CN103467605A - CD3 antigen and preparation method and application thereof - Google Patents
CD3 antigen and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a CD3 antigen and a preparation method and application thereof. The CD3 antigen comprises an Fc fragment, an extracellular domain CD3E ECD of the epsilon subunit CD3E of the CD3 and an extracellular domain CD3D ECD of the delta subunit CD3D of the CD3 or an extracellular domain CD3G ECD of the gamma subunit CD3G of the CD3, wherein the Fc fragment is formed by an Fc1 fragment and an Fc2 fragment through a disulfide bond; the antigen is a heterodimer formed by two fusion proteins; the C end of the CD3E ECD is connected with the N end of the Fc1 in the first fusion protein so that a fusion protein CD3E ECD-Fc1 is formed; the C end of the CD3D ECD is connected with the N end of the Fc2 in the second fusion protein so that a fusion protein CD3D ECD-Fc2 is formed, or the C end of the CD3G ECD is connected with the N end of the Fc2 in the second fusion protein so that a fusion protein CD3G ECD-Fc2 is formed; the amino acid sequences of the Fc1 fragment and the Fc2 fragment are both obtained by carrying out two or three amino acid point mutation on an amino acid sequence as shown in SEQ ID NO:1. The invention also discloses the preparation method and application of the CD3 antigen.
Description
Technical field
The present invention relates to biological technical field, particularly, relate to a kind of CD3 antigen and its production and use.
Background technology
CD3 is people's immune t-cell surface markers antigen, and six aggressiveness that are comprised of four kinds of subunits (subunit) are centered around φt cell receptor (T cell receptor, TCR) on every side by the electric charge adsorption.Four kinds of subunits of CD3 are respectively CD3 ε (CD3E), CD3 δ (CD3D), and CD3 γ (CD3G), CD3 ζ (CD3Z), be transmembrane protein; And CD3E and CD3D form dimer, CD3E and CD3G and form dimer (heterodimer), and their extracellular region can be used as the identification target site of anti-cd 3 antibodies usually.
The ectodomain that CD3E ECD is CD3E (extra-cellular domain), the ectodomain that CD3D ECD is CD3D, the ectodomain that CD3G ECD is CD3G.
OKT3 is as first business-like anti-cd 3 antibodies, and its target spot is CD3E and the dimeric extracellular region of CD3G.OKT3 not only can, in conjunction with CD3, also have the effect of activated T cell.Studies have found that to only have when CD3E and CD3G have formed dimer and just there is immunogenicity, can be by the OKT3 combination, and independent CD3E or CD3G all can't be by the OKT3 combinations.
UCHT1 is an anti-cd 3 antibodies commonly used, can either identify the dimeric ECD part of CD3E and CD3D, also can identify the dimeric ECD part of CD3E and CD3G, and independent CD3E ECD, CD3D ECD and CD3G ECD all can't be identified by UCHT1.
The preparation method of CD3 antigen has CD3E ECD and CD3G ECD fusion (CD3E-G ECD fusion) at present; CD3E ECD and CD3D ECD are merged to (CD3E-D ECD fusion); By the ECD of CD3E, CD3G and tri-kinds of subunits of CE3D and the Fc segment composition (CD3E/G/D-Fc) of Immunoglobulin IgG1; Perhaps at the C of fusion rotein end, add Flag label (CD3E/G/D-Fc-Flag) or His label (CD3E/G/D-Fc-His) etc.Wherein, CD3E-G ECD fusion rotein and CD3E-D ECD fusion rotein all adopt prokaryotic expression, are present in inclusion body, need to carry out renaturation after purifying, also there is CD3E/G/D ECD to use respectively prokaryotic expression, after purifying, CD3E and CD3G or CD3D equal proportion are mixed and carried out renaturation; Although prokaryotic expression CD3 antigen output is high, has increased the renaturation step, the immunogenic CD3 that has finally obtained only has original 10% even lower.
The CD3E/G/D-Fc recombination fusion protein is at 293 cells, there is immunogenicity, can be identified by OKT3 and UCHT1, but also have CD3E-Fc, CD3D-Fc and the impurity albumen such as the formed homodimer of CD3G-Fc (homodimer) and CD3D-Fc-CD3G-Fc heterodimer in the preparation process of this antigen, therefore form the efficiency of heterodimer generally below 50%.Although the preparation method of this antigen holds the technology of carrying different labels can obtain the antigen that purity is greater than 90% by adopting at different Fc fragment C, but in purge process, need to carry out at least three step affinity chromatographys, lose also often, cause the yield of antigen lower than 50%.
Due to the above-mentioned shortcoming of the middle existence of CD3 antigen and preparation method thereof at present, be necessary to develop the CD3 antigen that a kind of new heterodimer forms the heterodimer prepared by eukaryotic expression system that efficiency is high.
Summary of the invention
For this reason, the present invention proposes a kind of a kind of CD3 antigen that can address the above problem or that at least can partly address the above problem, wherein, described CD3 antigen comprises: the ectodomain CD3E ECD of the epsilon subunit CD3E of Fc fragment, CD3, and the ectodomain CD3G ECD of the γ subunit CD3G of the ectodomain CD3D ECD of the δ subunit CD3D of CD3 or CD3;
Wherein, described Fc fragment is formed by disulfide linkage by Fc1 fragment and Fc2 fragment;
Wherein, described antigen is the heterodimer formed by two fusion roteins, in first fusion rotein, the C end of CD3E ECD is connected with the N end of Fc1, forms fusion rotein CD3E ECD-Fc1, in second fusion rotein, the C end of CD3D ECD is connected with the N end of Fc2, form fusion rotein CD3D ECD-Fc2, or the C of CD3G ECD end is held and is connected, formation fusion rotein CD3G ECD-Fc2 with the N of Fc2;
Wherein, the aminoacid sequence of Fc1 fragment and Fc2 fragment is the aminoacid sequence shown in SEQ ID NO:1 is carried out to 2 or 3 amino acid whose point mutation acquisitions.
The present invention also provides a kind of preparation method of CD3 antigen, and described method comprises:
(1) obtain respectively recombinant vectors e and recombinant vectors d, or obtain respectively recombinant vectors e and recombinant vectors g;
(2) to described recombinant vectors e and d, or recombinant vectors e and g carry out point mutation, obtains sudden change recombinant vectors em and dm, or obtain sudden change recombinant vectors em and gm;
(3) transfection em and dm in eukaryotic cell; Perhaps transfection em and gm;
(4) utilize affinity chromatography method purifying to obtain CD3 antigens c D3E/CD3D-Fc1/Fc2 or CD3E/CD3G-Fc1/Fc2;
Wherein, the coding nucleotide sequence that described recombinant vectors e contains fusion rotein CD3E ECD-Fcw; The coding nucleotide sequence that described recombinant vectors d contains fusion rotein CD3D ECD-Fcw; The coding nucleotide sequence that described recombinant vectors g contains fusion rotein CD3G ECD-Fcw;
Wherein, Fcw is the coded polypeptide fragment of aminoacid sequence shown in SEQ ID NO:1.
In addition, the present invention also provides the purposes of CD3 antigen of the present invention in the screening of anti-cd 3 antibodies.
By technique scheme, CD3 antigen of the present invention has following advantage: the efficiency that (1) forms heterodimer is high; (2) can with the antibody generation specific combination of anti-CD3, there is very strong avidity; (3) by proteinA mono-step affinity chromatography, can obtain the albumen that purity is 95%, the yield of albumen is not less than the ratio of total content in ultimate capacity that 80%(purifies out from express supernatant and former supernatant); (4) adopt eukaryotic expression system, do not need to carry out the renaturation of protein.
The prepared CD3 antigen of the present invention can be for screening and the analysis of anti-cd 3 antibodies, and as the raw material of correlation detection test kit.
Other features and advantages of the present invention will partly be described in detail in embodiment subsequently.
The accompanying drawing explanation
By reading hereinafter detailed description of the preferred embodiment, various other advantage and benefits will become cheer and bright for those of ordinary skills.Accompanying drawing is only for the purpose of preferred implementation is shown, and do not think limitation of the present invention.And, in whole accompanying drawing, by identical reference symbol, mean identical parts.In the accompanying drawings:
The structural representation that Fig. 1 is CD3 antigen provided by the present invention
The plasmid map that Fig. 2 is recombinant vectors pcDNA3.1 (+) Hygro-CD3E-Fcw
The plasmid map that Fig. 3 is recombinant vectors pcDNA3.1 (+) Hygro-CD3G-Fcw
The plasmid map that Fig. 4 is recombinant vectors pcDNA3.1 (+) Hygro-CD3D-Fcw
The as a result figure of Fig. 5 for by Western Blot method, antigen provided by the present invention being detected
Fig. 6 detects the figure as a result of the purification effect of CD3 antigen of the present invention with 6%SDS-PAGE
Fig. 7 is that the CD3E/CD3D dimer SDS-PAGE after the prokaryotic expression Purification examines and dyes figure
Fig. 8 is for to detect by high performance liquid chromatography the efficiency that CD3 antigen preparation method provided by the present invention forms heterodimer
Fig. 9 for Dot blot analyzing and testing antigen 1 and 2 and antibody in conjunction with active figure as a result
Figure 10 for the ELISA method detect antigen provided by the present invention and OKT3 and UCHT1 antibody in conjunction with active figure as a result
Embodiment
The invention provides many applicable creative concepts, this creativeness concept can be reflected in a large number of in concrete context.The specific embodiment of describing in following embodiments of the present invention is only as the exemplary illustration of specific implementation of the present invention, and do not form limitation of the scope of the invention.
The invention provides a kind of CD3 antigen, wherein, described CD3 antigen comprises: the ectodomain CD3E ECD of the epsilon subunit CD3E of Fc fragment, CD3, and the ectodomain CD3G ECD of the γ subunit CD3G of the ectodomain CD3D ECD of the δ subunit CD3D of CD3 or CD3;
Wherein, described Fc fragment is formed by disulfide linkage by Fc1 fragment and Fc2 fragment;
Wherein, described antigen is the heterodimer formed by two fusion roteins, in first fusion rotein, the C end of CD3E ECD is connected with the N end of Fc1, forms fusion rotein CD3E ECD-Fc1, in second fusion rotein, the C end of CD3D ECD is connected with the N end of Fc2, form fusion rotein CD3D ECD-Fc2, or the C of CD3G ECD end is held and is connected, formation fusion rotein CD3G ECD-Fc2 with the N of Fc2;
Wherein, the aminoacid sequence of Fc1 fragment and Fc2 fragment is the aminoacid sequence shown in SEQ ID NO:1 is carried out to 2 or 3 amino acid whose point mutation acquisitions.
In the present invention, the coded polypeptide chain of aminoacid sequence shown in SEQ ID NO:1 is an Immunoglobulin IgG1 hinge area, Immunoglobulin IgG1 second a constant region CH2 and Immunoglobulin IgG1 the 3rd a constant region CH3 coding, wherein, the encoding sequence of hinge area, CH2 and CH3 holds the order of C end to be arranged in order according to the N of the aminoacid sequence from shown in SEQ ID NO:1.
In CD3 antigen provided by the present invention, the C of the peptide chain of CD3E ECD end is connected by forming peptide bond with the N end of the peptide chain of Fc1, forms fusion rotein CD3E ECD-Fc1; The C end of the peptide chain of CD3D ECD is connected by forming peptide bond with the N end of the peptide chain of Fc2, forms fusion rotein CD3D ECD-Fc2; The C end of the peptide chain of CD3G ECD is connected by forming peptide bond with the N end of the peptide chain of Fc2, forms fusion rotein CD3G ECD-Fc2;
In one embodiment of the invention, fusion rotein CD3E ECD-Fc1 and fusion rotein CD3D ECD-Fc2 are connected and form heterodimer CD3E/CD3D-Fc1/Fc2 with disulfide linkage between Fc2 by Fc1.
In one embodiment of the invention, fusion rotein CD3E ECD-Fc1 and fusion rotein CD3G ECD-Fc2 are connected and form heterodimer CD3E/CD3G-Fc1/Fc2 with disulfide linkage between Fc2 by Fc1.
In CD3 antigen provided by the present invention, the site that the aminoacid sequence shown in SEQ ID NO:1 is carried out to 2 or 3 amino acid whose point mutation be arranged in the aminoacid sequence shown in SEQ ID NO:1 the CH3 coding region.
In CD3 antigen provided by the present invention, the mutational site that the aminoacid sequence shown in SEQ ID NO:1 is carried out to 2 or 3 amino acid whose point mutation is selected from and the T of the 139th of aminoacid sequence shown in SEQ ID NO:1 is mutated into to W, the K of the 165th is mutated into D, the K of the 182nd and is mutated into D, the Y of the 180th and is mutated into the D that A, the D of the 129th be mutated into K and the 172nd and is mutated into 2 kinds or 3 kinds in K.
Wherein, T is Threonine, and W is that tryptophane, K are that Methionin, D are that aspartic acid, Y are tyrosine, and A is L-Ala.
In CD3 antigen provided by the present invention, the mode of under preferable case, the aminoacid sequence shown in SEQ ID NO:1 being carried out to 2 or 3 amino acid whose point mutation can be selected from a kind of in following sudden change mode:
The K that the T of the 139th is mutated into W and the 165th is mutated into D,
The K that the T of the 139th is mutated into W and the 182nd is mutated into D,
The D that the T of the 139th is mutated into W and the 129th is mutated into K,
The D that the T of the 139th is mutated into W and the 172nd is mutated into K,
The K that the Y of the 180th is mutated into A and the 165th is mutated into D,
The K that the Y of the 180th is mutated into A and the 182nd is mutated into D,
The D that the Y of the 180th is mutated into A and the 129th is mutated into K,
The D that the Y of the 180th is mutated into A and the 172nd is mutated into K,
The D that the D that the T of the 139th is mutated into W and the 129th is mutated into K and the 172nd is mutated into K,
The K that the K that the T of the 139th is mutated into W and the 165th is mutated into D and the 182nd is mutated into D,
The D that the D that the Y of the 180th is mutated into A and the 129th is mutated into K and the 172nd is mutated into K,
The K that the K that the Y of the 180th is mutated into A and the 165th is mutated into D and the 182nd is mutated into D.
In more preferred situation, when the aminoacid sequence of in Fc1 and Fc2 is mutated into for the D that the Y of the 180th of the aminoacid sequence shown in SEQ ID NO:1 is mutated into to A and the 129th sequence that K obtains, another aminoacid sequence is mutated into for the K that the T of the 139th of the aminoacid sequence shown in SEQ ID NO:1 is mutated into to W and the 165th sequence that D obtains;
When the aminoacid sequence of in Fc1 and Fc2 is mutated into for the T that the D of the 129th of the aminoacid sequence shown in SEQ ID NO:1 is mutated into to K and the 139th sequence that W obtains, another aminoacid sequence is mutated into for the K that the Y of the 180th of the aminoacid sequence shown in SEQ ID NO:1 is mutated into to A and the 165th sequence that D obtains;
When the aminoacid sequence of in Fc1 and Fc2 is mutated into for the D that the Y of the 180th of the aminoacid sequence shown in SEQ ID NO:1 is mutated into to A and the 172nd sequence that K obtains, another aminoacid sequence is mutated into for the K that the T of the 139th of the aminoacid sequence shown in SEQ ID NO:1 is mutated into to W and the 182nd sequence that D obtains, or the K that is mutated into D and the 182nd for the T of the 139th of the aminoacid sequence shown in SEQ ID NO:1 being mutated into to W, the K of the 165th is mutated into the sequence that D obtains;
When the aminoacid sequence of in Fc1 and Fc2 is mutated into for the D that the D of the 129th of the aminoacid sequence shown in SEQ ID NO:1 is mutated into to K and the 172nd sequence that K obtains, another aminoacid sequence is mutated into for the K that the K of the 182nd of the aminoacid sequence shown in SEQ ID NO:1 is mutated into to D and the 165th sequence that D obtains;
When the aminoacid sequence of in Fc1 and Fc2 is mutated into for the K of the 182nd of the aminoacid sequence shown in SEQ ID NO:1 being mutated into to D, the Y of the 180th sequence that A obtains, another aminoacid sequence is mutated into for the T that the D of the 172nd of the aminoacid sequence shown in SEQ ID NO:1 is mutated into to K and the 139th sequence that W obtains;
When the aminoacid sequence of in Fc1 and Fc2 is mutated into K and the 172nd D for the Y of the 180th of the aminoacid sequence shown in SEQ ID NO:1 being mutated into to A, the D of the 129th sports the sequence that K obtains, the K that another aminoacid sequence is mutated into D and the 182nd for the T of the 139th of the aminoacid sequence shown in SEQ ID NO:1 being mutated into to W, the K of the 165th sports the sequence that D obtains;
When the aminoacid sequence of in Fc1 and Fc2 is mutated into D and the 180th Y for the K of the 165th of the aminoacid sequence shown in SEQ ID NO:1 being mutated into to D, the K of the 182nd sports the sequence that A obtains, the T that another aminoacid sequence is mutated into K and the 139th for the D of the 129th of the aminoacid sequence shown in SEQ ID NO:1 being mutated into to K, the D of the 172nd sports the sequence that W obtains, or is mutated into the sequence that W obtains for the T that the D of the 172nd of the aminoacid sequence shown in SEQ ID NO:1 is mutated into to K and the 139th.
In CD3 antigen provided by the present invention, the aminoacid sequence of described CD3E ECD is as shown in SEQ ID NO:14, and the aminoacid sequence of described CD3D ECD is as shown in SEQ ID NO:15, and the aminoacid sequence of described CD3G ECD is as shown in SEQ ID NO:16.
CD3 antigen provided by the present invention can be combined with the antibodies specific of anti-CD3.Preferably, CD3 antigen provided by the present invention can with antibody OKT3, UCHT1, TRX4 and the L2K of anti-CD3 at least one specific binding.
The present invention also provides a kind of preparation method of CD3 antigen, and described method comprises:
(1) obtain respectively recombinant vectors e and recombinant vectors d, or obtain respectively recombinant vectors e and recombinant vectors g;
(2) to described recombinant vectors e and d, or recombinant vectors e and g carry out point mutation, obtains sudden change recombinant vectors em and dm, or obtain sudden change recombinant vectors em and gm;
(3) transfection em and dm in eukaryotic cell; Perhaps transfection em and gm;
(4) utilize affinity chromatography method purifying to obtain CD3 antigens c D3E/CD3D-Fc1/Fc2 or CD3E/CD3G-Fc1/Fc2;
Wherein, the coding nucleotide sequence that described recombinant vectors e contains fusion rotein CD3E ECD-Fcw; The coding nucleotide sequence that described recombinant vectors d contains fusion rotein CD3D ECD-Fcw; The coding nucleotide sequence that described recombinant vectors g contains fusion rotein CD3G ECD-Fcw;
Wherein, Fcw is the coded polypeptide fragment of aminoacid sequence shown in SEQ ID NO:1.
In method provided by the present invention, the method for preparing recombinant vectors e, d and g is had no particular limits, can adopt the preparation method of this area recombinant vectors commonly used to be built, for example can build recombinant vectors according to the method for introducing in " the molecular cloning experiment guide third edition ".
In method provided by the present invention, described recombinant vectors e can obtain by the coding nucleotide sequence of fusion rotein CD3E ECD-Fcw is imported to expression vector; Described recombinant vectors d can obtain by the coding nucleotide sequence of fusion rotein CD3D ECD-Fcw is imported to expression vector; Described recombinant vectors g can obtain by the coding nucleotide sequence of fusion rotein CD3G ECD-Fcw is imported to expression vector.
In the present invention, for the kind of expression vector, having no particular limits, can be this area carrier for expression of eukaryon commonly used, and for example, described expression vector can be pcDNA3.1 (+) Hygro, PSV2, CMV4 or pCMVp-NEO-BAN etc.; Preferably, described expression vector is pcDNA3.1 (+) Hygro carrier for expression of eukaryon.
Concrete, the construction process of described recombinant vectors can be, select suitable expression vector, and according to the coding nucleotide sequence of CD3D ECD, the coding nucleotide sequence of CD3G ECD, multiple clone site design primer in the coding nucleotide sequence of CD3G ECD and the coding nucleotide sequence of Fcw and expression vector, by PCR method, increased, then, utilize overlapping extension PCR method that the coding nucleotide sequence of CD3E ECD is connected with the coding nucleotide sequence of Fcw, and the coding nucleotide sequence of CD3D ECD is connected with the coding nucleotide sequence of Fcw or the coding nucleotide sequence of CD3G ECD is connected with the coding nucleotide sequence of Fcw.Purifying reclaims the product of overlapping extension PCR, utilizes the double digestion method that the product of overlapping PCR is inserted respectively to the multiple clone site in expression vector, obtains recombinant vectors e and d, or obtains recombinant vectors e and g.
In the present invention, PCR reacts the PCR reaction reagent that applied reagent can be this area routine use.
In method provided by the present invention, overlapping extension PCR method connects the condition of coding nucleotide sequence of each fragment and condition and the reagent of the overlapping extension PCR method that reagent can utilize this area routine carries out.
In the present invention, can utilize this area method commonly used for example DNA fragmentation reclaim test kit the PCR reaction product carried out to the purifying recovery.
In the preparation method of the CD3 antigen provided in this law invention, the method for recombinant vectors being carried out to point mutation has no particular limits, and can carry out according to the rite-directed mutagenesis method of this area routine, for example utilizes commercial test kit to carry out.
In the preparation method of CD3 antigen provided by the present invention, can utilize following primer pair to introduce the aminoacid sequence of encoded point sudden change in recombinant vectors e, d or g:
Primer pair SEQ ID NO:2 and SEQ ID NO:3 are for the base sequence at recombinant vectors e, d or g introducing encoded point sudden change D129K;
Primer pair SEQ ID NO:4 and SEQ ID NO:5 are for the base sequence at recombinant vectors e, d or g introducing encoded point mutation T 139W;
Primer pair SEQ ID NO:6 and SEQ ID NO:7 are for the base sequence at recombinant vectors e, d or g introducing encoded point sudden change K165D;
Primer pair SEQ ID NO:8 and SEQ ID NO:9 are for the base sequence at recombinant vectors e, d or g introducing encoded point sudden change D172K;
Primer pair SEQ ID NO:10 and SEQ ID NO:11 are for the base sequence at recombinant vectors e, d or g introducing encoded point sudden change Y180A;
Primer pair SEQ ID NO:12 and SEQ ID NO:13 are for the base sequence at recombinant vectors e, d or g introducing encoded point sudden change K182D.
In method provided by the present invention, the method that the plasmid of the recombinant vectors that contains mutational site is increased has no particular limits, and can be undertaken by the described recombinant vectors that contains mutational site is transformed to intestinal bacteria.
In the present invention, the transfection of the recombinant vectors that contains mutational site and expression can be carried out according to the method for this area routine, being preferably mammalian cell for the cell of expressing the described recombinant vectors that contains mutational site, can be for example HEK293,293E, 293F, COS7, CHO-S cell; More preferred, be the 293F cell.
In one embodiment of the invention, em and gm expressed fusion protein CD3E ECD-Fc1 and CD3G ECD-Fc2 respectively in host cell will suddenly change after recombinant vectors em and gm transfecting eukaryotic cells, different dimerization occurs in CD3E ECD-Fc1 and CD3G ECD-Fc2 in host cell, forms CD3 antigens c D3E/CD3G-Fc1/Fc2.
In one embodiment of the invention, em and dm expressed fusion protein CD3E ECD-Fc1 and CD3D ECD-Fc2 respectively in host cell will suddenly change after recombinant vectors em and dm transfecting eukaryotic cells, different dimerization occurs in CD3E ECD-Fc1 and CD3D ECD-Fc2 in host cell, forms CD3 antigens c D3E/CD3D-Fc1/Fc2.
In method provided by the present invention, method for antigen purification can be carried out according to following steps: by eccentric cell nutrient solution collecting cell nutrient solution supernatant and remove cell debris with membrane filtration, with after binding buffer liquid dilution supernatant liquor, the supernatant liquor after dilution being crossed to affinity column and obtained CD3 antigen with the elution buffer wash-out.In the present invention, can utilize SDS-PAGE to be detected the purity of the antigen of acquisition.
The present invention also provides the purposes of CD3 antigen in the screening of anti-cd 3 antibodies.
CD3 antigen provided by the present invention can be applied to the screening of anti-cd 3 antibodies, the humanization of mouse source CD3 antibody, the mensuration of CD3 affinity of antibody, the mensuration of CD3 antibody expression amount etc., CD3 antigen provided by the present invention also can be used as the raw material of correlation detection test kit.
Below will describe the present invention by embodiment.In following examples:
Then the DNA fragmentation of CD3E ECD gene, CD3G ECD gene and CD3D ECD gene utilizes reverse transcription PCR to obtain for extract mRNA from Jurkat cell (purchased from ATCC);
The gene of Fcw fragment (nucleic acid sequence encoding of the aminoacid sequence shown in SEQ ID NO:1), for to extract mRNA from human PBMC's cell, then utilizes reverse transcription PCR to obtain;
Intestinal bacteria TOP10, carrier for expression of eukaryon pcDNA3.1 (+) Hygro is purchased from Invitrogen company.
Affinity chromatography column packing rProtein A Sepharose HP is purchased from GE company;
The T4DNA ligase enzyme, Pyrobest archaeal dna polymerase, restriction enzyme are purchased from Takara company;
Plasmid extraction kit and DNA fragmentation reclaim test kit purchased from TianGen company;
Rite-directed mutagenesis test kit Quickchange Site-Directed Mutagenesis Kit is purchased from Aglient company;
Mammalian cell strain 293F, substratum 293freesytle and transfection reagent 293fectin are purchased from Invitrogen company;
The goat anti-human igg of horseradish peroxidase-labeled and sheep anti-mouse igg are all purchased from Sigma company;
OKT3 antibody is purchased from Thermo scientific pierce, and the trade mark is MA1-10176;
UCHT1 antibody is purchased from Merck, and the trade mark is 217570;
In following examples, the combination of CD3 antigen and antibody is measured by Dot blot analytical procedure.
In following examples, CD3 antigen and OKT3 and UCHT1 antibody utilizes the elisa assay method to be measured in conjunction with activity.
In following examples, the purity of CD3 antigen is detected by high performance liquid chromatography.
In following examples, the method for calculation of the yield of CD3 antigen are the sample that utilizes the rproteinA single step purification to obtain, difference according to molecular weight, carry out SDS-PAGE, SDS-PAGE is examined to the picture of taking pictures that dyes figure and carry out that swimming lane is chosen, band is chosen and gray analysis, obtain the ratio of each band at this swimming lane, thereby calculate the yield of CD3 antigen.
In following examples, avidity refers on antibody molecule between an antigen-combining site and corresponding antigenic determinant and adapts and the intensity of combination, testing method is according to document (cell and molecular immunology magazine, 2005,21(5)), the measuring method of the anti-PH2SA monoclonal antibody relative affinity of record carries out.
Preparation example 1
Preparation example 1 is for illustrating the preparation method of CD3 antigens c D3E/CD3G-Fc1/Fc2 provided by the present invention.
1, the structure of expression vector
Select pcDNA3.1 (+) Hygro as expression vector, according to the gene of CD3E ECD, CD3G ECD and Fcw and the multiple clone site design of amplification primers in pcDNA3.1 (+) Hygro carrier, the DNA fragmentation of CD3E ECD gene, CD3G ECD gene and Fcw gene of take is template, by the DNA fragmentation of pcr amplification CD3E ECD gene, CD3G ECD gene and Fcw gene, specific as follows respectively:
(1), primer:
Primer SEQ ID NO:17 and SEQ ID NO:18 are for the nucleic acid sequence encoding of the CD3E ECD that increases;
Primer SEQ ID NO:19 and SEQ ID NO:20 are for the nucleic acid sequence encoding of the CD3G ECD that increases;
Primer SEQ ID NO:21 and SEQ ID NO:22 are for the nucleic acid sequence encoding of the Fcw that increases.
(2), reaction system
Amount to: 25 μ L
(3), loop parameter condition:
95℃5min；
95 ℃ of 30sec, 56 ℃ of 30sec, 72 ℃ of 1min, totally 25 circulations;
72℃10min；
Purify and reclaim the PCR product with DNA fragmentation recovery test kit, obtain the DNA fragmentation of CD3E ECD gene, CD3G ECD gene and Fcw gene.
Adopt respectively overlapping extension PCR method to connect CD3E ECD fragment and Fcw fragment, CD3G ECD fragment and Fcw fragment.
Take the CD3E ECD of equivalent and Fcw as template primer each other, by following parameter circulation 2 times: 95 ℃ of sex change 2min, 55 ℃ of annealing 2min, 72 ℃ are extended 2min; Then add CD3E ECD upstream primer and Fcw downstream primer in system, by following condition, carry out the PCR reaction, connect CD3E ECD and Fcw fragment:
95℃5min；
95 ℃ of 1min, 56 ℃ of 1min, 72 ℃ of 2min, totally 25 circulations;
72℃10min；
Take the CD3G ECD of equivalent and Fcw as template primer each other, by following parameter circulation 2 times: 95 ℃ of sex change 2min, 55 ℃ of annealing 2min, 72 ℃ are extended 2min; Then add CD3G ECD upstream extremity primer and Fcw downstream primer in system;
Carry out the PCR reaction by following condition, connect CD3G ECD and Fcw fragment:
95℃5min；
95 ℃ of 1min, 56 ℃ of 1min, 72 ℃ of 2min, totally 25 circulations;
72℃10min；
Purify and reclaim overlapping extension PCR product with DNA fragmentation recovery test kit, with NheI enzyme and XhoI enzyme, overlapping extension PCR product is carried out to double digestion and by the CD3E ECD-Fcw fragment and the CD3G ECD-Fcw fragment that obtain after affinity chromatography method (affinity chromatography column packing rProtein A Sepharose HP is purchased from GE company) purifying, CD3E ECD-Fcw fragment and CD3G ECD-Fcw fragment are inserted respectively to multiple clone site acquisition collection of illustrative plates in pcDNA3.1 (+) Hygro carrier recombinant vectors pcDNA3.1 (+) Hygro-CD3E-Fcw and pcDNA3.1 (+) Hygr-CD3G-Fcw as shown in Figures 2 and 3.
2, the point mutation of recombinant vectors
Use rite-directed mutagenesis test kit Quickchange Site-Directed Mutagenesis Kit(purchased from stratagene company) expression vector pcDNA3.1 (+) Hygro-CD3E-Fcw and pcDNA3.1 (+) Hygro-CD3G-Fcw are carried out to point mutation.
In pcDNA3.1 (+) Hygro-CD3E-Fcw, introduce the base sequence of encoded point mutation T 139W with primer pair SEQ ID NO:4 and SEQ ID NO:5; Introduce the base sequence of encoded point sudden change D129K with primer pair SEQ ID NO:2 and SEQ ID NO:3, obtain recombinant vectors pcDNA3.1 (+) Hygro-CD3E-Fc1KW.
In pcDNA3.1 (+) Hygro-CD3G-Fcw, introduce the base sequence of encoded point sudden change Y180A with primer pair SEQ ID NO:10 and SEQ ID NO:11; Introduce the base sequence of encoded point sudden change K165D with primer pair SEQ ID NO:6 and SEQ ID NO:7, obtain recombinant vectors pcDNA3.1 (+) Hygro-CD3G-Fc2DA.
3, the plasmid amplification of recombinant vectors
Recombinant vectors pcDNA3.1 (+) Hygro-CD3E-Fc1KW and recombinant vectors pcDNA3.1 (+) Hygro-CD3G-Fc2DA are transformed into respectively in intestinal bacteria TOP10, be coated with dull and stereotyped also picking list colony inoculation in containing the LB substratum of 100mg/L penbritin, 37 ℃ of shaking culture 16 hours, the centrifugal 10min of 8000 * g collects thalline.Use Tiangen to go the large extraction reagent kit of intracellular toxin to extract plasmid.Plasmid is dissolved in 1ml elution buffer EB, add again 1.42ml Virahol and 0.42ml NaCl will precipitate plasmid, supernatant discarded, add twice of 70% ethanol 0.5ml washing precipitation, air-dry in super clean bench after supernatant discarded, measure the absorbancy under OD260/280 by microplate reader after adding the ultrapure water 1ml of sterilizing.The concentration of pcDNA3.1 (+) Hygro-CD3E-Fc1KW is 950 ug/ml, and OD260/280 is 1.8; The concentration of pcDNA3.1 (+) Hygro-CD3G-Fc2DA is 923 ug/ml, and OD260/280 is 1.8.
4, transfection, expression and the detection of antigen in mammalian cell 293F
First 24 hours of transfection inoculates 4 * 10 in the 125ml Erlenmeyer flask
7individual 293F cell, used 293freestyle substratum 45ml at 5%CO
237 ℃ of incubators in cultivate.
The 293fectin that first gets 50 microlitres during transfection joins in the OPtiMEM of 2.5ml, after fully mixing, and incubated at room 5 minutes; By recombinant vectors pcDNA3.1 (+) Hygro-CD3E-Fc1KW and each 12.5 micrograms of pcDNA3.1 (+) Hygro-CD3G-Fc2DA, the amount of total DNA is 25 micrograms, is dissolved in the OPtiMEM of 2.5ml simultaneously.Then DNA and transfection reagent 293fectin suspension are fully mixed, cumulative volume is 5ml, and incubated at room 20 minutes, then all add mixture in Erlenmeyer flask and mix, at 5%CO
237 ℃ of incubators in the 130rpm shaking table cultivate 5 days.
The collecting cell suspension, the centrifugal 5min of 12000g collects supernatant, and supernatant is carried out to Western Blot detection, and detected result is shown in Fig. 5, and detecting is that the goat anti-human igg is with the HRP mark with antibody.
5, the purifying of CD3E/CD3D-Fc antigen.
With the centrifugal transfection of 2000g the nutrient solution of cell of recombinant vectors, collect supernatant and, with the membrane filtration of 0.22 micron, with the binding buffer liquid of 10 times of volumes, dilute (9.5mM NaH
2pO
4+ 40.5mM Na
2hPO
4, pH7.0), cross rProteinA Sepharose Fast Flow affinity column, 100mM glycine, pH2.5 elution buffer wash-out.Elute protein is crossed the YM-30kD ultra-filtration membrane and is replaced by PBS damping fluid acquisition antigen 1, with 6%SDS-PAGE, detects protein purification, the results are shown in Figure 6.The yield of albumen and purity are in Table 1.
Preparation example 2
Adopt the method identical with preparation example 1 to prepare CD3 antigen, different, with the nucleic acid sequence encoding of primer SEQ ID NO:23 and SEQ ID NO:24 amplification CD3D ECD; Obtain collection of illustrative plates recombinant vectors pcDNA3.1 (+) Hygro-CD3D-Fcw as shown in Figure 4, introduce the base sequence of encoded point sudden change Y180A with primer pair SEQ ID NO:10 and SEQ ID NO:11; Introduce the base sequence of encoded point sudden change K165D with primer pair SEQ ID NO:6 and SEQ ID NO:7, obtain recombinant vectors pcDNA3.1 (+) Hygro-CD3D-Fc2DA and by itself and recombinant vectors pcDNA3.1 (+) Hygro-CD3E-Fc1KKW transfection mammalian cell 293F, obtain antigen 2, with western blot, antigen 2 is detected, be the results are shown in Figure 5.Detect protein purification with 6%SDS-PAGE, purifying the results are shown in Figure 6.The yield of albumen and purity are in Table 1.
In Fig. 5: M is protein marker, the 1st swimming lane be pcDNA3.1 (+) Hygro-CD3D-Fc2DDA and with the 293F cell of recombinant vectors pcDNA3.1 (+) Hygro-CD3E-Fc1KKW cotransfection; The 293F cell that the 2nd swimming lane is pcDNA3.1 (+) Hygro-CD3E-Fc1KW and pcDNA3.1 (+) Hygro-CD3G-Fc2DA cotransfection; The 293F cell that the 3rd swimming lane is the transfection of pcDNA3.1 (+) Hygro-CD3E-Fc1KW simple substance grain; The 293F cell that the 4th swimming lane is the transfection of pcDNA3.1 (+) Hygro-CD3D-Fc2DA simple substance grain; The 293F cell that the 5th swimming lane is the transfection of pcDNA3.1 (+) Hygro-CD3G-Fc2DA simple substance grain; The 293F cell blank contrast that the 6th swimming lane is any plasmid of untransfected, the 293F cell that the 7th swimming lane is pcDNA3.1 (+) Hygro-CD3E-Fc1KW and pcDNA3.1 (+) Hygro-CD3D-Fc2DA cotransfection; The 293F cell that the 8th swimming lane is pcDNA3.1 (+) Hygro-CD3E-Fc1KW and pcDNA3.1 (+) Hygro-CD3G-Fc2DA cotransfection; Wherein, the 1-5 swimming lane is non-reduced SDS-PAGE; The 7-8 swimming lane is SDS-PAGE.
In Fig. 6: the supernatant that swimming lane 1 is CD3E/CD3D-Fc; The supernatant that swimming lane 2 is CD3E/CD3G-Fc; Swimming lane 3 is the CD3E/CD3D-Fc after purifying; Swimming lane 4 is the CD3E/CD3G-Fc after purifying.All samples is non-reduced SDS-PAGE.
Preparation example 3-12
Preparation example 3 is for illustrating the preparation method of CD3 antigen provided by the present invention.
Adopt the method identical with embodiment 1 to prepare CD3 antigen 3-12, different is, mutational site in the structure of CD3 antigen and Fc1 and Fc2 fragment is as shown in table 1, and, in antigen 3,5-8, what with the Fc2 fragment, be connected is CD3G ECD fragment, in antigen 4,9-12, what with the Fc2 fragment, be connected is CD3D ECD fragment, and purity and the yield of the CD3 antigen antigen of acquisition the results are shown in Table 1.
In preparation example 3-12, utilize following primer pair Fcw to carry out rite-directed mutagenesis:
Primer pair SEQ ID NO:2 and SEQ ID NO:3 are for introducing the base sequence of encoded point sudden change D129K at recombinant vectors; Primer pair SEQ ID NO:4 and SEQ ID NO:5 are for introducing the base sequence of encoded point mutation T 139W at recombinant vectors; Primer pair SEQ ID NO:6 and SEQ ID NO:7 are for introducing the base sequence of encoded point sudden change K165D at recombinant vectors; Primer pair SEQ ID NO:8 and SEQ ID NO:9 are for introducing the base sequence of encoded point sudden change D172K at recombinant vectors; Primer pair SEQ ID NO:10 and SEQ ID NO:11 are for introducing the base sequence of encoded point sudden change Y180A at recombinant vectors; Primer pair SEQ ID NO:12 and SEQ ID NO:13 are for introducing the base sequence of encoded point sudden change K182D at recombinant vectors.
Table 1
Comparative Examples 1
This Comparative Examples is for illustrating the preparation method of existing CD3 antigen
Prepare CD3 antigen according to the method for putting down in writing in (PNAS, 2004, Crystal Structure Of Human Cd3-ED dimer in complex with A Ucht1Single-Chain Antibody Fragment).
1, protein expression
Respectively CD3E ECD and CD3D ECD are building up in prokaryotic expression carrier pET28a, and be transformed into respectively cell BL21(DE3), each (is cultured to logarithmic phase in (20g/liter tryptone_10g/liter yeast extract_5g/liter NaCl_2%glycerol_50mM K2HPO4_10mM MgCl2_1%glucose_100mg/liter ampicillin), then adds final concentration 1mM IPTG abduction delivering 2-4 hour at 1 liter of rich substratum.The centrifugal 30min of 10000X g, abandon supernatant, cell precipitation is resuspended in containing 50mM Tris-HCl, 25%sucrose, 1mM EDTA, 0.1% sodium azide, in the damping fluid of 10mM DTT and pH8.0, adds Lysozyme(1mg/ml simultaneously), DNase I(25 μ g/ml) and MgCl
2(5mM).Cell lysis buffer solution is containing 50mM Tris-HCl, 1%(v/v) TritonX-100,1%(w/v) Sodium desoxycholate, 100mM NaCl, 0.1% sodium azide, 10mM DTT and pH8.0, add 2.5 milliliters of lysates in every ml cells suspension during use.After the solution viscosity reduces, add 10mM EDTA, the frozen-thawed cell suspension is once.Add final concentration 10mM MgCl
2with 10mM EDTA, high speed centrifugation is abandoned supernatant.Cell precipitation is resuspended in containing 50mM Tris-HCl, 0.5%TritonX-100,100mM NaCl, 1mM EDTA, 0.1% sodium azide, and in the damping fluid of 1mM DTT and pH8.0, then the centrifugal supernatant of abandoning, repeat four times; The inclusion body finally obtained (inclusion bodies) is resuspended in containing 50mM Tris-HCl, 1mM EDTA, 0.1% sodium azide, in the damping fluid of 1mM DTT and pH8.0, centrifugal, abandon supernatant, precipitation is dissolved in 10 milliliters containing 25mM2-(N-morpholino) ethanesulfonic acid, 8M urea, 10mM EDTA, in the damping fluid of 0.1mM DTT and pH6.0.The 140000Xg ultra-high speed is centrifugal, gets supernatant, obtains CD3E and the CD3D of sex change, by the BCA method, surveys protein concentration.CD3E concentration is 9.8mg/ml, and CD3D concentration is 9.6mg/ml.
2, protein renaturation
CD3E and CD3D respectively get 20mg, are added to fast the ice-cold renaturation buffer of 100ml (1M L-Arg, 100mM Tris-pH8.3,2mM EDTA, 3.6mM cystamine, 6.7mM cysteamine) after mixing, hatch 48h.The recombinant protein mixed solution is inserted to dialysis twice in the dialyzate (10mM Tris, pH8.0) that is not less than 10 times of volumes, each 24h.Use DEAE anion-exchange chromatography post (GE company) and SEC post to carry out protein purification.CD3E/CD3D antigen after purifying concentrates and changes to the 1XPBS damping fluid, and it is 5mg/ml that the BCA method is surveyed concentration, and output is 10mg.The yield of albumen is 25 % by weight, and purity is examined and dyed figure (see Fig. 7, the CD3E/CD3D dimer SDS-PAGE after the prokaryotic expression Purification examines and dyes figure according to SDS-PAGE.Marker is protein labeling, and sample is the CD3E/CD3D dimer after Purification.) analyze, the dimer ratio is higher than 50%, contains in addition 30% high polymer and 20% not polymeric protein.
This test case is for illustrating the efficiency of the heterodimer that CD3 antigen preparation method provided by the present invention obtains.
Use high performance liquid chromatography to preparation in preparation example 2 to antigen 2 detected (instrument is Aglient HPLC, and SEC post model is BioSEC-3,7.8 * 300mm, 3 μ m, moving phase is PBS).Utilize respectively fusion rotein CD3E-Fc1, the CD3D ECD of rproteinA purifying CD3E ECD and Fc1 and fusion rotein CD3D-Fc2 and the heterodimer antigens c D3E/CD3D-Fc1/Fc2 of Fc1, dialyse to PBS, concentration all is adjusted into 0.5mg/ml.With SEC-HPLC, detected, applied sample amount is respectively 10 μ L.The main component of fusion rotein CD3E-Fc1 and fusion rotein CD3D-Fc2 is monomer (molecular weight is about 35kD, and ratio is 80%) and contains a small amount of homodimer and high polymer; The main component of CD3E/CD3D-Fc1/Fc2 is dimer (molecular weight is 70kD, and ratio is 95%) and contains a small amount of high polymer, monomer-free.The dimer that CD3E/CD3D-Fc1/Fc2 forms is the heterodimer form, and forming efficiency is 95%.The results are shown in Figure 8.
This test case is for illustrating that CD3 antigen provided by the present invention can specific combination occur with CD3 antibody.
Adopt Dot blot analyzing and testing antigen 1 and 2 and the combination of antibody.
Respectively the sample spot of the purifying of 10 microlitres (500 ug/ml) is added on the NC film, then 5% skimmed milk seals 1h, use respectively again (A) UCHT1(0.5 ug/ml), (B) OKT3(0.5 ug/ml) and (C) goat anti-human igg is with the anti-1h of hatching respectively of HRP bis-, then (A) is (B) with the anti-1h of hatching of sheep anti-mouse igg band HRP bis-, finally with TBST, wash three times, exposure is taken pictures.The results are shown in Figure 9.
In Fig. 9: the 1st classifies antigen 2 as; The 2nd classifies antigen 1 as; The 3rd classifies fusion rotein CD3E ECD-Fc1 as; The 4th classifies fusion rotein CD3D ECD-Fc2 as; The 5th classifies fusion rotein CD3G ECD-Fc2 as; The primary antibodie that the A group is used is UCHT1, and two resist the mark for sheep anti-mouse igg band HRP; The primary antibodie that the B group is used is OKT3, and two resist the mark for sheep anti-mouse igg band HRP; The antibody that the C group is used is with the HRP mark for the goat anti-human igg.
This test case for illustrate CD3 antigen 1 provided by the present invention and 2 with OKT3 and UCHT1 antibody in conjunction with the activity.By the antigen 1 of purifying and 2, be coated in respectively in enzyme plate and 4 ℃ of lower overnight incubation.The OKT3(that is 1 μ g/ml by concentration respectively again is dissolved in PBS) and UCHT1(be dissolved in PBS) join in hole, hatch 1 hour for 37 ℃, wash plate 3 times with PBS, adding sheep anti-mouse igg band HRP mark two to resist 37 ℃ hatches half an hour, PBS washes plate 3 times, add the TMB incubated at room 5 minutes, after ending with 1M hydrochloric acid, use 450nm wavelength reading result in microplate reader.The results are shown in Figure 10.
In Figure 10, the negative contrast of NC, the primary antibodie OKT3 of use and UCHT1 are mouse IgG (1 μ g/ml), and two resist the mark for sheep anti mouse band HRP; As shown in the figure, CD3 antigen of the present invention has than commercialization antigen (CD3D& CD3E heterodimer protein(is purchased from Sino Biological Inc, article No. CT026-H0323H)) lower background value, and CD3 antigen of the present invention and OKT3 antibody in conjunction with highly significant, and commercialization antigen and OKT3 antibody do not have combination.
Test case 4-15
Test case 4-15 is for illustrating the relative affinity of CD3 antigen 1-12 provided by the invention and anti-cd 3 antibodies.Testing method is carried out according to the measuring method of the anti-PH2SA monoclonal antibody relative affinity of record in document (cell and molecular immunology magazine, 2005,21(5)).
The antigen 1 of purifying-12 are coated in to enzyme plate and spend night 4 with the consumption in 10ng/ hole respectively.By antibody OKT3 to be measured from 160nM with 2 times of dilutions, 11 gradients of serial dilution are to 0.08nM.The OKT3 solution of 12 different concns is joined respectively in the enzyme plate of coated CD3 antigen 1-12, hatch 1 hour for 37 ℃, with PBS, wash plate 3 times, adding sheep anti-mouse igg band HRP mark two to resist 37 ℃ hatches half an hour, PBS washes plate 3 times, adds the TMB incubated at room 5 minutes, with 1M hydrochloric acid, ends.Use 450nm wavelength reading result in microplate reader.Take antibody concentration as X-axis, and the OD450 photoabsorption is Y-axis, draws binding curve, and calculates the avidity of antigen 1-12 and OKT3.The results are shown in Table 2.
Comparative Examples 2
Comparative Examples 2 is for illustrating the relative affinity of existing CD3 antigen and anti-cd 3 antibodies.
Adopt the method test CD3 antigen identical with test case 4-15 and the avidity of anti-cd 3 antibodies OKT3 and UCHT1, different, the reference antigens c D3 used is commercially available commercialization CD3 antigens c D3D& CD3E heterodimer protein(is purchased from Sino Biological Inc, article No. CT026-H0323H), calculate CD3D& The relative affinity of CD3E heterodimer protein and UCHT1, the results are shown in Table 2.
Table 2
It should be noted, the present invention will be described rather than limit the invention for above-described embodiment, and those skilled in the art can design alternative embodiment in the situation that do not break away from the claims scope.In the claims, any reference symbol between bracket should be configured to limitations on claims.Word " comprises " not to be got rid of existence and is not listed in element or the step in claim.
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Claims (10)
1. a CD3 antigen, it is characterized in that described CD3 antigen comprises: the ectodomain CD3E ECD of the epsilon subunit CD3E of Fc fragment, CD3, and the ectodomain CD3G ECD of the γ subunit CD3G of the ectodomain CD3D ECD of the δ subunit CD3D of CD3 or CD3;
Wherein, described Fc fragment is formed by disulfide linkage by Fc1 fragment and Fc2 fragment;
Wherein, described antigen is the heterodimer formed by two fusion roteins, in first fusion rotein, the C end of CD3E ECD is connected with the N end of Fc1, forms fusion rotein CD3E ECD-Fc1, in second fusion rotein, the C end of CD3D ECD is connected with the N end of Fc2, form fusion rotein CD3D ECD-Fc2, or the C of CD3G ECD end is held and is connected, formation fusion rotein CD3G ECD-Fc2 with the N of Fc2;
Wherein, the aminoacid sequence of Fc1 fragment and Fc2 fragment is the aminoacid sequence shown in SEQ ID NO:1 is carried out to 2 or 3 amino acid whose point mutation acquisitions.
2. CD3 antigen according to claim 1, wherein carry out the aminoacid sequence shown in SEQ ID NO:1 sporting of 2 or 3 amino acid whose point mutation and the T of the 139th of aminoacid sequence shown in SEQ ID NO:1 is mutated into to W, the K of the 165th is mutated into D, the K of the 182nd and is mutated into D, the Y of the 180th and is mutated into the D that A, the D of the 129th be mutated into K and the 172nd and is mutated into 2 kinds or 3 kinds in K.
3. CD3 antigen according to claim 1 and 2, the mode of wherein aminoacid sequence shown in SEQ ID NO:1 being carried out to 2 or 3 amino acid whose point mutation is selected from a kind of in following sudden change mode:
The K that the T of the 139th is mutated into W and the 165th is mutated into D,
The K that the T of the 139th is mutated into W and the 182nd is mutated into D,
The D that the T of the 139th is mutated into W and the 129th is mutated into K,
The D that the T of the 139th is mutated into W and the 172nd is mutated into K,
The K that the Y of the 180th is mutated into A and the 165th is mutated into D,
The K that the Y of the 180th is mutated into A and the 182nd is mutated into D,
The D that the Y of the 180th is mutated into A and the 129th is mutated into K,
The D that the Y of the 180th is mutated into A and the 172nd is mutated into K,
The D that the D that the T of the 139th is mutated into W and the 129th is mutated into K and the 172nd is mutated into K,
The K that the K that the T of the 139th is mutated into W and the 165th is mutated into D and the 182nd is mutated into D,
The D that the D that the Y of the 180th is mutated into A and the 129th is mutated into K and the 172nd is mutated into K,
The K that the K that the Y of the 180th is mutated into A and the 165th is mutated into D and the 182nd is mutated into D.
4. CD3 antigen according to claim 3, wherein, when the aminoacid sequence of in Fc1 and Fc2 is mutated into for the D that the Y of the 180th of the aminoacid sequence shown in SEQ ID NO:1 is mutated into to A and the 129th sequence that K obtains, another aminoacid sequence is mutated into for the K that the T of the 139th of the aminoacid sequence shown in SEQ ID NO:1 is mutated into to W and the 165th sequence that D obtains;
When the aminoacid sequence of in Fc1 and Fc2 is mutated into for the T that the D of the 129th of the aminoacid sequence shown in SEQ ID NO:1 is mutated into to K and the 139th sequence that W obtains, another aminoacid sequence is mutated into for the K that the Y of the 180th of the aminoacid sequence shown in SEQ ID NO:1 is mutated into to A and the 165th sequence that D obtains;
When the aminoacid sequence of in Fc1 and Fc2 is mutated into for the D that the Y of the 180th of the aminoacid sequence shown in SEQ ID NO:1 is mutated into to A and the 172nd sequence that K obtains, another aminoacid sequence is mutated into for the K that the T of the 139th of the aminoacid sequence shown in SEQ ID NO:1 is mutated into to W and the 182nd sequence that D obtains, or the K that is mutated into D and the 182nd for the T of the 139th of the aminoacid sequence shown in SEQ ID NO:1 being mutated into to W, the K of the 165th is mutated into the sequence that D obtains;
When the aminoacid sequence of in Fc1 and Fc2 is mutated into for the D that the D of the 129th of the aminoacid sequence shown in SEQ ID NO:1 is mutated into to K and the 172nd sequence that K obtains, another aminoacid sequence is mutated into for the K that the K of the 182nd of the aminoacid sequence shown in SEQ ID NO:1 is mutated into to D and the 165th sequence that D obtains;
When the aminoacid sequence of in Fc1 and Fc2 is mutated into for the K of the 182nd of the aminoacid sequence shown in SEQ ID NO:1 being mutated into to D, the Y of the 180th sequence that A obtains, another aminoacid sequence is mutated into for the T that the D of the 172nd of the aminoacid sequence shown in SEQ ID NO:1 is mutated into to K and the 139th sequence that W obtains;
When the aminoacid sequence of in Fc1 and Fc2 is mutated into K and the 172nd D for the Y of the 180th of the aminoacid sequence shown in SEQ ID NO:1 being mutated into to A, the D of the 129th sports the sequence that K obtains, the K that another aminoacid sequence is mutated into D and the 182nd for the T of the 139th of the aminoacid sequence shown in SEQ ID NO:1 being mutated into to W, the K of the 165th sports the sequence that D obtains;
When the aminoacid sequence of in Fc1 and Fc2 is mutated into D and the 180th Y for the K of the 165th of the aminoacid sequence shown in SEQ ID NO:1 being mutated into to D, the K of the 182nd sports the sequence that A obtains, the T that another aminoacid sequence is mutated into K and the 139th for the D of the 129th of the aminoacid sequence shown in SEQ ID NO:1 being mutated into to K, the D of the 172nd sports the sequence that W obtains, or is mutated into the sequence that W obtains for the T that the D of the 172nd of the aminoacid sequence shown in SEQ ID NO:1 is mutated into to K and the 139th.
5. according to the described CD3 antigen of any one in claim 1,2 and 4, wherein, the aminoacid sequence of CD3E ECD is as shown in SEQ ID NO:14, and the aminoacid sequence of CD3D ECD is as shown in SEQ ID NO:15, and the aminoacid sequence of CD3G ECD is as shown in SEQ ID NO:16.
6. CD3 antigen according to claim 1, wherein, described CD3 antigen can be combined with the antibodies specific of anti-CD3.
7. CD3 antigen according to claim 6, wherein, described CD3 antigen can with antibody OKT3, UCHT1, TRX4 and the L2K of anti-CD3 at least one specific binding.
8. the preparation method of the described CD3 antigen of claim 1-7, described method comprises:
(1) obtain respectively recombinant vectors e and recombinant vectors d, or obtain respectively recombinant vectors e and recombinant vectors g;
(2) to described recombinant vectors e and d, or recombinant vectors e and g carry out point mutation, obtains sudden change recombinant vectors em and dm, or obtain sudden change recombinant vectors em and gm;
(3) transfection em and dm in eukaryotic cell; Perhaps transfection em and gm;
(4) utilize affinity chromatography method purifying to obtain CD3 antigens c D3E/CD3D-Fc1/Fc2 or CD3E/CD3G-Fc1/Fc2;
Wherein, the coding nucleotide sequence that described recombinant vectors e contains fusion rotein CD3E ECD-Fcw; The coding nucleotide sequence that described recombinant vectors d contains fusion rotein CD3D ECD-Fcw, the coding nucleotide sequence that described recombinant vectors g contains fusion rotein CD3G ECD-Fcw;
Wherein, Fcw is the coded polypeptide fragment of aminoacid sequence shown in SEQ ID NO:1.
9. the preparation method of CD3 antigen according to claim 8, wherein,
Primer pair SEQ ID NO:2 and SEQ ID NO:3 are for the base sequence at recombinant vectors e, d or g introducing encoded point sudden change D129K;
Primer pair SEQ ID NO:4 and SEQ ID NO:5 are for the base sequence at recombinant vectors e, d or g introducing encoded point mutation T 139W;
Primer pair SEQ ID NO:6 and SEQ ID NO:7 are for the base sequence at recombinant vectors e, d or g introducing encoded point sudden change K165D;
Primer pair SEQ ID NO:8 and SEQ ID NO:9 are for the base sequence at recombinant vectors e, d or g introducing encoded point sudden change D172K;
Primer pair SEQ ID NO:10 and SEQ ID NO:11 are for the base sequence at recombinant vectors e, d or g introducing encoded point sudden change Y180A;
Primer pair SEQ ID NO:12 and SEQ ID NO:13 are for the base sequence at recombinant vectors e, d or g introducing encoded point sudden change K182D.
10. the purposes of the described CD3 antigen of claim 1-7 in the screening of anti-cd 3 antibodies.
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| CN110172100A (en) * | 2019-05-06 | 2019-08-27 | 北京智仁美博生物科技有限公司 | Anti-human CD3E antibody and application thereof |
| CN112961251A (en) * | 2021-03-26 | 2021-06-15 | 吴江近岸蛋白质科技有限公司 | Preparation method and application of CD3 antigen |
| CN112961251B (en) * | 2021-03-26 | 2022-04-29 | 苏州近岸蛋白质科技股份有限公司 | Preparation method and application of CD3 antigen |
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