the culture medium of host cell growth.
It can be isolated and purified according to the characteristic of antibody, it, will in the method saltoutd such as according to the difference of protein hydrophobic
Antibody is separated with other albumen, and the difference of charge is carried according to antibody, with ion exchange, the separation of the technologies such as electrophoresis.It isolates and purifies
Method include precipitate, saltout, membrane technology, electrophoresis, chromatography etc..It is preferably anti-using albumin A or the purifying of protein g affinity chromatography method
Body.
The purpose of the present invention is to provide application of the above-mentioned high-throughput human antibody preparation method in preparing antibody.
Preferably, the antibody of preparation is to treat the anti-tumour antibody of tumour or treat resisting for the infectious disease caused by microorganism
Body.
Tumour preferably is selected from following group：Including hematologic malignancies such as lymthoma, leukaemia, myeloma；Cancer such as gland cancer,
Its original site is in esophagus, lung, breast, ovary, liver, endometrium, cervix, colon, pancreas, prostate, stomach, small intestine,
Rectum and uterus；Squamous cell carcinoma, original site is in oral cavity, tongue, throat, esophagus, lung, skin, wing moon bright, cervix,
Eyelid, eye conjunctiva, vagina etc..Other tumours for the treatment of include:Meat cytoma, such as fibrosarcoma, muscle-derived sarcoma, nerve cell
Tumor, melanoma, trophocyte's tumor, gonioma, neuroendocrine cell tumor, neuroectodermal cells tumor.Particularly,
Tumour is selected from following group, including:Metastatic breast cancer, non-Hodgkin's lymphocytoma, chronic lymphocytic leukemia are acute
Myeloid leukemia.
Microorganism preferably is selected from following group：HIV, hepatitis type B virus (HBV), Hepatitis C Virus (HCV), the thermophilic T leaching of people
Bar cell virus 1 and 2, parvovirus, herpes virus hominis include herpes simplex virus (HSV) 1 and 2, EBV viruses), cell fertilizer
Big virus (CMV), human papilloma virus (HPV), varicella zoster virus (VZV) and Human herpesviryus 6 (HHV-6), can
Antibody is prepared with method provided by the invention to prevent, treat or alleviate.Other pathogen infections that can be replicated in the cell are for example
Cause a disease protobiont such as bullet, malaria, toxoplasm；Bacterium such as mycobacterium, salmonella, chlamydia trachomatis, Li Siteshi clothing
Substance, fungi such as candida albicans, can also develop into chronic disease, and therefore, they are also prepared by method provided by the invention
The ideal candidate of antibody prophylactic treatment.
The present invention also provides the human antibodies prepared by using the above method.
The present invention also provides the use that nucleic acid construct according to the present invention is used to generate univalent antibody according to the present invention
On the way.
The present invention also provides the host cell containing nucleic acid according to the present invention, the host cell is not have differentiation
Totipotency.
The present invention also provides host cells according to the present invention for generating unit price, divalent or multivalence according to the present invention
The purposes of antibody.
The present invention also provides the monomeric iggs according to the present invention as drug.
Purposes the present invention also provides univalent antibody according to the present invention as drug.
The present invention also provides use pharmaceutical composition of the Antibody preparation according to the present invention for treating disease or illness.
The present invention also provides antibody according to the present invention in preparing the pharmaceutical composition for treating disease or illness
Purposes.
The present invention also provides the methods for treating disease or illness, wherein the method includes to it is in need for the treatment of by
Examination person applies the univalent antibody according to the present invention of therapeutically effective amount, the pharmaceutical composition comprising the antibody, includes described anti-
The immune conjugate of body or nucleic acid construct according to the present invention, whereby treat disease or illness.
The present invention also provides pharmaceutical compositions, and it includes univalent antibody according to the present invention and one or more drugs
Acceptable excipient, diluent or carrier.
The object of the present invention is to provide a set of amplimer for human antibody single cell clone, amplimer includes
Heavy chain variable region gene amplimer group, light chain λ variable region gene amplimers group, light chain κ variable region gene amplimers group,
Weight chain constant area gene amplimer group, light chain λ constant region gene amplimers group, light chain κ constant region gene amplimer groups.
Heavy chain variable region gene amplimer group is specifically shown in Table 5.
Light chain κ variable region gene amplimer groups are specifically shown in Table 6.
Light chain λ variable region gene amplimer groups are specifically shown in Table 7.
Further, amplimer also includes that reverse transcription synthesizes cDNA primer sets, is specifically shown in Table 1.
Preferably, amplimer also include heavy chain full-length gene amplimer group, light chain λ full-length gene amplimers group,
Light chain κ full-length gene amplimer groups.
Heavy chain full-length gene amplimer group is specifically shown in Table 2.
Light chain κ full-length gene amplimer groups are specifically shown in Table 3.
Light chain λ full-length gene amplimer groups are specifically shown in Table 4.
The present invention relates to part nominal definition：
Constant region (CR) refers to the constant region of light chain (LCCR) or heavy chain constant region (HCCR) of antibody.General CR includes with two
1 to 4 immunoglobulin domains that the ring structure that sulfide linkage is stablized is characterized.Preferred CR is the CR of immunoglobulin, preferably
The CR of human immunoglobulin(HIg), wherein further preferably the immunoglobulin, the preferably described human immunoglobulin(HIg) be selected from IgG1,
IgG2, IgG3, IgG4, IgA, IgE, IgM and IgD.Particularly preferred CR be include the amino acid that can be obtained from public database
The people CR that sequence or the amino acid sequence by being obtained from public database form.
Specifically κ LCCR or λ LCCR, the natural κ or lambda light chain C- of general proxy natural antibody are last for constant region of light chain (LCCR)
Half section of end.LCCR generally comprises about 110 amino acid for representing an immunoglobulin domains.
Heavy chain constant region (HCCR) includes about 3/4ths or more of heavy chain of antibody, and is located at its ends C-.One
As, HCCR includes three or four immunoglobulin domains.Preferred HCCR be selected from γ HCCR, α HCCR, ε HCCR,
MyHCCR and δ HCCR.
Variable region (VR) refers to variable region or the variable domains of antibody, refers specifically to heavy chain variable region (HCVR) or refers to light chain
Variable region (LCVR).Preferably, VR includes single immunoglobulin domains.Preferred VR is the VR of immunoglobulin, excellent
Choose the VR of immunoglobulin, wherein the further preferably immunoglobulin, the preferably described human immunoglobulin(HIg) be selected from IgG1,
IgG2, IgG3, IgG4, IgA, IgE, IgM and IgD.
Each VR includes the binding characteristic for determining antibody and the so-called complementary determining region (CDR) of embedded so-called frame.It is preferred that
Ground, VR include three kinds of CDR of embedded frame (FR1-4), preferably CDR1, CDR2 and CDR3.Thus, in preferred embodiment
In, VR includes the following elements by the following ends N- to C- terminal orders：FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.It is logical
Normal VR includes polypeptide or is preferably made of polypeptide, wherein the polypeptide is V- genetic fragments family member and other gene piece
Section, such as such as D and J genetic fragments (HCVR) or the product of J genetic fragments (LCDR) combination.
Light chain variable region is κ LCVR or λ LCVR by the nucleic acid molecule encoding reset.Humanized κ LCVR include more
Peptide, wherein the polypeptide is the product of the 1st to 7 family member of humanized's κ V- genetic fragments.Humanized λ LCVR include more
Peptide, wherein the polypeptide is the product of 1 to 11, the ground family member of humanized's λ V- genetic fragments.Preferred λ LCVR are people source
Property λ LCVR, preferably by the humanized λ LCVR of the DNA encoding expanded from human B cell.
Heavy chain variable region (HCVR) is by the heavy chain variable region for the nucleic acid molecule encoding reset.Humanized HCVR includes polypeptide,
The wherein described polypeptide is the product of humanized hV- the 1st to 7 family member of genetic fragment.Preferred HCVR is humanized HCVR,
It is preferred that the humanized HCVR of the DNA encoding by human B cell amplification.
The beneficial effects of the present invention are：
The present invention provides the method provided based on gene clone technology from single bone-marrow-derived lymphocyte separation antibody gene,
And prepare high-throughput human antibody.This method remains the natural pairing of light chain heavy chain variable region, good with gene diversity,
Potency height, Quan Renyuan, affinity of antibody be good, high specificity, without xenogenesis seroreaction, do not propagate the danger of other communicable diseases
The advantages such as danger, for the prevention and treatment of the full human monoclonal antibody of development communicable disease, autoimmune disease and tumour
Property drug provides a wide approach.
Description of the drawings
Fig. 1 is specific plasma cell sorting figure of the present invention；
Fig. 2 is specific memory B-cell sorting figure of the present invention；
Fig. 3 is the variable region gene fragment electrophoretic figure of heavy chain immunoglobulin and light chain of the present invention；
Fig. 4 is linear Ig expression vector schematic diagrams in the embodiment of the present invention 4；
Fig. 5 is that the expression quantity of linear carrier and plasmid gene of the present invention compares figure.
Specific implementation mode
The preferred embodiment of the present invention is described in detail below in conjunction with embodiment.It will be appreciated that following real
Providing merely to play the purpose of explanation for example is applied, is not used to limit the scope of the present invention.The skill of this field
Art personnel can carry out various modifications and replace to the present invention without departing substantially from spirit of the invention and spirit.
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
The separation of embodiment 1 sample collection and PBMC
Literature survey is first passed through for different experiment purposes, understanding causes these communicable diseases, autoimmune disease
Disease or the pathogen information of tumour, pathogenesis, disease lapse to process, the immune response of body, main antigenic structure
And neutralizing epitope and corresponding treatment the case where being researched and developed with vaccine.Then the blood 5- at neutralizing antibody titers highest time point is extracted
10ml carries out PBMC separation.
PBMC is detached and is frozen：The blood sample of fresh acquisition, room temperature 2400rpm centrifuge 7min, take another centrifuge tube, be added etc.
Volume Ficoll (GE) is spare；After centrifugation, upper plasma is transferred to new -80 DEG C of preservations of centrifuge tube, middle level leucocyte is inhaled
It takes into another centrifuge tube, 1640 culture mediums (Gibco) dilution containing 5%FBS in equal volume is added, piping and druming is uniform, carefully slowly
It is added in Ficoll, liquid level is kept to understand；20 DEG C of 2200rpm of room temperature centrifuge 30min, draw middle layer, i.e. PBMC cells, are added
For 1640 culture mediums containing 5%FBS to 50ml, 1800rpm centrifuges 8min, washes twice；Flicking test tube bottom keeps cell distribution uniform,
Detect cell number and motility rate；With cells frozen storing liquid (fresh serum：DMEM=9:1) cell, liquid nitrogen cryopreservation have been hanged.
Embodiment 2
1, thick liquid cell is sorted
In view of human body, antigen-specific plasma cell dramatically increases after receiving immunity inoculation, therefore, to there is clearly immune connect
The sample in kind and appropriate sampling time, will use the single thick liquid cell of selected by flow cytometry apoptosis.PBMC is dyed：According to Staining Protocol mark
Remember streaming pipe, single dye positive control, negative control and Isotype controls are set；Thick liquid cell Staining Protocol is：
Group unification：AqVD-Amycan-/CD19-BV605+/CD3/14/16/235a-PE-Cy5-/CD20-PerCP-/
IgD-PE-/CD138-FITC-/CD38-AF647+/CD27-BV421+；
Combination two：AqVD-Amycan-/CD19-PE+/CD3/14/16/235a-PE-Cy5-/CD20-PerCP-/IgD-
BB515-/CD138-PE-CF594-/CD38-AF647+/CD27-BV421+；
Combination three：AqVD-Amycan-/CD19-PerCP+/CD3/14/16/235a-PE-Cy5-/CD20-PE-Cy7-/
IgD-PE-/CD138-FITC-/CD38-BV605+/CD27-BV421+。
Thick liquid cell sorting is as shown in Figure 1.
2, memory B lymphocytes are sorted
Since thick liquid cell is restored to foundation level after immunity inoculation several weeks, there is cell using memory B lymphocytes at this time
The characteristics of surface antibody, contains full people source Dan Ke using the specific antigen of fluorescent marker as bait using selected by flow cytometry apoptosis
The antigen specific memory bone-marrow-derived lymphocyte of grand antibody gene, the fluorescent marker of use：
Group unification：AqVD-AmCyan-/CD16-PE-Cy7-/CD14-BV570-/CD3-PerCP5.5-/CD20-
FITC-/IgD-PE-/CD27+/specific antigen-AF647+/specific antigen-BV421+；
Combination two：AqVD-AmCyan-/CD3/CD16/CD235a-PE-Cy5-/CD14-FITC-/IgD-PE-/IgM-
APC-/CD20-BV605+/CD27-APC-H7+/specific antigen-PE-Cy7+/specific antigen-BV421+；
Combination three：AqVD-AmCyan-/CD3/CD16/CD235a-PE-Cy5-/CD14-PerCP-/IgD-PE-/IgM-
FITC-/CD20-PE-CF594+/CD27-APC-H7+/specific antigen-AF647+/specific antigen-BV421+.
Specific memory B-cell sorting is as shown in Figure 2.
Experimental method：The cell of recovery, is added 2mlPBS/1%BSA, mixing, and 22 DEG C of 450g centrifuge 5min.Supernatant is abandoned, weight
It is multiple to move into streaming pipe twice, 200 μ l/ pipes；It is protected from light and corresponding antibody (BD, Biolegend) is added, 4 DEG C of placement 45min；Add
Enter 2mlPBS/1%BSA, light mixing, 4 DEG C of 450g centrifuge 5min, and repetition is washed 3 times；It is resuspended with 200-1000 μ lPBS/1%BSA,
Upper machine sorting after 0.22 μm of membrane filtration.Single B cell is set to be sorted into 96 orifice plates (Eppendorf).It is slow containing 20 μ l in 96 orifice plates
Rush system：5 μ l 5x PCR Buffer, 1.25 μ l DTT, 0.5 μ l RNAase out, 13.25 μ l water (Invitrogen).One
Rapid sealer (Axygen) after the completion of the sorting of 96 orifice plate of block, is put into -80 DEG C of refrigerators and preserves.
3 single bone-marrow-derived lymphocyte gene magnification of embodiment
Isolated single periphery nucleus carries out the cloning of V_H gene of heavy chain immunoglobulin and light chain.
1, reverse transcription synthesizes cDNA：
CDNA, the Random hexamer Primers of 50ng/ μ l are synthesized with 20 μ l reaction systems in the PCR plate of the holes 96-,
25mMdNTPs the and 50U Superscript III reverse transcriptase (Invitrogen, Carlsbad, CA) of 1.5 μ l, 65 DEG C
It is reacted 1 hour in PCR instrument；
2, nested PCR amplification antibody variable gene：
Antibody variable gene is carried out using nest-type PRC primer provided by the present invention.PCR reactions are in 96 orifice plates with 50
μ l reaction systems are respectively synthesized IgH, Ig λ and Ig κ variable region genes, first round PCR：RT reactions containing 5 μ l in 50 μ l systems
Product, the HotStarTaq Plus enzymes (QIAGEN) of 5 units, 0.2mM dNTPs and 0.5 μM of IgH (VH1.1-
VH7.1), Ig κ (VK0.1-VK0.5) or Ig λ (VL1.1-VL10) variable region primers, IgM, IgG, IgD, IgA1 and IgA2, or
Person's Ig κ or Ig λ antibody constant region primers, reaction condition：Then 95 DEG C of 5min of pre-degeneration carry out 35 PCR cycles, each to recycle
For：95 DEG C × 30sec, (V λ × 60sec, finally use 72 DEG C and extend 7min by 72 DEG C × 90sec for 55 DEG C (VH with V κ) or 50 DEG C.
Second wheel PCR：Contain the first round PCR reaction product of 3 μ l in 50 μ l systems as masterplate, 5 units
HotStarTaq Plus enzymes ((QIAGEN)), 0.2mM dNTPs and 0.5 μM of IgH (VH1-VH7), Ig κ (VK1-VK6) or
Ig λ (VL1-VL10) variable region primers, IgM, IgG, IgD, IgA1 and IgA2 either Ig κ or the antibody variable regions Ig λ primer,
Reaction condition：95 DEG C of 5min of pre-degeneration, then carry out 35 PCR cycles, and each cycle is：95 DEG C × 30sec, 58 DEG C (VH),
60 DEG C (V κ) or 64 DEG C of (V λ) × 60sec, 72 DEG C × 90sec, finally with 72 DEG C of extension 7min.The VH of amplification, V κ and V λ's
Variable region PCR product is identified with 1.2% agarose gel electrophoresis.All reactions total serum IgE extracted from PBMC
As the positive control of PCR reaction mass control, with being not added with the PCR reaction systems of masterplate as negative control.
The variable region gene electrophoresis detection for detaching Cloning of Immunoglobulin heavy chain and light chain, is shown in Fig. 3.
3, heavy chain immunoglobulin and chain variable region gene sequence analysis
1) heavy chain immunoglobulin and chain variable region gene product expanded carries out primitive sequencer sequence with Sequcher5.0
Row cleaning, splicing, output fasta formats preserve.The bad part of sequencing quality is cleared up automatically with Trime ends, can be excluded
The bimodal sequence of multi-ribbon.It excludes after a plurality of primer sequencing result is spliced and one by one, regard the correct sequence of reading frame
For positive sequence, finally exports and preserve with fasta formatted files.
2) areas antibody sequence V genetic analysis.With IMGT databases (http://www.imgt.org/) it completes.Use IMGT/
" Analyse your immunoglob μ lin (IG) or antibody nucleotide sequences " work(under VQUEST
It can complete.The areas the V hypotype and identity of heavy chain of antibody and light chain, CDR1/CDR2/CDR3 length etc. can be analyzed,
And it can confirm that whether the reading frame of sequence and sequence translation are functional.According to database analysis as a result, picking from same
Functional (productive) sequence of the H/K or H/L of cDNA, are combined pairing, are used for follow-up overlapping
PCR。
As a comparison, primer of the present invention is replaced with delivered primer (Liao, H.X., Levesque, M.C.,
Nagel,A.,Dixon,A.,Zhang,R.,Walter,E.,Parks,R.,Whitesides,J.,Marshall,D.J.,
Hwang,K.K.,et al.(2009).High-throughput isolation of immunoglobulin genes
from single human B cells and expression as monoclonal
Antibodies.J.Virol.Methods 158,171-179), compare amplification difference.
As a result as follows：
Since heavy chain is longer, amplification difficulty is bigger than light chain, so generally we count heavy chain amplification positive rate.Single B is thin
In the PCR results of born of the same parents, the average positive rate of I group primer heavy chains is 36, and the average positive rate of 50%, II group primer heavy chains is
53.20%.It with rate is 52.31% that I group primers, which are 34.65%, II group primers with rate,.II groups primer ratio I groups primer expands in heavy chain
It is all significantly increased in terms of increasing positive rate and last heavy chain light chain pairing rate.
Average positive rate=(30.47%+32.14%+46.88%)/3=36.50% of I group primer heavy chains；
Average positive rate=(54.17%+54.64%+50.78%)/3=53.20% of II group primer heavy chains；
I group primers match rate=(41+48+69)/(128+168+160)=34.65%；
II group primers match rate=(129+98+67)/(240+194+128)=52.31%.
It can be seen that using primer sets provided by the invention, primer has a clear superiority with rate, and the heavy chain amplified is average
Positive rate is significantly improved compared with primer sets disclosed in the prior art.
Embodiment 4 builds heavy chain of antibody, the linear expression system of light chain gene
The variable region gene of the sequence analysis positive, which is put on linear expression vector, expresses.
One, linear Ig expression vectors constitute (see Fig. 4)：
1, the linear Ig expression vectors of heavy chain are constituted：CMV promoter, the boot sections Ig, heavy chain variable region, heavy chain constant region and
BGH poly (A) signal peptide fragment.
2, the linear Ig expression vectors of light chain kappa (κ) are constituted：CMV promoter, the boot sections Ig, the variable regions light chain κ, light chain κ
Constant region and BGH poly (A) signal peptide fragment.
3, the linear Ig expression vectors of light chain lambda (λ) are constituted：CMV promoter, the boot sections Ig, the variable regions light chain λ, light chain
λ constant regions and BGH poly (A) signal peptide fragment.
4, the above segment is assemblied together with OverlappingPCR respectively, Pyrobest archaeal dna polymerases
(Takara), 50 μ l systems, the 1 μ l containing PCR product, each 1 μ l, C-H heavy chain of forward and reverse primer, C-K light chains kappa, C-L are light
51 μ l of μ l, dNTPs of chain lambda and B-H, B-K, B-L each 1 μ l, 10 × Buffer, remaining uses ddH2O is supplied.Reaction condition：
94 DEG C of 3min of pre-degeneration, then carry out 30 PCR cycles, and each cycle is：94 DEG C × 30sec, 63 DEG C (VH) or 55 DEG C of (V κ
With V λ) × 30sec, 72 DEG C × 120sec, finally with 72 DEG C of extension 10min.Then use PCR purification kits (Qiagen) pure
Change PCR product, is then transfected.
5, by the heavy chain of pairing and light chain gene expression vector FuGENE (promega E2692) PolyFect
(Qiagen, Valencia, CA) transfection reagent cotransfection human embryonic kidney cell's HEK 293i or 293F cells, 6-8 after transfection
Hour changes fresh culture, and in 37 DEG C of 5%CO2It is cultivated in incubator 72-96 hours and collects cell conditioned medium.
Two, ELISA is detected
1, it is coated with：Prepare elisa plate (costar), dilutes antigen with coating buffer solution, 100 μ l antigen diluents are added per hole
Liquid.4 DEG C overnight.
2, it closes：It is washed 5 times using board-washing machine (PBST), confining liquid is added per 250 μ l of hole, 37 DEG C are incubated 1 hour.
3, add primary antibody：It is washed 5 times using board-washing machine (PBST), cells and supernatant is added, per 100 μ l of hole, 37 DEG C of incubations 1 are small
When.
4, add secondary antibody：It is washed 5 times using board-washing machine (PBST), secondary antibody (goat anti-human igg of HRP labels) every 100 μ l of hole is added,
37 DEG C are incubated 1 hour.
5, it develops the color：It is washed 5 times using board-washing machine (PBST), developing solution (developing solution A and developing solution B1 is added:1 mixing, it is current
Now match) per 100 μ l of hole.
6, it terminates：5 minutes are placed at room temperature for, terminate liquid is added, per 50 μ l of hole.
7, microplate reader is read, dual wavelength 450-630nm.
The positive Overlapping PCR product digestions of ELISA detections, are connected to antibody dedicated expression vector therefor
pcDNATM3.3-TAKit(InvitrogenTMArticle No.：K830001).
5 antibody producing of embodiment and purifying
Transfection by the heavy chain of pairing and light chain gene expression vector calcium phosphate precipitation or PolyFect (Qiagen,
Valencia, CA) transfection reagent cotransfection HEK 293 or 293T cells, replace fresh culture, training within 8-12 hours after transfection
It supports base DMEM (Gibco) and heat 10% fetal calf serum of inactivation (Gibco) and penicillin 100U/ml is added, and in 37 DEG C of 5%CO2Training
It supports and is cultivated 48-72 hours in case.
Purified pool expresses supernatant, and centrifugation discards cell fragment, 1mL pH 8.0 is added, 0.1moL/L phosphate buffers are simultaneously
PH to 9.0 is adjusted with pH 9.0,1moL/L TRIS-HCL.0.1moL/L phosphate buffers pH has been used in cell conditioned medium addition
In 8.0 Protein G or Protein the A Sepharose CL 4B albumen columns balanced (GE Healthcare), use
It states buffer solution and washes pillar, until can't detect foreign protein in efflux.It is eluted, is collected with the citrate buffer solution of pH 3.0
Efflux, and use 1moL/L pH 8.5TRIS-HCL buffer solutions to neutralize immediately, with pH 7.2,0.01M PBS dialysis 72h.Sampling
OD260, OD280 are surveyed on ultraviolet specrophotometer, are calculated protein content, are subsequently placed in 4 DEG C of preservations,
Antibody expression detects：
1, preparation of samples：20 μ l antibody-solutions are taken, 5 × sample buffer, 5 μ l are added, 5min is boiled so that albumen in boiling water bath
Denaturation.
2, TGS buffer solutions are added in electrophoresis tank, the protein adhesive made (4%-15%) installation is put well on the top of the shelf
In electrophoresis tank, fresh TGS is added in inside groove to sample-adding hole site is flooded, pulls out comb.
3, point sample successively in sequence, MARKER pre-dyed albumen MARKER (BIO-RAD).Then it is run in 200V
30min is so that protein band is well separated.
4, after running glue, the glue for being SDS cuts away the upper right corner and is put into dyeing liquor, and room temperature dyes 2h on shaking table, then takes off
In color liquid overnight or until glue background color take off to that can see band, take pictures.
5, the glue for being Western cuts away the upper right corner, and the NC films of handle, glue, conductive sheet, filter paper are put on transferring film instrument successively, note
Meaning cuts the corresponding film angle in the glue upper right corner, makes sandwich interlayer bubble-free.Transferring film 30min.Then it removes film and is put into confining liquid
In be placed on shaking table room temperature and close 2h or 4 DEG C overnight.Closing terminates to be rinsed twice with PBST, 5 minutes every time.Adding primary antibody, (AP is marked
The goat anti-human igg H+K of note) it is placed on shaking table and is incubated at room temperature 2h.It is rinsed twice with PBST, 5 minutes every time.Add chromogenic substrate, sets
It develops the color 20 minutes, takes pictures at dark.
The carrier antibody expression quantity that linear expression vector and plasmid gene are calculated by excel calibration curve methods compares figure
(see Fig. 5).
The preparation of 6 full people source anti-hepatitis c virus neutralizing antibody of embodiment
Hepatitis C Virus (HCV) is the known RNA virus that can cause chronic infection only in addition to retrovirus.Control
HCV is replicated and the key factor of immune clearance is to generate wide spectrum neutralizing antibody to be prepared for using the above method provided by the invention
A kind of monoclonal neutralizing antibody of the full people source HCV-Ab IgG of combination HCV Surface env proteins E2, the antibody can be used for prevention third
Liver and can be also used for identification HCV.
Preparation method：
(1) detection finds HCV infection patient；
(2) monocyte in HCV infection Venous Blood is detached, carries out memory B cell using flow cytometry later
Sorting sub-elects the memory B cell containing E2 antibody using the unification of fluorescent marker group and enters in 96 hole PCR plates, makes every Kong Hanyi
A B cell；
(3) antibody light chain and heavy chain variable region in the single B cell of single-cell RT-PCR amplification step (2) acquisition are utilized
Nucleotide fragments；
(4) nucleotide fragments of antibody light chain and heavy chain variable region that step (3) obtains are fused to containing human antibodies
Recombinant expression carrier is formed in the expression vector of constant region, imports host cell expression later；
(5) antibody screening Platform Screening obtains the full people source HCV-Ab IgG with the present invention for combining activity and neutralization activity
Monoclonal neutralizing antibody.
The antibody binding activity test experience of preparation is the results show that carry out the antibody of expression and purification to be more than 2000 times of dilutions
Antibody still can have extremely strong combination activity with antigen binding afterwards.Affine Activity determination the results show that antibody affinity
Up to 2.03*10-8mol.Antibody shows from the neutralization activity testing result of different HCV virus strains, with H77, JFH1, S52,
The neutralization efficiency of ED43, SA13 Strain is 0.5117 μ g/mL, 0.01038 μ g/mL, 0.002745 μ g/mL, 0.08102 respectively
μ g/mL and 0.8507 μ g/mL.
The preparation of 7 full human source anti-rabies virus neutralizing antibody of embodiment
Preparation method：
(1) monocyte in volunteer's venous blood of rabies vaccine has been injected in separation, utilize later flow cytometry into
The single thick liquid cell sorting of row, is unified using thick liquid cell fluorescent marker group, and PBMC sortings obtain single thick liquid cell and enter 96 hole PCR plates
In；
(2) antibody light chain and weight chain variable in the single B cell of single-cell RT-PCR amplification step (1) acquisition are utilized
The nucleotide fragments in area；
(3) nucleotide fragments of antibody light chain and heavy chain variable region that step (2) obtains are fused to containing human antibodies
Recombinant expression carrier is formed in the expression vector of constant region, imports host cell expression later；
(4) antibody screening Platform Screening obtains the full people source rabies with the present invention for combining activity and neutralization activity
The monoclonal neutralizing antibody of poison.
The antibody binding activity test experience of preparation is the results show that carry out the antibody of expression and purification dilute more than 50000 times
Release rear antibody still can have extremely strong combination activity with antigen binding.Antibody and neutralization activity testing result show, antibody
It is about 1700IU/ml to protect potency, illustrates that the recombinant antibodies of expression are the full people with rabies viruses neutralization activity of high-purity
Resource monoclonal antibody.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (7)
1. a kind of preparation method of high throughput human antibody, which is characterized in that the described method comprises the following steps：
(1) separating peripheral blood mononuclear cells：The blood sample or autoimmune disease of communicable disease self-healing patient or
Person's tumor patient generates the blood sample of autoantibody, detaches PBMC；
(2) single B cell is sorted：The single B cell is antigen-specific plasma cell and/or antigen specific memory B lymphs
Cell；
Sort the one kind of the fluorescent marker of antigen-specific plasma cell use in following three combinations：
Group unification：AqVD-Amycan-/CD19-BV605+/CD3/14/16/235a-PE-Cy5-/CD20-PerCP-/IgD-
PE-/CD138-FITC-/CD38-AF647+/CD27-BV421+；
Combination two：AqVD-Amycan-/CD19-PE+/CD3/14/16/235a-PE-Cy5-/CD20-PerCP-/IgD-
BB515-/CD138-PE-CF594-/CD38-AF647+/CD27-BV421+；
Combination three：AqVD-Amycan-/CD19-PerCP+/CD3/14/16/235a-PE-Cy5-/CD20-PE-Cy7-/IgD-
PE-/CD138-FITC-/CD38-BV605+/CD27-BV421+；
Sort the one kind of the fluorescent marker of antigen specific memory bone-marrow-derived lymphocyte use in following three combinations：
Group unification：AqVD-AmCyan-/CD16-PE-Cy7-/CD14-BV570-/CD3-PerCP5.5-/CD20-FITC-/
IgD-PE-/CD27+/specific antigen-AF647+/specific antigen-BV421+；
Combination two：AqVD-AmCyan-/CD3/CD16/CD235a-PE-Cy5-/CD14-FITC-/IgD-PE-/IgM-APC-/
CD20-BV605+/CD27-APC-H7+/specific antigen-PE-Cy7+/specific antigen-BV421+；
Combination three：AqVD-AmCyan-/CD3/CD16/CD235a-PE-Cy5-/CD14-PerCP-/IgD-PE-/IgM-
FITC-/CD20-PE-CF594+/CD27-APC-H7+/specific antigen-AF647+/specific antigen-BV421+；
(3) single B cell gene magnification and analysis：Reverse transcription is carried out to single B cell and synthesizes cDNA, amplification heavy chain of antibody and light
Chain variable region gene；
(4) the linear expression system containing heavy chain of antibody, light chain gene is built：By the method for overlapping PCR by step
(3) isolated heavy chain of antibody, chain variable region gene are connected into respectively in linear carrier expression system；
(5) antibody isolates and purifies：The linear carrier expression system that step (4) obtains is imported host cell to express, is received
Collect the supernatant of expression, antibody purification；
Wherein, step (3) passes through nested PCR amplification heavy chain of antibody and chain variable region gene：First round nested PCR amplification weight
Chain, light chain λ and light chain κ full-length genes, the primer is as shown in table 2, table 3, table 4；Second wheel nest-type PRC expand respectively heavy chain,
Light chain λ and light chain κ variable region genes, the primer table 5, table 6, shown in table 7；One terminal sequence of heavy chain variable region amplified production and portion
Ig guiding region sequences are divided to overlap, another terminal sequence is overlapped with the constant-region sequences of Partial heavy；The variable regions light chain λ amplified production one
Terminal sequence is overlapped with part Ig guiding region sequences, and another terminal sequence is overlapped with the constant-region sequences of part light chain λ；Light chain κ is variable
Amplified production one terminal sequence in area's is overlapped with part Ig guiding region sequences, the constant-region sequences weight of another terminal sequence and part light chain κ
It closes.
2. preparation method according to claim 1, which is characterized in that be with the part Ig sequences for guiding region sequence to overlapCTGGGTTCCAGGTTCCACTGGTGAC；
The sequence overlapped with the constant-region sequences of Partial heavy isGCGGCCCTGGGCTGCCTGGTCAAG；
The sequence overlapped with the constant-region sequences of part light chain λ isAGGAGAGTGTCACAGAGCAGGACAG；
The sequence overlapped with the constant-region sequences of part light chain κ isCTTCAAGCCAACAAGGCCACACTGGTG。
3. preparation method according to claim 1, which is characterized in that the linear expression systemic vectors are by two parts group
At first part is made of promoter and the boot sections Ig, and second part is by heavy chain fragment or light chain segments constant region and BGH
PolyA signal peptide fragments form.
4. preparation method according to claim 3, which is characterized in that the promoter is CMV promoter, the Ig guiding
Region sequence is that sequence is：MKVRGIQRNYPQWWIWSMLGLWMLMICNG.
5. according to Claims 1 to 4 any one of them preparation method, which is characterized in that high throughput prepared by the method is complete
Human antibody is antiviral antibody or immunotherapeutic antibodies.
6. the primer of nested PCR amplification heavy chain of antibody and light chain gene combines, which is characterized in that draw including first round nest-type PRC
Object, as shown in table 2, table 3, table 4 and the second wheel nest-type PRC primer expands heavy chain, light chain λ respectively as shown in Table 5, Table 6, Table 7
With light chain κ variable region genes.
7. the reagent or kit that are combined containing primer described in claim 6.
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| CN111378650A (en) * | 2018-12-28 | 2020-07-07 | 先声生物医药科技有限公司 | Combined primer for nested amplification and application thereof |
| CN111378671A (en) * | 2018-12-28 | 2020-07-07 | 先声生物医药科技有限公司 | Method for high-throughput construction of monoclonal antibody expression vector |
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| CN109097382A (en) * | 2018-08-28 | 2018-12-28 | 东北农业大学 | A method of for screening specific antibody variable region |
| CN110386983B (en) * | 2019-08-12 | 2021-02-05 | 西南医科大学 | Fully human anti-CD 20 recombinant antibody |
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| CN101451134A (en) * | 2007-11-29 | 2009-06-10 | 上海凯勃生物技术有限公司 | Method for amplifying human heavy chain and light chain of antibody from human trace B cells |
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| CN111378650A (en) * | 2018-12-28 | 2020-07-07 | 先声生物医药科技有限公司 | Combined primer for nested amplification and application thereof |
| CN111378671A (en) * | 2018-12-28 | 2020-07-07 | 先声生物医药科技有限公司 | Method for high-throughput construction of monoclonal antibody expression vector |
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