CN112778426A - Precise antibody nucleic acid directional connection method - Google Patents
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Abstract
The invention discloses a method for directionally connecting nucleic acid of an accurate antibody, which comprises the following steps: (1) HUH endonuclease and Protein G are connected through flexible Linker molecules to obtain fusion Protein; (2) synthesizing a single-stranded DNA containing a site recognized by an endonuclease of the HUH type; (3) mixing the fusion protein and the single-stranded DNA for reaction to obtain a protein-nucleic acid compound; (4) mixing the protein nucleic acid compound with a target antibody, and reacting under the condition of photocatalysis by light to realize the directional connection of the target antibody and the single-chain DNA; the steps (1) and (2) can be interchanged; the Protein G gene specific site is inserted with the codon of the unnatural amino acid which induces site-specific crosslinking. The method does not need to carry out any chemical or functional modification on the antibody and the nucleic acid, and does not influence the antigen recognition capability of the antibody and the properties and functions of nucleic acid molecules; and the obtained antibody nucleic acid complex has only one nucleic acid molecule connected to one antibody.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to a method for directionally connecting nucleic acid of an accurate antibody.
Background
Immunoassay converts information of target molecules into signals which can be quantitatively interpreted through specific immune recognition, is widely applied to qualitative and quantitative detection of proteins, and is an important analysis method in the fields of clinical diagnosis, microbial detection, food detection and the like. However, the traditional immunoassay method is limited by the catalytic activity of enzyme molecules, and it is difficult to realize highly sensitive and accurate quantitative analysis for some trace analytes, such as rare cell surface receptors, trace disease markers of circulatory system, and the like. immuno-PCR is a highly sensitive assay established in 1992 by professor Sano, university of California, which combines the high specificity of immune recognition with the high sensitivity of nucleic acid amplification to become the most sensitive immunoassay at present.
The core technical link of immuno-PCR is to connect antibody and nucleic acid, and the existing connection technology can be mainly divided into two categories of non-covalent connection and covalent connection. The non-covalent linking system mainly comprises non-covalent linking based on the interaction between streptavidin and biotin, non-covalent linking based on the interaction between NTA and histidine, non-covalent linking based on the interaction between aptamer and ligand, and non-covalent linking based on the interaction between antibody binding protein and antibody. The covalent linking system mainly comprises chemical crosslinking, DNA and antibody linking mediated by expressed protein linking, DNA template mediated antibody and DNA linking, enzymatic linking and the like. However, these ligation methods are based on random chemical cross-linking, and have low cross-linking efficiency, significant loss of molecular activity and inability to achieve ligation products with controllable stoichiometry, which significantly limits the application of immuno-PCR technology. Therefore, there is a need to develop an efficient, stable, and precise stoichiometric antibody nucleic acid ligation technique in response to the urgent need of trace immunoassay.
Disclosure of Invention
The invention aims to provide an antibody nucleic acid connection technology with high efficiency, stability and accurate stoichiometric ratio aiming at the problems of low crosslinking efficiency, obvious molecular activity loss and uncontrollable stoichiometric ratio of the existing connection technology of antibodies and nucleic acids in the prior art.
The technical scheme of the invention is as follows:
the invention provides a method for directionally connecting nucleic acid of a precise antibody, which comprises the following steps:
(1) HUH endonuclease and Protein G are connected through flexible Linker molecules to obtain fusion Protein;
(2) synthesizing a single-stranded DNA containing a site recognized by an endonuclease of the HUH type;
(3) mixing the fusion protein with the single-stranded DNA, and reacting in the presence of divalent metal ions to obtain a protein-nucleic acid compound;
(4) mixing the Protein nucleic acid compound with a target antibody, reacting under the condition of photocatalysis, and directionally crosslinking Protein G in the Protein nucleic acid compound and a heavy chain Fc section of the target antibody to realize directional connection of the target antibody and single-chain DNA;
wherein, the steps (1) and (2) are not in sequence;
the gene sequence of the Protein G needs to be modified by inserting codons of non-natural amino acids specifically crosslinked with light-induced sites into a specific site;
the HUH endonuclease refers to endonuclease with histidine-hydrophobic domain-histidine structure, and can be selected from TYLCV, TOPO I, IS608, A or Tra I.
As an optional specific embodiment, the fusion Protein obtained in step (1) may be obtained by fusing the HUH endonuclease gene and the Protein G gene together through a Linker gene, cloning the fused Protein onto a vector, and expressing the fused Protein.
Alternatively, or preferably, in the above-mentioned connection method,
when the HUH endonuclease is TYLCV, the single-stranded DNA of step (2) has the sequence of CAACTTGATA (SEQ ID NO: 1);
when the HUH endonuclease is TOPO I, the single-stranded DNA in the step (2) contains a sequence of CCCTT;
when the HUH endonuclease IS IS608, the single-stranded DNA of step (2) contains a sequence of TATGTTAC;
when the HUH endonuclease is A, the single-stranded DNA of step (2) contains the sequence of CAACTTGATAT (SEQ ID NO: 2);
when the HUH endonuclease is TraI, the single-stranded DNA of step (2) has the sequence of GTTTTTGCGTGGGGTGT (SEQ ID NO: 3).
In the step (3), the divalent metal ion may be selected from Mn ion or Mg ion.
Alternatively or preferably, in the above-mentioned connection method, the divalent metal ion in the step (3) satisfies the following requirements:
when the HUH endonuclease is TYLCV, the concentration of the divalent metal ions is 4 mM;
when the HUH endonuclease is TOPO I, the concentration of the divalent metal ions is 1 mM;
when the HUH endonuclease IS IS608, the concentration of the divalent metal ion IS 20 mM;
when the HUH endonuclease is A, the concentration of the divalent metal ions is 4 mM;
when the HUH endonuclease is TraI, the concentration of the divalent metal ion is 4 mM.
Optionally or preferably, in the connection method, the reaction condition in the step (3) is 36-38 ℃, and the reaction time is 25-35 min.
Alternatively or preferably, in the above method, the unnatural amino acid is L-p-benzoylphenylalanine, and the codon is the amber codon TAG.
Alternatively or preferably, in the above method, the irradiation condition of step (4) is 365nm ultraviolet irradiation. Optionally or preferably, in the above method, after the photocatalytic reaction in step (4) is completed, the product is purified by nickel affinity chromatography.
The invention also provides a detection antibody compound for immune PCR, which comprises a target antibody and single-chain DNA, wherein the target antibody is covalently connected with the single-chain DNA through a connecting Protein, and the connecting Protein is HUH endonuclease and Protein G which are connected together through flexible Linker molecules; wherein the single-stranded DNA contains a site recognized by an endonuclease of the HUH type; protein G in the connexin is covalently cross-linked to the Fc fragment of the antibody of interest.
The invention has the following beneficial effects:
the nucleic acid antibody covalent linking method does not need to carry out any chemical or functional modification on the antibody and the nucleic acid, and does not influence the antigen recognition capability of the antibody and the properties and functions of nucleic acid molecules; the antibody nucleic acid complex obtained by the method has an accurate stoichiometric ratio, namely, only one nucleic acid molecule is connected to one antibody.
The breakthrough of the technology provides a key technical basis for the practicability of the immune PCR, and has important scientific value and application prospect. In addition, the technology can be used together with digital PCR to realize absolute quantitative detection of protein; the method can also be combined with high-throughput sequencing to analyze the protein map of the cancer single cell, so that more valuable individual cell information is provided, thereby monitoring the tumor dynamics in real time, evaluating the treatment effect, better guiding individualized medical treatment and improving the treatment effect.
Drawings
FIG. 1 is a schematic diagram of the precise nucleic acid-antibody directed ligation strategy of the present invention (the precise antibody-nucleic acid directed ligation principle).
FIG. 2 shows the modification efficiency of fusion protein and nucleic acid at different divalent metal ion concentrations, wherein A IS the modification efficiency of TYLCV protein and nucleic acid, B IS the modification efficiency of Topo I protein and nucleic acid, and C IS the modification efficiency of IS608 protein and nucleic acid.
FIG. 3 is an SDS-PAGE of SPG-HUH fusion protein reacted with single-stranded DNA of different lengths, lane 1 is Marker, lane 2 is pure SPG-HUH protein, lane 3 is a single-stranded DNA reaction product of SPG-HUH and 59nt, and lane 4 is a single-stranded DNA reaction product of SPG-HUH and 107 nt.
FIG. 4 is the separation of protein nucleic acid complex, wherein a is anion exchange chromatography elution chromatogram, and b is SDS-PAGE analysis protein separation and purification. Lane 1 is pure protein SPG-HUH, lane 2 is the product of the reaction between protein and nucleic acid, lane 3 is the peak product of graph a, lane 4 is the peak product of graph a, lane 5 is the peak product of graph a, lane 6 is the peak product of graph a, and lane 7 is the peak product of graph a.
FIG. 5 shows a SDS-PAGE pattern of the directional modification of the universal antibody, wherein the protein G and the antibody are subjected to site-specific cross-linking under 365nm light induction, and a band close to 85kDa is an antibody heavy chain with a modified protein.
FIG. 6 shows the precise labeling of antibody-nucleic acid (TYLCV protein for example), in the left panel, Marker in lane M, SPG-TYLCV + DNA in the reaction of SPG-HUH with DNA, and SPG-TYLCV-DNA in the ligation product of SPG-HUH with DNA (protein-nucleic acid complex for short); in the right panel, lane M shows Marker, lane SPG-TYLCV-DNA shows protein nucleic acid complex alone, lane SPG-TYLCV-DNA + IgG shows protein nucleic acid complex reaction with antibody, and lane SPG-TYLCV-DNA-IgG shows protein nucleic acid complex cross-linking with antibody, finally achieving the connection of antibody IgG and nucleic acid DNA.
FIG. 7 shows three sites on Protein G Protein (i.e., SPG) that can be inserted.
Detailed Description
The inventive concepts of the present invention are explained and illustrated in detail below with reference to specific embodiments so that those skilled in the art can better practice the invention.
Example 1 antibody nucleic acid directed ligation
1. Preparing SPG-HUH fusion protein, connecting with single-stranded DNA to obtain protein-nucleic acid compound
Firstly, the gene Sequence of Protein G (SPG) is modified at fixed point
The SPG gene sequence is artificially synthesized, a specific site is selected on the SPG, such as 12 th leucine, 18 th threonine, 20 th alanine, 23 th alanine, 26 th alanine, 28 th arginine, 32 th glutamine and the like, and the sites are respectively mutated into amber codon TAG which can code unnatural amino acid L-p-benzoyl phenylalanine by utilizing a site-specific genetic modification technology (only one site needs to be modified, but different sites can be modified to obtain a plurality of different modified Protein G). Referring to FIG. 7, three sites on the Protein G Protein (i.e., SPG) at which insertion can occur are shown.
Secondly, artificially synthesizing a gene sequence of the HUH endonuclease and a flexible Linker gene sequence according to the published sequence information. See Table 1 for combinations of HUH and Linker
TABLE 1
The obtained HUH gene and the SPG subjected to site transformation are connected through a flexible Linker gene sequence by a gene engineering means (a double enzyme digestion and connection mode) to obtain a fusion protein SPG-HUH, and the fusion protein has the function of combining an antibody and nucleic acid.
Thirdly, artificially synthesizing single-stranded DNA corresponding to the HUH endonuclease.
Mixing the SPG-HUH fusion protein with the single-stranded DNA of the specific sequence in a molar ratio of 1:1, carrying out water bath reaction at 37 ℃ for 30 min under the action of divalent metal ions (TYLCV protein-Mn ions, 4 mM; A. protein-Mg ions, 4 mM; TOPO I protein-Mn ions, 1 mM; IS608 protein-Mn ions, 20 mM), and then realizing covalent linkage of protein nucleic acid to obtain the protein nucleic acid compound SPG-HUH-ssDNA.
The concentration of the divalent metal ions required for different proteins varied, with the most reactive divalent metal ion concentration for TYLCV protein being 4mM (FIG. 2), the most reactive divalent metal ion concentration for TOPO I being 1mM (FIG. 3), and the most reactive divalent metal ion concentration for IS608 being 20mM (FIG. 4).
FIG. 3 is an SDS-PAGE of SPG-HUH fusion protein reacted with single-stranded DNA of varying lengths, showing the results of protein-nucleic acid covalent linkage (TYLCV protein as an example), lane 1 being Marker, lane 2 being pure SPG-HUH protein, lane 3 being a single-stranded DNA reaction product of SPG-HUH and 59nt, and lane 4 being a single-stranded DNA reaction product of SPG-HUH and 107 nt. The SDS-PAGE analysis shows that only one DNA is connected to each fusion protein, namely the connection ratio of the protein nucleic acid is 1: 1.
59nt DNA sequence:
CGTATAATATTACCGCAGTGCTCACGACAGCGACAACTACCGACACTCCGAGCAGCACC（SEQ ID NO:4）。
107nt DNA sequence:
CGTATAATATTACCGCAGTGACCATCTACACATGACCCTCTATGAGCACAATAGTCAAAAGCTAACACTGTCAAAAACCTAAATGGCTATAGGGGCGTATTGTGACC（SEQ ID NO:5）。
and fourthly, separating and purifying the protein nucleic acid compound by using an anion exchange column HiTrap Q HP.
After the reaction of the SPG-HUH fusion protein and the single-stranded DNA is finished, free protein and free nucleic acid are contained in the solution besides the protein nucleic acid compound SPG-HUH-ssDNA, and the unnecessary components are preferably removed, so that the purity of the target product is improved, and the subsequent purification of the target pathogen antibody is facilitated.
In the case of TYLCV protein, the covalent linkage between TYLCV protein and a nucleic acid molecule of a specific sequence is realized after the reaction is carried out at 37 ℃ for 30 min in a molar ratio of 1:1 in the presence of 4mM divalent metal ions, but in this case, free protein and free nucleic acid exist in addition to the protein-nucleic acid complex in the solution, and if the free protein and the free nucleic acid are not removed, the subsequent application is influenced.
In the research process, the separation of protein-nucleic acid complexes was attempted by using gel exclusion chromatography, nickel affinity chromatography and magnetic bead method, but the separation effect was poor and the loss was significant. In consideration of the isoelectric point of the TYLCV protein, the TYLCV protein is positively charged and the nucleic acid molecule is negatively charged in a buffer solution of pH 5.0, and thus, can be separated and purified using an anion exchange column to obtain a protein-nucleic acid complex with high purity. As shown in FIG. 4A, the reaction solution was eluted through an anion exchange column to generate a plurality of elution peaks, and SDS-PAGE analysis was performed on protein samples of the peaks (FIG. 4B), indicating that the method can successfully separate and purify the high-purity protein-nucleic acid complex SPG-HUH-ssDNA. The corresponding nucleic acid sequence of FIG. 4 is 59nt in length.
SPG-HUH-ssDNA and target antibody IgG are covalently linked
FIG. 5 shows the result of a simple directional crosslinking reaction between SPG and an antibody, wherein the SPG is not linked to a nucleic acid. Protein and antibody are directionally modified, and SDS-PAGE picture analysis shows that Protein G inserted with unnatural amino acid and antibody can realize fixed-point crosslinking under 365nm light induction, and a band close to 85kDa is an antibody heavy chain for modifying the Protein. That is, the SPG can cross-link with the antibody without the ssDNA being attached.
Mixing the protein-nucleic acid compound SPG-HUH-ssDNA and the target antibody IgG in a molar ratio of 1:1, and reacting under 365nm ultraviolet light irradiation to realize the fixed-point crosslinking reaction between the two.
Due to the fact that the specific site of the SPG is inserted with the unnatural amino acid which induces site specific cross-linking, after the Protein G is specifically combined with the antibody, the Protein G and the heavy chain region of the antibody are directionally cross-linked through photocatalysis by utilizing the proximity effect between the amino acids, and a product IgG-SPG-HUH-ssDNA of the accurate antibody nucleic acid directional connection is obtained.
IgG-SPG-HUH-ssDNA purification
The inventive concept is explained in detail herein using specific examples, which are given only to aid in understanding the core concepts of the invention. It should be understood that any obvious modifications, equivalents and other improvements made by those skilled in the art without departing from the spirit of the present invention are included in the scope of the present invention.
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<211> 107
<212> DNA
<213> Artificial sequence
<400> 5
cgtataatat taccgcagtg accatctaca catgaccctc tatgagcaca atagtcaaaa 60
gctaacactg tcaaaaacct aaatggctat aggggcgtat tgtgacc 107
Claims (10)
1. A method for the directional ligation of nucleic acids of a precision antibody, comprising the steps of:
(1) HUH endonuclease and Protein G are connected through flexible Linker molecules to obtain fusion Protein;
(2) synthesizing a single-stranded DNA containing a site recognized by an endonuclease of the HUH type;
(3) mixing the fusion protein with the single-stranded DNA, and reacting in the presence of divalent metal ions to obtain a protein-nucleic acid compound;
(4) mixing the Protein nucleic acid compound with a target antibody, reacting under the condition of photocatalysis, and directionally crosslinking Protein G in the Protein nucleic acid compound and a heavy chain Fc section of the target antibody to realize directional connection of the target antibody and single-chain DNA;
wherein, the steps (1) and (2) are not in sequence;
the gene sequence of the Protein G needs to be modified by inserting codons of non-natural amino acids which are specifically crosslinked with light induction sites into at least one specific site;
the HUH endonuclease IS selected from TYLCV, TOPO I, IS608, A or Tra I.
2. The connecting method according to claim 1,
when the HUH endonuclease is TYLCV, the single-stranded DNA of the step (2) contains CAACTTGATA sequence;
when the HUH endonuclease is TOPO I, the single-stranded DNA in the step (2) contains a sequence of CCCTT;
when the HUH endonuclease IS IS608, the single-stranded DNA of step (2) contains a sequence of TATGTTAC;
when the HUH endonuclease is a ″, the single-stranded DNA of step (2) contains the sequence of CAACTTGATAT;
when the HUH endonuclease is TraI, the single-stranded DNA obtained in step (2) has the sequence of GTTTTTGCGTGGGGTGT.
3. The connecting method according to claim 2, wherein the divalent metal ions in the step (3) satisfy the following requirements:
when the HUH endonuclease is TYLCV, the concentration of the divalent metal ions is 4 mM;
when the HUH endonuclease is TOPO I, the concentration of the divalent metal ions is 1 mM;
when the HUH endonuclease IS IS608, the concentration of the divalent metal ion IS 20 mM;
when the HUH endonuclease is A, the concentration of the divalent metal ions is 4 mM;
when the HUH endonuclease is TraI, the concentration of the divalent metal ion is 4 mM.
4. The connecting method according to claim 1, wherein the reaction in the step (3) is carried out at 36 to 38 ℃ for 25 to 35 min.
5. The ligation process according to claim 1, wherein the product obtained in step (3) is further isolated and purified by passing through an anion exchange column HiTrap Q HP.
6. The method according to claim 1, wherein the Protein G gene sequence has a specific position selected from the group consisting of leucine at position 12, threonine at position 18, alanine at position 20, alanine at position 23, alanine at position 26, arginine at position 28 and glutamine at position 32.
7. The method of claim 1, wherein the unnatural amino acid is L-p-benzoylphenylalanine and the codon is the amber codon TAG.
8. The joining method according to claim 1, wherein the light irradiation condition in the step (4) is 365nm ultraviolet light irradiation.
9. The linking method according to claim 1, wherein the product is purified by nickel affinity chromatography after the photocatalytic reaction in step (4) is completed.
10. A detection antibody compound for immune PCR is characterized by comprising a target antibody and single-chain DNA, wherein the target antibody is covalently connected with the single-chain DNA through a connecting Protein, and the connecting Protein is HUH endonuclease and Protein G which are connected together through flexible Linker molecules; wherein
The single-stranded DNA contains a site recognized by an endonuclease of the HUH type;
protein G in the connexin is covalently cross-linked to the Fc fragment of the antibody of interest.
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Citations (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20070224620A1 (en) * | 2006-02-08 | 2007-09-27 | Promega Corporation | Compositions and methods for capturing and analyzing cross-linked biomolecules |
| US20090298088A1 (en) * | 2008-05-30 | 2009-12-03 | Belyaev Alexander S | Cleavable catalytic binding and detection system |
| US20100143259A1 (en) * | 2006-09-18 | 2010-06-10 | Institute Of Process Engineering, Chinese Academy Of Sciences | Fluorescent microspheres, the preparation method and uses thereof |
| US20110143955A1 (en) * | 2009-10-11 | 2011-06-16 | Weiner Michael P | Protein Capture, Detection and Quantitation |
| CN102159592A (en) * | 2008-06-30 | 2011-08-17 | 财团法人东京都医学研究机构 | Antibody complex, antigen detection method, and method for production of antibody complex |
| CN102994638A (en) * | 2012-11-29 | 2013-03-27 | 深圳市达科为生物工程有限公司 | Sandwich immuno-PCR (polymerase chain reaction) detection method and kit for to-be-detected object in biological samples |
| CN103454427A (en) * | 2012-06-03 | 2013-12-18 | 河北省健海生物芯片技术有限责任公司 | Method for detecting trace protein in blood by utilizing antibody-nucleic acid combined amplification technology |
| CN105510591A (en) * | 2014-09-22 | 2016-04-20 | 程永升 | Detection kit and detection method using antibody to modify immuno-PCR reaction |
| US20160289306A1 (en) * | 2013-08-30 | 2016-10-06 | Kaneka Corporation | Fab REGION-BINDING PEPTIDE |
| CN106591436A (en) * | 2016-11-24 | 2017-04-26 | 湖南大学 | Proximity nucleic acid probe for detecting low-abundance antibody in human body, kit, and preparation method thereof |
| WO2017181339A1 (en) * | 2016-04-19 | 2017-10-26 | 廖世奇 | Method and kit for simultaneous detection of protein ligand and gene |
| US20180344871A1 (en) * | 2015-05-12 | 2018-12-06 | The Trustees Of The University Of Pennsylvania | Compositions and methods for making antibody conjugates |
| CN110452303A (en) * | 2019-08-08 | 2019-11-15 | 中国科学院武汉病毒研究所 | It is covalently attached the method and application of nucleic acid and peptide or protein |
-
2021
- 2021-01-06 CN CN202110010916.5A patent/CN112778426B/en active Active
Patent Citations (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20070224620A1 (en) * | 2006-02-08 | 2007-09-27 | Promega Corporation | Compositions and methods for capturing and analyzing cross-linked biomolecules |
| US20100143259A1 (en) * | 2006-09-18 | 2010-06-10 | Institute Of Process Engineering, Chinese Academy Of Sciences | Fluorescent microspheres, the preparation method and uses thereof |
| US20090298088A1 (en) * | 2008-05-30 | 2009-12-03 | Belyaev Alexander S | Cleavable catalytic binding and detection system |
| CN102159592A (en) * | 2008-06-30 | 2011-08-17 | 财团法人东京都医学研究机构 | Antibody complex, antigen detection method, and method for production of antibody complex |
| US20110143955A1 (en) * | 2009-10-11 | 2011-06-16 | Weiner Michael P | Protein Capture, Detection and Quantitation |
| CN103454427A (en) * | 2012-06-03 | 2013-12-18 | 河北省健海生物芯片技术有限责任公司 | Method for detecting trace protein in blood by utilizing antibody-nucleic acid combined amplification technology |
| CN102994638A (en) * | 2012-11-29 | 2013-03-27 | 深圳市达科为生物工程有限公司 | Sandwich immuno-PCR (polymerase chain reaction) detection method and kit for to-be-detected object in biological samples |
| US20160289306A1 (en) * | 2013-08-30 | 2016-10-06 | Kaneka Corporation | Fab REGION-BINDING PEPTIDE |
| CN105510591A (en) * | 2014-09-22 | 2016-04-20 | 程永升 | Detection kit and detection method using antibody to modify immuno-PCR reaction |
| US20180344871A1 (en) * | 2015-05-12 | 2018-12-06 | The Trustees Of The University Of Pennsylvania | Compositions and methods for making antibody conjugates |
| WO2017181339A1 (en) * | 2016-04-19 | 2017-10-26 | 廖世奇 | Method and kit for simultaneous detection of protein ligand and gene |
| CN106591436A (en) * | 2016-11-24 | 2017-04-26 | 湖南大学 | Proximity nucleic acid probe for detecting low-abundance antibody in human body, kit, and preparation method thereof |
| CN110452303A (en) * | 2019-08-08 | 2019-11-15 | 中国科学院武汉病毒研究所 | It is covalently attached the method and application of nucleic acid and peptide or protein |
Non-Patent Citations (2)
| Title |
|---|
| ANNA LUIZA F. V. ASSUMPÇÃO: "Immuno PCR in cancer and non cancer related diseases a review", 《VETERINARY QUARTERLY》 * |
| ANNA LUIZA F. V. ASSUMPÇÃO: "Immuno PCR in cancer and non cancer related diseases a review", 《VETERINARY QUARTERLY》, vol. 36, no. 2, 28 March 2016 (2016-03-28), pages 63 - 70, XP055333700, DOI: 10.1080/01652176.2016.1164912 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112851771A (en) * | 2021-02-09 | 2021-05-28 | 中国科学院武汉病毒研究所 | Method for linking nucleic acids to proteins or peptides |
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|---|---|
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