Disclosure of Invention
Based on the above description, the current total Tau detection system has many disadvantages. Early detection of AD and dynamic tracking of therapeutic effects is therefore limited. In order to overcome the defects in the prior art, the application provides a recombinant antibody targeting Tau protein, which is used for obtaining a recombinant antigen of the recombinant antibody and a nucleic acid molecule for encoding the recombinant antigen.
The specific technical scheme of the application is as follows:
1. A recombinant antibody targeting Tau protein, wherein the recombinant antibody comprises three heavy chain complementarity determining regions (CDR-H1, CDR-H2, CDR-H3) and three light chain complementarity determining regions (CDR-L1, CDR-L2, CDR-L3), wherein:
the amino acid sequence of CDR-H1 is shown as SEQ ID No. 1 or SEQ ID No. 2;
The amino acid sequence of CDR-H2 is shown as SEQ ID No. 3 or SEQ ID No. 4;
The amino acid sequence of CDR-H3 is shown as SEQ ID No. 5 or SEQ ID No. 6;
The amino acid sequence of CDR-L1 is shown as SEQ ID No. 7 or SEQ ID No. 8;
The amino acid sequence of CDR-L2 is shown as SEQ ID No. 9 or SEQ ID No. 10;
The amino acid sequence of CDR-L3 is shown as SEQ ID No. 11 or SEQ ID No. 12.
2. The recombinant antibody according to item 1, wherein,
The amino acid sequence of the CDR-H1 is shown as SEQ ID No. 1;
the amino acid sequence of the CDR-H2 is shown as SEQ ID No. 3;
the amino acid sequence of the CDR-H3 is shown as SEQ ID No. 5;
the amino acid sequence of the CDR-L1 is shown as SEQ ID No. 7,
The amino acid sequence of the CDR-L2 is shown as SEQ ID No. 9;
the amino acid sequence of the CDR-L3 is shown as SEQ ID No. 11.
3. The recombinant antibody according to item 1, wherein,
The amino acid sequence of the CDR-H1 is shown as SEQ ID No. 2;
The amino acid sequence of the CDR-H2 is shown as SEQ ID No. 4;
The amino acid sequence of the CDR-H3 is shown as SEQ ID No. 6;
The amino acid sequence of the CDR-L1 is shown as SEQ ID No. 8;
the amino acid sequence of the CDR-L2 is shown as SEQ ID No. 10;
the amino acid sequence of the CDR-L3 is shown as SEQ ID No. 12.
4. The recombinant antibody according to any one of claims 1-3, wherein the recombinant antibody comprises a heavy chain variable region and a light chain variable region, wherein:
The amino acid sequence of the heavy chain variable region is shown as SEQ ID No. 13 or SEQ ID No. 14, or the amino acid sequence has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% identity with SEQ ID No. 13 or SEQ ID No. 14;
The amino acid sequence of the light chain variable region is shown as SEQ ID No. 15 or SEQ ID No. 16, or the amino acid sequence has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% identity with SEQ ID No. 15 or SEQ ID No. 16.
5. The recombinant antibody according to item 4, wherein,
The amino acid sequence of the heavy chain variable region is shown as SEQ ID No. 13 or has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% identity with SEQ ID No. 13;
the amino acid sequence of the light chain variable region is shown as SEQ ID No. 15 or the amino acid sequence which has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% identity with SEQ ID No. 15.
6. The recombinant antibody according to item 4, wherein the heavy chain variable region has an amino acid sequence as shown in SEQ ID No. 14 or an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID No. 14;
The amino acid sequence of the light chain variable region is shown as SEQ ID No. 16 or the amino acid sequence which has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% identity with SEQ ID No. 16.
7. A nucleic acid molecule encoding the recombinant antibody according to any one of claims 1 to 6.
8. A Tau protein recombinant antigen, wherein the amino acid sequence of the recombinant antigen is shown as SEQ ID No. 17.
9. A nucleic acid molecule encoding the Tau protein recombinant antigen of claim 8, wherein the nucleotide sequence of said nucleic acid molecule is shown as SEQ ID No. 18.
10. The Tau protein recombinant antigen of claim 8 or the nucleic acid molecule of claim 9 for use in obtaining the recombinant antibody of any one of claims 1 to 6.
11. A kit for detecting Tau protein, wherein the kit comprises:
Magnetic beads coated with a first antibody targeting the Tau protein, and
A chemiluminescent-labeled second antibody targeting Tau protein;
Wherein the first antibody and the second antibody targeting Tau protein are recombinant antibodies according to any one of items 1 to 6.
12. The kit of item 11, wherein it is used for diagnosing Alzheimer's disease.
13. The recombinant antibody according to any one of claims 1 to 6, or the nucleic acid molecule according to claim 7, for use in the preparation of a kit for diagnosing Alzheimer's disease.
ADVANTAGEOUS EFFECTS OF INVENTION
The application provides a novel recombinant antibody targeting Tau protein, a nucleic acid molecule for encoding a recombinant antigen used for obtaining the antibody and a kit containing the recombinant antibody, wherein the nucleic acid molecule for encoding the recombinant antigen can obviously improve the expression quantity of the recombinant antibody, and the total Tau recombinant antibody obtained by using the recombinant antigen as an immunogen has excellent and stable performance, high affinity and high sensitivity, and meanwhile, the production period of the total Tau recombinant antibody is obviously shortened, is more suitable for being applied to the field of in vitro diagnostic reagents as a core raw material, and can bring obvious social benefit and economic benefit.
Detailed Description
The present application is described in further detail below in conjunction with the specific embodiments which are presented to provide a more thorough understanding of the present application and to fully convey the scope of the application to those skilled in the art.
Technical and scientific terms used in the present specification have the same meaning as commonly understood by one of ordinary skill in the art, and if so conflict, the present specification will control.
The main function of Tau protein in normal human body is to induce and promote tubulin aggregation into microtubules and maintain its stability. There are 6 Tau protein subtypes in the adult brain, three of which have three microtubule-binding repeat regions (2N 3R, 1N3R, 0N 3R) and the other three have four microtubule-binding repeat regions (2N 4R, 1N4R, 0N 4R). 2N4R/Tau-441 is the full-length Tau protein, and K18 and K19 are truncated Tau proteins, containing only microtubule-binding repeat regions. The microtubule binding capacity of Tau proteins is controlled by posttranslational modifications, of which phosphorylation modification is one of the main modes. 20% of the amino acids of the Tau protein can be potential sites for phosphorylation. Typically, the microtubule binding region of Tau protein is positively charged and thus attracts negatively charged microtubules. When this microtubule binding region is hyperphosphorylated, it loses its positive charge, detaches from the tubulin and no longer has the ability to bind stable microtubules. The subsequent failure of tubulin and the highly phosphorylated Tau aggregate precipitate, toxic to neural cells, which together contribute to the etiology of neural degeneration, which makes the pathogenesis of AD very complex, since Tau proteins have multiple phosphorylation sites and are involved in the regulation of more than one protein kinase and phosphatase. Tau precursor fibrinogen (PFFs) can induce aggregation of Tau protein in artificially cultured cells and animal living bodies, causing aggregation of soluble monomers to form insoluble fibers. The incorporation of small amounts TauPFFs into cells expressing Tau protein can cause large amounts of Tau to aggregate to form filamentous inclusion similar to NFTs. Synthetic TauPFFs can also enter into non-neural cells to aggregate Tau protein to form NFTs.
In some embodiments, the Tau protein of the present application is the Tau full-length protein 2N4R/Tau-441.
As used herein, the term "recombinant antigen" refers to an antigen that is purified by recombinant expression in vitro, after ligation of an antigen gene to a vector, and transfer into a prokaryotic or eukaryotic cell.
As used herein, the term "recombinant antibody" is an antibody obtained by cloning immunospecific heavy and light chain antibody genes into highly efficient expression vectors, and introducing these vectors into an expression host (e.g., bacteria, yeast or mammal) for antibody expression. The antibody of the present application may be a fully humanized antibody, a chimeric antibody, or the like.
In some embodiments, the recombinant antibodies of the application are antibodies obtained after immunization of an animal with a recombinant antigen of the application.
In some embodiments, the antibodies of the application are full length antibodies, which typically refer to antibodies consisting of two "heavy chains" and two "light chains. A "heavy chain" is typically a polypeptide consisting of a heavy chain variable region (VH), a heavy chain constant region 1 (CH 1), a Hinge Region (HR), an antibody heavy chain constant region 2 (CH 2), and a heavy chain constant region 3 (CH 3), abbreviated as VH-CH1-HR-CH2-CH3, in the N-to C-terminal direction, and in some embodiments, a "full-length antibody heavy chain" is a polypeptide consisting of VH, CH1, HR, CH2, and CH3 in the N-to C-terminal direction. A "full length antibody light chain" is generally a polypeptide consisting of a light chain variable region (VL) in the N-to C-terminal direction, and a light chain constant region (CL), abbreviated VL-CL.
In the present application, the light chain constant region and the heavy chain constant region may be any light chain constant region and heavy chain constant region. The light chain constant region (CL) may be kappa (kappa) or lambda (lambda). The heavy chain constant region may be any of the heavy chain constant regions of IgG, igM, igA, igE, igD.
It is well known to those skilled in the art that each heavy chain variable region may be composed of three Complementarity Determining Regions (CDRs) and four Framework Regions (FR) and each light chain variable region may be composed of three Complementarity Determining Regions (CDRs) and four Framework Regions (FR) with the complementarity determining regions (CDRs, typically CDR1, CDR2 and CDR 3) being the regions of the variable regions that have the greatest influence on the affinity and specificity of the antibody. In some embodiments, from N-terminal to C-terminal, the heavy chain variable region and the light chain variable region each comprise FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The CDR sequences of the heavy chain variable region or the light chain variable region are defined in two general ways, namely by Kabat definition and by Chothia definition, see for example Kabat et al.,"Sequences of Proteins ofImmunological Interest",National Institutes of Health,Bethesda,MD.(1991);Al-Lazikani et al.,J MolBiol 273:927-948(1997); and Martin et al, proc.Natl. Acad.Sci.USA86:9268-9272 (1989). For a given antibody variable region sequence, the CDR sequences in the heavy chain variable region or light chain variable region sequence can be determined according to the Kabat definition or Chothia definition. In embodiments of the application, the CDR sequences are determined using the Chothia definition.
In this context, the term "identity" or "identity" is defined as the percentage of identical residues in an amino acid or nucleotide sequence variant after sequence alignment and introduction of gaps. Methods and computer programs for alignment are well known in the art. In this context, reference to an amino acid sequence having a percent identity to the amino acid sequence refers to a sequence having said percent identity over the entire length of the amino acid sequence referred to.
In this context, the term "vector" generally refers to a nucleic acid molecule capable of self-replication in a suitable host, which transfers the inserted nucleic acid molecule into and/or between host cells. The vector may include a vector mainly used for inserting DNA or RNA into a cell, a vector mainly used for replicating DNA or RNA, and a vector mainly used for expression of transcription and/or translation of DNA or RNA. The carrier also includes a carrier having a plurality of functions as described above. The vector may be a polynucleotide capable of transcription and translation into a polypeptide when introduced into a suitable host cell. Typically, the vector will produce the desired expression product by culturing a suitable host cell comprising the vector.
In this context, the term "nucleic acid" or "polynucleotide" or "nucleic acid molecule" generally refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form. Unless specifically limited, the term may include nucleic acids comprising analogs of natural nucleotides that have similar binding properties as the reference nucleic acid (e.g., sequence information is shown) and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, the sequence of a nucleic acid may include variants thereof that are conservatively modified, such as degenerate codon substitutions, alleles, orthologs, SNPs, and complementary sequences, as well as the sequences explicitly indicated.
As used herein, the term "EC50 value" refers to half maximal effect concentration (concentration for 50%ofmaximal effect,EC50), which refers to the concentration that causes 50% of the maximal effect.
Herein, "affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, "binding affinity" as used herein refers to an inherent binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be expressed by the equilibrium dissociation constant (K D). Affinity can be determined by common methods known in the art.
Herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homologous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind to the same epitope, except for possible variant antibodies (e.g., containing naturally occurring mutations or produced during production of monoclonal antibody preparations), which are typically present in minor amounts. Unlike polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being derived from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
In this context, the term "recombinant antibody targeting a Tau protein" means a recombinant antibody capable of binding a Tau protein with sufficient affinity, capable of being used as a diagnostic and/or therapeutic agent targeting a Tau protein.
The recombinant antibody targeting Tau protein of the present application does not bind to target independent proteins. Here, "unrelated proteins" means other proteins than the Tau protein as a target, and "unbound" means that the recombinant antibody targeting the Tau protein of the present application has a binding capacity to the unrelated protein of less than 10%, for example, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0, when the binding capacity of the recombinant antibody targeting the Tau protein of the present application to the Tau protein as a target is 100%.
Tau protein suitable for antibody production can be produced by any of a variety of standard protein purification or recombinant expression techniques known in the art. Other forms of beta-amyloid may also include Tau protein expressing cells, preparations or cell extracts or fractions containing Tau protein, partially purified Tau protein.
In some embodiments, the application adopts a magnetic particle chemiluminescence immunoassay method to determine the level of Tau protein in a sample, and the specific principle is that a chemiluminescent agent is used for directly labeling an antigen or an antibody (a chemiluminescent agent label), the antigen or the antibody reacts with the corresponding antibody or antigen in the sample to be tested, the antigen or the antibody with magnetic granularity (magnetic beads), the chemiluminescent agent label in a combined state (a precipitation part) and a free state is separated through a magnetic field, then a luminescent promoter (a luminescent substrate) is added for luminescence reaction, and quantitative or qualitative detection is carried out on detection of luminescence intensity.
As used herein, ELISA (enzyme linked immune sorbent assay), an ELISA, refers to a detection method that uses the characteristic of an antibody molecule that specifically binds to an antigen molecule to bind free hetero-protein to a target protein bound to a solid support and uses a specific label to perform qualitative or quantitative analysis. The principle is that the antigen or antibody can be physically adsorbed on the surface of solid carrier and retain its immunological activity, the antigen or antibody can form enzyme conjugate with enzyme by means of covalent bond, at the same time retain its immunological activity or enzyme activity, after the enzyme conjugate is combined with correspondent antigen or antibody, the immunological reaction can be defined by means of colour reaction of added substrate, and the depth of colour reaction is proportional to the quantity of correspondent antigen or antibody in the specimen.
In a first aspect, the application provides a recombinant antibody targeting Tau protein, which comprises three heavy chain complementarity determining regions (CDR-H1, CDR-H2 and CDR-H3) and three light chain complementarity determining regions (CDR-L1, CDR-L2 and CDR-L3), wherein the amino acid sequence of the CDR-H1 is shown as SEQ ID No. 1 or SEQ ID No. 2, the amino acid sequence of the CDR-H2 is shown as SEQ ID No. 3 or SEQ ID No. 4, the amino acid sequence of the CDR-H3 is shown as SEQ ID No. 5 or SEQ ID No. 6, the amino acid sequence of the CDR-L1 is shown as SEQ ID No. 7 or SEQ ID No. 8, the amino acid sequence of the CDR-L2 is shown as SEQ ID No. 9 or SEQ ID No. 10, and the amino acid sequence of the CDR-L3 is shown as SEQ ID No. 11 or SEQ ID No. 12.
In some embodiments, the amino acid sequence of CDR-H1 is shown as SEQ ID No.1 (GFTFTDY), the amino acid sequence of CDR-H2 is shown as SEQ ID No.3 (RNKANGYT), the amino acid sequence of CDR-H3 is shown as SEQ ID No. 5 (EEDYRHGFPY), the amino acid sequence of CDR-L1 is shown as SEQ ID No. 7 (NSSQSLLNTGNQKNYLA), the amino acid sequence of CDR-L2 is shown as SEQ ID No. 9 (GALTRES), and the amino acid sequence of CDR-L3 is shown as SEQ ID No. 11 (QNDHSYPLT).
In some embodiments, the amino acid sequence of CDR-H1 is shown as SEQ ID No. 2 (GFVFIDY), the amino acid sequence of CDR-H2 is shown as SEQ ID No. 4 (RNKANGYR), the amino acid sequence of CDR-H3 is shown as SEQ ID No. 6 (EGSYPYGFPD), the amino acid sequence of CDR-L1 is shown as SEQ ID No. 8 (KSSQSLLHSGNQKNYLA), the amino acid sequence of CDR-L2 is shown as SEQ ID No. 10 (GASTRES), and the amino acid sequence of CDR-L3 is shown as SEQ ID No. 12 (QNDHSYPFT).
In some embodiments, the recombinant antibody comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 13 or SEQ ID No. 14, or is an amino acid sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID No. 15 or SEQ ID No. 16.
In some embodiments, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No:13(EVMLQESGGGLVQPGSSLRLTCATSGFTFTDYYMSWVRQPPGKAL EWLGVIRNKANGYTTEYSASVKGRFTISRDNTQGILYLHMNTLSAEDSAT YYCAREEDYRHGFPYWGQGTLVTVSA), or is an amino acid sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID No.13, or the amino acid sequence of the light chain variable region is as shown in SEQ ID No:15(DIIMTQSPSSLSVSVGEKVTMNCNSSQSLLNTGNQKNYLAWYQQK PGQTPKLLIHGALTRESGIPDRFTGRGSGTDFTLTISSVQAEDLAVYYCQN DHSYPLTFGTGTKLELRRAD), or is an amino acid sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID No. 15.
In some embodiments, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No:14(EVKLIESGGGLMQPGSSLRLSCASSGFVFIDYYMSWVRQPPGKALE WLGLVRNKANGYRTEYSSTVKGRFTISRDNSQGILYLHMNTLSAEDSATY YCAREGSYPYGFPDWGQGTLVTVSA), or is an amino acid sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID No. 14, or the amino acid sequence of the light chain variable region is as shown in SEQ ID No:16(DIVMTQSPSSLSASVGERVTMNCKSSQSLLHSGNQKNYLAWYQQK PGQQPKLLIHGASTRESGVPDRFTGRGSGTDFTLTISSVQAEDLALYYCQN DHSYPFTFGTGTKLELRRAN), or is an amino acid sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID No. 16.
In a second aspect, the application provides a nucleic acid molecule encoding any one of the recombinant antibodies described above.
In a third aspect, the present application provides a Tau protein recombinant antigen, the amino acid sequence of which is shown as SEQ ID No:17(MHHHHHHLAEAKVLANRELDKYGVSDYHKNLINNAKTVEGVKD LQAQVVESAKKARISEATDGLSDFLKSQTPAEDTVKSIELAEAKVLANRELDKYGVSDYYKNLINNAKTVEGVKALIDEILAALPDDDDKGTMAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTEDGSEEPGSETSDAKSTPTAEDVTAPLVDEGAPGKQAAAQPHTEIPEGTTAEEAGIGDTPSLEDEAAGHVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPKSPSSAKSRLQTAPVPMPDLKNVKSKIGSTENLKHQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHVPGGGSVQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQLATLADEVSASLAKQGL).
In some embodiments, the recombinant antigens of the application are prepared by identifying the ABP tag amino acid sequence (UniPort, P19909) and the Tau-F amino acid sequence (UniPort, P10636-8) (2N 4R/Tau-441 form) by literature and database, and constructing the recombinant antigens into pET30a vectors in tandem after codon optimization. After the vector is sequenced correctly, the vector is transformed into escherichia coli BL21 (DE 3) competence and recombinant antigen is prepared and purified. Recombinant antigen was obtained by purification through Ni-NTA Agarose (QIAGEN, cat. No. 30210).
Albumin binding proteins (ABP, albumin Binding Protein) derived from streptococcal protein G are a protein of about 15kDa that can be used as a folding tool in protein engineering. The existing Tau recombinant antigen is often prepared by an escherichia coli expression system, and the antigen with higher stability, immunogenicity and solubility can be obtained by means of an ABP label, so that an antibody with higher affinity can be obtained.
In a fourth aspect, the application provides a nucleic acid molecule encoding the Tau protein recombinant antigen, wherein the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID No:18(ATGCACCATCATCACCACCACTTAGCTGAAGCTAAAGTGTTAGCT AACCGTGAACTTGATAAATACGGCGTGAGTGACTATCATAAAAACCTGATTAATAACGCTAAAACCGTAGAAGGTGTTAAGGACCTGCAGGCGCAGGTTGTTGAAAGCGCGAAAAAAGCGCGTATCAGCGAAGCGACCGATGGTCTGAGCGATTTCCTGAAAAGCCAGACCCCGGCGGAAGATACCGTTAAAAGCATCGAACTGGCGGAAGCGAAAGTTCTGGCGAACCGTGAACTGGATAAATACGGTGTTAGCGATTACTACAAAAACCTGATCAACAACGCGAAAACCGTTGAAGGCGTTAAAGCGCTGATCGATGAAATCCTGGCGGCGCTGCCGGATGATGATGATAAAGGTACCATGGCGGAACCGCGCCAAGAATTTGAAGTGATGGAAGATCATGCGGGCACCTATGGCCTGGGCGATCGCAAAGATCAAGGCGGCTATACCATGCATCAAGATCAAGAAGGCGATACCGATGCGGGCCTGAAAGAAAGCCCGCTGCAGACCCCGACCGAAGATGGCAGCGAAGAACCGGGCAGCGAAACGAGCGATGCGAAAAGCACCCCGACCGCGGAAGATGTGACCGCGCCGTTAGTGGATGAAGGCGCGCCGGGCAAACAAGCGGCCGCGCAGCCGCATACCGAAATTCCGGAAGGCACCACCGCGGAAGAAGCGGGCATTGGCGATACGCCAAGCTTAGAAGATGAAGCGGCGGGCCATGTGACCCAAGCGCGCATGGTGAGCAAAAGCAAAGATGGCACCGGCAGCGATGATAAAAAAGCGAAAGGCGCGGATGGTAAGACCAAAATCGCGACCCCGCGCGGTGCCGCGCCGCCGGGTCAGAAAGGTCAAGCCAATGCCACGCGTATTCCGGCCAAGACCCCACCGGCGCCAAAGACCCCGCCGAGCAGCGGCGAACCGCCGAAAAGTGGCGATCGCAGTGGCTATAGTAGCCCGGGCAGCCCGGGCACCCCGGGCAGTCGTAGCCGCACCCCAAGCCTGCCGACCCCGCCGACCCGCGAACCGAAAAAAGTGGCGGTGGTGCGCACCCCACCAAAAAGCCCGAGCAGCGCCAAAAGCCGCTTACAGACCGCGCCGGTGCCGATGCCGGATCTGAAAAACGTGAAAAGCAAAATTGGCAGCACGGAAAACCTGAAACATCAGCCGGGCGGTGGCAAGGTTCAGATTATTAACAAAAAACTGGATCTGAGCAACGTGCAGAGCAAGTGCGGCAGTAAAGACAACATTAAACATGTTCCGGGCGGTGGCAGCGTGCA GATTGTTTACAAACCGGTGGATCTGAGTAAGGTTACGAGCAAATGCGGCAGCCTGGGCAACATTCATCATAAACCGGGTGGCGGCCAAGTGGAAGTGAAAAGCGAAAAACTGGATTTTAAAGATCGCGTTCAGAGCAAGATCGGCAGCCTGGATAACATTACCCATGTGCCGGGCGGTGGCAATAAAAAAATTGAAACCCATAAACTGACCTTTCGCGAAAACGCGAAAGCGAAAACCGATCATGGCGCGGAAATCGTGTATAAGAGCCCGGTGGTGAGCGGCGATACGAGCCCGCGCCATCTGAGCAATGTTAGCAGCACCGGCAGCATTGATATGGTGGATAGCCCGCAGCTGGCGACCCTGGCGGATGAAGTGAGCGCGAGCCTGGCGAAACAAGGCCTGTGA). Analysis by the golden line codon analysis tool (https:// www.genscript.com/tools/rare-codon-analysis) showed that the CAI (codon adaptation index ) of the optimized DNA sequence on the E.coli expression platform was 0.92, the GC content was 55.76% and the CFD (low frequency codon percentage) was 1%. The current sequence has the theoretical basis of smooth expression in an escherichia coli system. The final average amount of recombinant antigen of the present application per 1L LB medium was 15.1mg.
In a fifth aspect, the application also provides the use of the Tau protein recombinant antigen of the third aspect or the nucleic acid molecule encoding the Tau protein recombinant antigen of the fourth aspect for obtaining a recombinant antibody targeting a Tau protein of the application.
In a sixth aspect, the application also provides a kit for detecting Tau protein, which comprises magnetic beads coated with a first antibody targeting Tau protein, and a second antibody targeting Tau protein, which is marked by a chemiluminescent agent, wherein the first antibody and the second antibody targeting Tau protein are recombinant antibodies according to any one of the first aspect respectively.
In some embodiments, the kit further comprises a sample standard to be detected, a substrate, a coating buffer solution, a washing solution, a sealing solution, a sample diluent and the like, wherein the coating buffer solution provides a high pH environment for magnetic bead coating, the coating efficiency is improved, and the sealing solution is used for sealing uncoupling vacancies and preventing later non-specific binding. In some embodiments, the substrate comprises luminescence excitation solution A and luminescence excitation solution B, wherein luminescence excitation solution A is nitric acid and hydrogen peroxide aqueous solution, luminescence excitation solution B is sodium hydroxide aqueous solution, coating buffer solution is TBS-T buffer solution, washing solution is PBS and Tween-20, blocking solution is BSA blocking solution, and sample dilution solution is PBS buffer solution.
In the above embodiment, the kit further comprises a preservation buffer for providing a stable pH environment, reducing antibody degradation, preserving, and the like, and a quenching buffer for quenching off the label not bound to the antibody. In some embodiments, the preservation buffer is TBS-T buffer and the quenching buffer is 5% DL-lysine.
The kit provided by the application can be used for diagnosing Alzheimer's disease.
In a seventh aspect, the application also provides the use of any one of the recombinant antibodies, or any one of the nucleic acid molecules, as defined above, in the preparation of a kit for diagnosing alzheimer's disease.
In some embodiments, the kit may be a magnetic particle chemiluminescent kit, a magnetic particle electrochemiluminescent kit, an ELISA kit, a POCT detection kit, a SERS detection kit, a mass spectrometry kit, a microfluidic kit, a photochemical kit, and the like, preferably a magnetic particle chemiluminescent kit.
Examples
The present application will be described with reference to specific examples, but the scope of the application is not limited thereto. Unless otherwise specified, reagents and equipment used in the following examples are all conventional in the art and are commercially available. The methods used are all routine experimental methods, which can be carried out without any doubt by a person skilled in the art on the basis of the examples and with corresponding results.
EXAMPLE 1 preparation of recombinant antigen Tau-ABP
The recombinant antigen was prepared by confirming the ABP tag amino acid sequence (UniPort, P19909) and the Tau-F amino acid sequence (UniPort, P10636-8) by means of literature and databases, and constructing the recombinant antigen in series to pET30a vector after codon optimization. After the vector is sequenced correctly, the vector is transformed into escherichia coli BL21 (DE 3) competence and recombinant antigen is prepared and purified. Recombinant antigen was obtained by purification through Ni-NTA Agarose (QIAGEN, cat. No. 30210). The amino acid sequence of the recombinant antigen Tau-ABP is shown as SEQ ID No. 17, and the nucleotide sequence of the nucleic acid molecule encoding the recombinant antigen Tau-ABP after codon optimization is shown as SEQ ID No. 18.
EXAMPLE 2 preparation of recombinant antibodies rTau-A and rTau-B
The recombinant antibody is prepared by immunizing mice with the recombinant antigen Tau-ABP prepared in the embodiment, combining the mouse monoclonal antibody cell line with highest sensitivity, sequencing the variable region sequence of the antibody, constructing chimeric vectors according to the variable region sequence, and transferring the chimeric vectors into a Hek293T cell line for recombinant antibody expression, wherein the light chain vectors are (pFUSE 2ss-CLIg-mk, invivoGen company) and the heavy chain vectors are (pFUSEss-CHIg-mG 1, invivoGen company), the two vectors are commercial vectors and have constant region sequences without additional treatment, and the recombinant antibodies are expressed in the Hek293T cell line and purified recombinant antibodies rTau-A and rTau-B through Protein A fillers. Wherein the amino acid sequence of the rTau-A heavy chain variable region is shown as SEQ ID No. 13, the amino acid sequence of the rTau-A light chain variable region is shown as SEQ ID No. 15, the amino acid sequence of the rTau-B heavy chain variable region is shown as SEQ ID No. 14, and the amino acid sequence of the rTau-B light chain variable region is shown as SEQ ID No. 16.
EXAMPLE 3 Performance verification of recombinant antibodies rTau-A and rTau-B
(1) Recombinant antibody concentration determination
Concentration measurements were performed on batches of different recombinant antibodies by means of BCA (BicinchoninicAcidAssay) technique, the concentration statistics are shown in table 1 below, and the purified recombinant antibody concentrations are shown in table 1. The BCA protein concentration assay kit (enhanced) (bi yun day, cat No. P0010) was used. In particular according to the instruction of the kit.
TABLE 1
Concentration (mg/mL) |
20220801 |
20220802 |
rTau-A |
12.3 |
13.2 |
rTau-B |
10.9 |
12.1 |
(2) Recombinant antibody concentration determination
Multiple batches of recombinant antibodies were affinity tested by means of the Elisa technique. The elisa plate was coated with 100ng Tau antigen per well and incubated for 1 hour at 37 ℃ with the double diluted recombinant antibody. After the incubation, adding goat anti-human secondary antibody with HRP label, finally adding TMB to develop color, adding 2M sulfuric acid to stop the reaction, placing the wavelength of the enzyme label at 450nM, and detecting, wherein the detection results are shown in tables 2-5 respectively.
20220801 Batch rTau-A:
TABLE 2
20220802 Batch rTau-A:
TABLE 3 Table 3
20220801 Batch rTau-B:
TABLE 4 Table 4
20220802 Batch rTau-B:
TABLE 5
The EC50 of each batch of recombinant antibodies was calculated according to sigmaplot13.0 software and the data are summarized in table 6 below, the results of table 6 showing that the recombinant antibodies described above have higher affinity. The K D value of the recombinant antibodies of the application can reach pM class, about one thousandth of the prior art, relative to the K D value of nM class in the prior art, which is far higher than the prior art.
TABLE 6
Example 4 preparation of kit for detecting Tau protein and detection procedure
(1) Preparation of Tau protein antibody-coated magnetic bead working solution the magnetic beads were selected from toluene sulfonyl activated magnetic beads (ThermoDynabeads TM M-280 Tosylated, cat#30110D) at an initial concentration of 100mg/mL. After vortexing, sonication was carried out for 5min and resuspended well. The fully resuspended mother-of-magnetic beads solution was aspirated with a pipette 100. Mu.L (about 10mg of magnetic beads) and the solution was removed by magnetic separation for 5 min. 165. Mu.L of 0.1M borate buffer (pH 9.5) and 200. Mu.g of rTau-A recombinant antibody were added, thoroughly mixed and incubated overnight at 37 ℃. After incubation, the liquid was removed by magnetic separation for 5 min. Blocking was performed for 3h at 37℃with 10% BSA. After closing, the liquid was removed by magnetic separation for 5 min. Finally, TBS-T buffer (0.1M phosphate buffer, pH7.4 supplemented with 0.1% BSA,0.5% Tween-20 and 0.1% Proclin 300) was added and stored at 4 ℃. In this example, the mass ratio of the recombinant antibody to the magnetic beads was 1:50. The mother solution after coating is further diluted by 20 times by TBS-T buffer solution to prepare working solution for use.
(2) A Tau protein labelled antibody working solution was prepared by pipetting 0.25mg of rTau-B recombinant antibody and mixing well with 8.25. Mu.L of a 4mM NSP-SA-NHS (Heliosense, cat#HS-11015005 in DMF) solution for 2h. 200 μl of 5% DL-lysine was added to the solution and mixing was continued for 30min. Desalting was performed using Sephadex G-25, TBS-T buffer (0.1M phosphate buffer system, pH7.4, supplemented with 0.1% BSA,0.5% Tween-20 and 0.1% Proclin 300). Adding the glycerol with the same volume, fully and uniformly mixing, and preserving at-20 ℃ for later use. The mass ratio of recombinant antibody to NSP-SA-NHS in this example was 1:20. The marked mother solution is further diluted 4000 times by TBS-T buffer solution to prepare working solution for use.
(3) Sample tracing inG1200 platform is used according to instructionThe G Total Tau (Fujirebio, cat. Number 231302) traces the Tau antigen.
(4) And subpackaging the calibrator and the quality control product, wherein the calibrator and the quality control product of Tau are prepared from antigen and sample diluent. Wherein the concentration of the Tau calibrator is 300pg/mL and 1200pg/mL, and the concentration of the Tau quality control is 300pg/mL and 1500pg/mL. The sample dilution was PBS buffer at pH7.4, and the sample dilution also contained 1% BSA and 0.1% Proclin-300 per liter. The sample dilution was PBS buffer pH7.4, and the sample dilution also contained 1% BSA and 0.1% Proclin300 per liter.
(5) And (3) assembling the kit, namely subpackaging the coated antibodies and the labeled antibodies produced by the 3 batches of the small test in a reagent bin, and assembling the coated antibodies and the labeled antibodies, the calibrator and the quality control product into the kit. Specific information is shown in table 7 below:
TABLE 7
(6) On-machine detection
The detection flow takes a full-automatic chemiluminescence immunoassay analyzer (Smart 500s full-automatic chemiluminescence analyzer, chongqing Ke Si mai biotechnology Co., ltd.) as a detection tool, and the self-detection is completed after the machine is started. The parameters and reagent lot number required by the total Tau test are input manually or by scanning codes, and the reagent is placed in the corresponding reagent position. And calibrating the main curve by using a Tau calibrator, and then performing quality control on the detection system by using a Tau quality control product. And after the quality control is qualified, sequentially adding samples to be detected into the sample positions for detection. And after the detection is finished, carrying out data output, analysis and arrangement.
The reaction system is that a machine automatically absorbs 150 mu L of a sample, fully and uniformly mixes the sample with 100 mu L of a magnetic bead working solution and 100 mu L of a labeled antibody working solution, and carries out magnetic separation after incubation for 20min at 37 ℃. After the supernatant was discarded, the immunoassay kit washing solution (PBS and Tween-20) was added, and the washing was repeated 3 times. The reaction was then sent to a darkroom, and 100. Mu.L each of luminescence excitation solution A (nitric acid and hydrogen peroxide aqueous solution) and luminescence excitation solution B (sodium hydroxide aqueous solution) for the immunoassay instrument was added to perform luminescence reaction, and luminescence values were recorded. And calculating the corresponding concentration value of the sample by combining the recorded and calibrated calculation curves, wherein the result is shown in the following table 8.
TABLE 8
Antigen concentration pg/mL |
Mean value of |
Deviation of |
5 |
6.661 |
33.22% |
10 |
10.244 |
2.44% |
20 |
19.461 |
-2.70% |
50 |
51.6 |
3.20% |
100 |
98.92 |
-1.08% |
200 |
196.768 |
-1.62% |
500 |
511.728 |
2.35% |
1000 |
958.845 |
-4.12% |
1500 |
1528.521 |
1.90% |
2000 |
1919.408 |
-4.03% |
3000 |
3146.019 |
4.87% |
4000 |
3436.819 |
-14.08% |
5000 |
3990.926 |
-20.18% |
As can be seen from Table 8, the kit of the present application has a lower detection limit, high detection sensitivity, and a deviation of detection values of antigen concentrations within a range of 10 to 3000pg/mL of within + -5%.
Example 5 evaluation of kit Performance
(1) Precision of
According to the protocol of the American clinical laboratory standards Committee (NCCLS/CLSI) document EP5-A2, the in-batch precision and inter-laboratory precision are tested by adopting a multi-factor integrated nested design, different operators, different equipment and different places are tested once each day in the morning and afternoon, each sample is tested repeatedly for 2 times, the test is continuously carried out for 20 days, 160 data results are collected for each batch of the kit, and the precision is calculated. Meanwhile, the traced Tau-ABP antigen is configured into 200pg/mL and 1500pg/mL solutions, and detection is repeated for 3 times and data analysis is performed. The test results are shown in Table 9 below:
TABLE 9
In conclusion, the detection results of the kit for the low-concentration sample and the high-concentration sample are repeatedly displayed, and the precision CV between the batch and the batch is smaller than 10%, which indicates that the kit meets the precision performance evaluation requirement.
(2) Linear range
1 Cerebrospinal fluid supplemented with Tau-ABP antigen was selected, diluted with diluent at 12 concentration levels, and each diluted sample was tested 3 times repeatedly to calculate the mean. The corresponding linear relationship was calculated from the concentration points at which the results gradually decreased, and the widest linear range of the kit was determined, and the detection results of the Tau kit are shown in Table 10 below (concentration units are pg/mL).
Table 10
Table 10 shows that the Tau kit has a correlation coefficient r of not less than 0.9900 and no outlier in the range of [10 to 3000] pg/mL. Therefore, the linear range of the Tau kit is [ 10-3000 ] pg/mL.
(3) Sensitivity of
And (5) detecting the blank sample by using the kit, and repeating the detection for 20 times to determine the sensitivity. The results of the Tau kit assay are shown in Table 11 below (concentration units are pg/mL):
TABLE 11
Project |
Parameters (parameters) |
Mean |
627.4 |
SD |
27.0524 |
Mean+2SD |
681.505 |
Corresponding concentration value |
6.951pg/mL |
The average value of the average luminescence value of the blank sample measured by the Tau kit is 627.4, and the value is brought into a reaction curve, so that the sensitivity of the total Tau kit is 6.951pg/mL.
(4) Sample detection results
Detecting warpSamples of the G1200 platform assignment 14 cases to determine the consistency of the kit with the platform product. And detecting the part beyond the linear range of the target reagent by adopting a dilution and loading mode. The analysis results are shown in fig. 1 (the concentration unit is pg/mL), and regression analysis data show that y= 1.0366x-38.861 and R 2 = 0.8724, so that the product has good consistency with commercial products.
The embodiments show that the Tau kit has good stability, high accuracy, high precision and high sensitivity, the linear range of the kit is [ 10-3000 ] pg/mL, the linear range of the kit is far wider than that of the prior art, the upper detection limit of the kit is at least 6 times of the upper detection limit of the prior art, and meanwhile, the kit has good consistency with commercial products.
The above description is only a preferred embodiment of the present application, and is not intended to limit the application in any way, and any person skilled in the art may make modifications or alterations to the disclosed technical content to the equivalent embodiments. However, any simple modification, equivalent variation and variation of the above embodiments according to the technical substance of the present application still fall within the protection scope of the technical solution of the present application.