CN116948034A - Active type heterophagy antibody blocker Block-5S and preparation method thereof - Google Patents
Active type heterophagy antibody blocker Block-5S and preparation method thereof Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
- C07K2317/14—Specific host cells or culture conditions, e.g. components, pH or temperature
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/106—Plasmid DNA for vertebrates
- C12N2800/107—Plasmid DNA for vertebrates for mammalian
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses an active type heterophagy antibody blocker Block-5S and a preparation method thereof, wherein the blocker Block-5S comprises a light chain Block-L and a heavy chain Block-H; wherein the CDR sequences of the 6 complementarity determining regions of the light chain Block-L are respectively: CDR1: RASESISNNIH, CDR2: RANQSIS, CDR3: LQYDSWPLT, CDR4: HASQNLSGFLS, CDR5: AASTLD, CDR6: LQYTSFPITFG; the 6 complementarity determining region CDR sequences of heavy chain Block-H are: CDR1: fsday, CDR2: TIGNGGSNHYTIYVESV, CDR3: AYYGNYGDSTRLAY, CDR4: FSIKDAYIH, CDR5: WIYPANGYTKYNEKFRG, CDR6: EPLPHNYAMD. The blocker Block-5S provided by the invention can be specifically combined with the autophagic antibody or the rheumatoid factor, is obtained by an in-vitro recombination method, and has the characteristics of simple production, good blocking effect, low use concentration and the like.
Description
Technical Field
The invention relates to an active type heterophagic antibody blocker Block-5S and a preparation method thereof, belonging to the technical field of biomedicine.
Background
In the in vitro diagnostic industry (IVD industry), an antibody-antigen binding detection method based on the immunological principle is widely used, because the antibody-antigen binding has high affinity and specificity, so that the antibody-antigen has high accuracy in clinical detection, for example, a specific antibody is used for detecting a certain pathological antigen or characteristic antigen in a patient such as cancer, inflammation, liver fibrosis, infectious disease and the like.
However, in immunological tests, the presence of certain interferents in a sample of a patient's body fluid (typically blood/plasma) can interfere with the reaction between the test antibody and the analyte by non-specific binding to the test antibody, resulting in a bias in the test value. In particular in detection kits based on the double-antibody sandwich method, such interferents inhibit on the one hand the binding of the target analyte to the detection antibody, giving rise to false negatives; on the other hand, it can bind to the coated and detected antibodies in the absence of the target analyte, thereby producing false positives. The interference can greatly influence the detection result, so that the research and development difficulty of the kit is increased.
There are two general classes of interferents that commonly cause detection bias, one class being amphotropic antibodies, which are found in patient serum/plasma, that bind to immunoglobulins of other species, including detection antibodies that are reagents for immunoassays, such as murine IgG, sheep IgG, and rabbit IgG. Such allophilic antibodies include Human Anti-mouse antibodies (HAMA), human Anti-sheep (HAGA), and Human Anti-rabbit (HARA), among others. For example, in some patients who have been exposed to murine antibodies or injected with human-murine chimeric antibody drugs, human anti-murine (HAMA) antibodies are present in their body fluids, and when both pairs of antibodies used in sandwich immunoassays are murine antibodies, the human anti-murine antibodies contained in the serum sample can bind the coated antibodies and the detection antibodies in the absence of analyte, thereby affecting the normal double antibody sandwich reaction of the serum sample resulting in erroneous detection results. Another class of interferents are rheumatoid factors, which react with the Fc-segment epitopes of self-IgG molecules, and are contained in part in the body fluids of patients suffering from rheumatoid arthritis or autoimmune diseases. Some rheumatoid factors recognize common antigenic determinants of human and animal IgG and thus can cause cross-reactions.
A blocking agent is an agent that eliminates immunodetection interference by blocking an interfering substance. In the development of immunodiagnosis kit, adding animal serum and purifying animal IgG with high efficiency is an effective method for eliminating interference caused by heterophagic antibody, and in order to improve blocking effect, animal IgG is often crosslinked to obtain crosslinked IgG polymer. Methods for crosslinking IgG into a polymer include physical methods, which often employ high temperature treatment, low pH treatment, etc., and chemical methods, which have poor controllability, limited ability to form a polymer, and some damage to the antibody. The usual chemical approach is to crosslink IgG monomers into polymers by forming intermolecular covalent bonds using some chemical reagent that reacts with the amino groups on the lysine residues of the antibody. Typical chemical reagents are bis-succinimidyl suberate (DSS), glutaraldehyde and suberate. IgG multimers formed by physical or chemical cross-linking will greatly increase the ability of animal IgG to eliminate interference from the detection of antibodies that are heterophagic in the sample.
Whether animal serum or high purity animal IgG is used as a blocking agent or cross-linked animal IgG is used as a blocking agent, such blocking agents are passive and are added in very large amounts and at very high concentrations during use.
Disclosure of Invention
The invention aims to provide an active type heterophagic antibody blocker Block-5S and a preparation method thereof, wherein the blocker Block-5S can be specifically combined with heterophagic antibodies or rheumatoid factors and is obtained by an in-vitro recombination method, and the blocker Block-5S has the characteristics of simple production, good blocking effect, low use concentration and the like.
The technical scheme adopted by the invention is as follows:
an active heterophagic antibody blocker Block-5S comprises two recombinant protein components, namely Block-L and Block-H; wherein, the Block-L is connected with the Block-L1 and the Block-L2 through flexible short peptide NAAAANAAAA; block-H is formed by connecting Block-H1 and Block-H2 through a flexible short peptide NAAAANAAAA; finally, block-L shares six complementarity determining regions, corresponding to the CDR sequences:
CDR1：RASESISNNIH，
CDR2：RANQSIS，
CDR3：LQYDSWPLT，
CDR4：HASQNLSGFLS，
CDR5：AASTLD，
CDR6：LQYTSFPITFG；
Block-H also has six complementarity determining regions corresponding to the CDR sequences:
CDR1：FSDAYMY，
CDR2：TIGNGGSNHYTIYVESV，
CDR3：AYYGNYGDSTRLAY，
CDR4：FSIKDAYIH，
CDR5：WIYPANGYTKYNEKFRG，
CDR6：EPLPHNYAMD。
preferably, the amino acid sequences of six complementarity determining regions CDR1, CDR2, CDR3, CDR4, CDR5 and CDR6 of the Block-L are respectively shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO. 6;
the amino acid sequences of the 6 complementary determining regions CDR1, CDR2, CDR3, CDR4, CDR5 and CDR6 of the Block-H are respectively shown as SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO. 12.
Preferably, the amino acid sequence of the Block-L1 is shown as SEQ ID NO. 13; the amino acid sequence of the Block-L2 is shown as SEQ ID NO. 14.
Preferably, the amino acid sequence of the Block-H1 is shown as SEQ ID NO. 15; the amino acid sequence of the Block-H2 is shown as SEQ ID NO. 16.
Preferably, the amino acid sequence of the Block-L is shown as SEQ ID NO. 17; the nucleotide sequence of the Block-L is shown as SEQ ID NO. 18.
Preferably, the amino acid sequence of the Block-H is shown as SEQ ID NO. 19; the nucleotide sequence of the Block-H is shown as SEQ ID NO. 20.
The preparation method of the active type autophagy antibody blocker Block-5S specifically comprises the following steps: the nucleotide sequences of the Block-L and the Block-H are correctly expressed through artificial synthesis, the nucleotide is cloned to a proper plasmid vector through a gene cloning technology, and the recombinant plasmid is transfected to a host expression cell, and the nucleotide is transcribed and translated in the host cell and expresses the recombinant proteins of the Block-L and the Block-H; purifying the two recombinant proteins, and mixing or purifying after mixing.
The invention has the beneficial effects that:
1. the blocker Block-5S protein is obtained through recombinant expression, the blocker Block-5S can specifically bind with the autophagic antibody or the rheumatoid factor, and is obtained through an in vitro recombination method, the production is simple, the use concentration is low, the blocking capability of blocking the autophagic antibody is good, and the interference caused by factors such as the autophagic antibody and the rheumatoid factor can be well eliminated.
2. The purpose of using the flexible peptide NAAAANAAAA is to form better connection between the Block-L1 and the Block-L2 or between the Block-H1 and the Block-H2, so that the formed Block-L or Block-H protein has a good flexible stable structure. The active protein can be well folded when expressed in cells, and cysteine on the Block-L and the Block-H can form disulfide bonds in the expression process, so that the multifunctional blocker Block-5R is assembled in the cells, and the finished blocker Block-5S is obtained through affinity or ion exchange purification.
Drawings
FIG. 1 is a schematic diagram showing the elimination degree of the false positive phenomenon of a serum sample after no blocking agent is added in the false positive sample, a passive blocking agent HBR-6 is added, and the blocking agent Block-5S of the invention is added.
Detailed Description
The invention is described in detail below with reference to the drawings and examples.
Example 1
The embodiment is an active type autophagy antibody blocker Block-5S, which comprises two recombinant protein components, namely Block-L and Block-H; wherein, the light chain Block-L is an antibody light chain Block-L1 and a light chain Block-L2 which are connected by a flexible short peptide NAAAANAAAA and have different blocking effects; the heavy chain Block-H is an antibody heavy chain Block-H1 and Block-H2 which are connected through a flexible short peptide NAAAANAAAA and have different blocking effects; finally, the light chain Block-L shares six complementarity determining regions, corresponding to the CDR sequences:
CDR1：RASESISNNIH，
CDR2：RANQSIS，
CDR3：LQYDSWPLT，
CDR4：HASQNLSGFLS，
CDR5：AASTLD，
CDR6：LQYTSFPITFG；
the heavy chain Block-H also has six complementarity determining regions corresponding to the CDR sequences:
CDR1：FSDAYMY，
CDR2：TIGNGGSNHYTIYVESV，
CDR3：AYYGNYGDSTRLAY，
CDR4：FSIKDAYIH，
CDR5：WIYPANGYTKYNEKFRG，
CDR6：EPLPHNYAMD。
the amino acid sequences of the six complementarity determining regions CDR1, CDR2, CDR3, CDR4, CDR5, CDR6 of light chain Block-L are:
CDR1：SEQ ID NO.1：ArgAlaSerGluSerIleSerAsnAsnIleHis；
CDR2：SEQ ID NO.2：ArgAlaAsnGlnSerIleSer；
CDR3：SEQ ID NO.3：LeuGlnTyrAspSerTrpProLeuThr；
CDR4：SEQ ID NO.4：HisAlaSerGlnAsnLeuSerGlyPheLeuSer；
CDR5：SEQ ID NO.5：AlaAlaSerThrLeuAsp；
CDR6：SEQ ID NO.6：LeuGlnTyrThrSerPheProIleThrPheGly。
the amino acid sequences of the 6 complementarity determining regions CDR1, CDR2, CDR3, CDR4, CDR5, CDR6 of heavy chain BlockH are respectively:
CDR1：SEQ ID NO.7：PheSerAspAlaTyrMetTyr；
CDR2：SEQ ID NO.8：ThrIleGlyAsnGlyGlySerAsnHisTyrThrIleTyrValGluSerVal；
CDR3：SEQ ID NO.9：AlaTyrTyrGlyAsnTyrGlyAspSerThrArgLeuAlaTyr；
CDR4：SEQ ID NO.10：PheSerIleLysAspAlaTyrIleHis；
CDR5：SEQ ID NO.11：TrpIleTyrProAlaAsnGlyTyrThrLysTyrAsnGluLysPheArgGly；
CDR6：SEQ ID NO.12：GluProLeuProHisAsnTyrAlaMetAsp。
the CDR is the active center of the blocker Block-5S, and is the core sequence which enables the Block-5S to have blocking effect.
The amino acid sequence of the light chain Block-L1 is: SEQ ID NO.13:
Block-L1-amino acid sequence
AspIleValMetThrGlnSerProAlaThrMetSerValSerLeuGlyGluArgValSer
IleSerCysArgAlaSerGluSerIleSerAsnAsnIleHisTrpPheGlnGlnLysPro
GlyLysSerProLysThrLeuIleTyrArgAlaAsnGlnSerIleSerGlyValProSer
ArgPheSerGlySerGlySerGlyGlnAspTyrThrLeuSerIleSerSerLeuGluThr
GluAspMetGlyIleTyrTyrCysLeuGlnTyrAspSerTrpProLeuThrPheGlyAla
GlyThrLysLeuGluIleIleArgAla
The amino acid sequence of the light chain Block-L2 is: SEQ ID NO.14:
Block-L2-amino acid sequence
AspIleGlnMetAsnGlnSerProSerSerLeuSerValSerLeuGlyAspArgValSer
LeuThrCysHisAlaSerGlnAsnLeuSerGlyPheLeuSerTrpTyrGlnGlnLysPro
GlyAsnIleProLysLeuLeuIleTyrAlaAlaSerThrLeuAspThrGlyAspProSer
ArgIleSerGlySerThrSerGlySerAspTyrSerLeuThrIleSerSerLeuGlnPro
GluAspIleAlaThrTyrTyrCysLeuGlnTyrThrSerPheProIleThrPheGlyAla
GlyThrLysLeuGluIleLysArgAla
The amino acid sequence of the heavy chain Block-H1 is: SEQ ID NO.15:
Block-H1-amino acid sequence
GluValGlnGlyGluGluSerGlyGlyGlyLeuValGlnProGlyGlySerLeuLysLeu
SerCysAlaAlaPheGlyPheThrPheSerAspAlaTyrMetTyrTrpValArgGlnSer
ProGluLysGlyLeuGluTrpValAlaThrIleGlyAsnGlyGlySerAsnHisTyrThr
IleTyrValGluSerValLysGlyArgPheThrIleSerArgAspAspSerLysSerSer
ValTyrLeuGlnMetAsnSerLeuArgThrGluAspThrAlaMetTyrTyrCysAlaTyr
TyrGlyAsnTyrGlyAspSerThrArgLeuAlaTyrTrpGlyGlnGlyThrLeuValThr
ValSerSer
The amino acid sequence of the heavy chain Block-H2 is: SEQ ID NO.16:
Block-H2-amino acid sequence
GluValGlnLeuGlnGlnSerGlyProGluLeuValLysProGlyAlaLeuValLysIle
SerCysLysAlaSerAspPheSerIleLysAspAlaTyrIleHisTrpValLysGlnArg
ProLysGlnGlyLeuGluTrpIleGlyTrpIleTyrProAlaAsnGlyTyrThrLysTyr
AsnGluLysPheArgGlyLysAlaThrLeuThrAlaAspLysSerSerSerThrAlaTyr
MetGlnLeuSerSerLeuThrSerGluAspSerAlaValTyrPheCysValArgGluPro
The amino acid sequence of LeuProHisnTyrAlaMetAspTyrTrpGlyGlnGlyThrValThrValSerSer light chain Block-L: SEQ ID NO.17:
Block-L-amino acid sequence
AspIleValMetThrGlnSerProAlaThrMetSerValSerLeuGlyGluArgValSer
IleSerCysArgAlaSerGluSerIleSerAsnAsnIleHisTrpPheGlnGlnLysPro
GlyLysSerProLysThrLeuIleTyrArgAlaAsnGlnSerIleSerGlyValProSer
ArgPheSerGlySerGlySerGlyGlnAspTyrThrLeuSerIleSerSerLeuGluThr
GluAspMetGlyIleTyrTyrCysLeuGlnTyrAspSerTrpProLeuThrPheGlyAla
GlyAsnAlaAlaAlaAlaAsnAlaAlaAlaAlaAspIleGlnMetAsnGlnSerProSer
SerLeuSerValSerLeuGlyAspArgValSerLeuThrCysHisAlaSerGlnAsnLeu
SerGlyPheLeuSerTrpTyrGlnGlnLysProGlyAsnIleProLysLeuLeuIleTyr
AlaAlaSerThrLeuAspThrGlyAspProSerArgIleSerGlySerThrSerGlySer
AspTyrSerLeuThrIleSerSerLeuGlnProGluAspIleAlaThrTyrTyrCysLeu
GlnTyrThrSerPheProIleThrPheGlyAlaGlyThrLysLeuGluIleLysArgAla
The nucleotide sequence of the light chain Block-L is: SEQ ID NO.18:
Block-L-nucleotide sequence
GATATTGTGatgACCCAGAGCCCGGCGACCatgAGCGTGAGCCTGGGCGAACGTGTG
AGCATTAGCTGCCGTGCGAGCGAAAGCATTAGCAACAACATTCATTGGTTTCAGC
AGAAACCGGGCAAAAGCCCGAAAACCCTGATTTATCGTGCGAACCAGAGCATTAG
CGGCGTGCCGAGCCGTTTTAGCGGCAGCGGCAGCGGCCAGGATTATACCCTGAGC
ATTAGCAGCCTGGAAACCGAAGATatgGGCATTTATTATTGCCTGCAGTATGATAGC
TGGCCGCTGACCTTTGGCGCGGGCAACGCGGCGGCGGCGAACGCGGCGGCGGCG
GATATTCAGatgAACCAGAGCCCGAGCAGCCTGAGCGTGAGCCTGGGCGATCGTGT
GAGCCTGACCTGCCATGCGAGCCAGAACCTGAGCGGCTTTCTGAGCTGGTATCAG
CAGAAACCGGGCAACATTCCGAAACTGCTGATTTATGCGGCGAGCACCCTGGATA
CCGGCGATCCGAGCCGTATTAGCGGCAGCACCAGCGGCAGCGATTATAGCCTGAC
CATTAGCAGCCTGCAGCCGGAAGATATTGCGACCTATTATTGCCTGCAGTATACCA
The amino acid sequence of GCTTTCCGATTACCTTTGGCGCGGGCACCAAACTGGAAATTAAACGTGCG heavy chain Block-H is: SEQ ID NO.19:
Block-H-amino acid sequence
GluValGlnGlyGluGluSerGlyGlyGlyLeuValGlnProGlyGlySerLeuLysLeu
SerCysAlaAlaPheGlyPheThrPheSerAspAlaTyrMetTyrTrpValArgGlnSer
ProGluLysGlyLeuGluTrpValAlaThrIleGlyAsnGlyGlySerAsnHisTyrThr
IleTyrValGluSerValLysGlyArgPheThrIleSerArgAspAspSerLysSerSer
ValTyrLeuGlnMetAsnSerLeuArgThrGluAspThrAlaMetTyrTyrCysAlaTyr
TyrGlyAsnTyrGlyAspSerThrArgLeuAlaTyrTrpGlyGlnGlyThrLeuValAsn
AlaAlaAlaAlaAsnAlaAlaAlaAlaGluValGlnLeuGlnGlnSerGlyProGluLeu
ValLysProGlyAlaLeuValLysIleSerCysLysAlaSerAspPheSerIleLysAsp
AlaTyrIleHisTrpValLysGlnArgProLysGlnGlyLeuGluTrpIleGlyTrpIle
TyrProAlaAsnGlyTyrThrLysTyrAsnGluLysPheArgGlyLysAlaThrLeuThr
AlaAspLysSerSerSerThrAlaTyrMetGlnLeuSerSerLeuThrSerGluAspSer
AlaValTyrPheCysValArgGluProLeuProHisAsnTyrAlaMetAspTyrTrpGly
GlnGlyThrSerValThrValSerSer
The nucleotide sequence of the heavy chain Block-H is: SEQ ID NO.20:
Block-H-nucleotide sequence
GAAGTGCAGGGCGAAGAAAGCGGCGGCGGCCTGGTGCAGCCGGGCGGCAGCCTG
AAACTGAGCTGCGCGGCGTTTGGCTTTACCTTTAGCGATGCGTATatgTATTGGGTG
CGTCAGAGCCCGGAAAAAGGCCTGGAATGGGTGGCGACCATTGGCAACGGCGGC
AGCAACCATTATACCATTTATGTGGAAAGCGTGAAAGGCCGTTTTACCATTAGCCG
TGATGATAGCAAAAGCAGCGTGTATCTGCAGatgAACAGCCTGCGTACCGAAGATA
CCGCGatgTATTATTGCGCGTATTATGGCAACTATGGCGATAGCACCCGTCTGGCGT
ATTGGGGCCAGGGCACCCTGGTGAACGCGGCGGCGGCGAACGCGGCGGCGGCGG
AAGTGCAGCTGCAGCAGAGCGGCCCGGAACTGGTGAAACCGGGCGCGCTGGTGA
AAATTAGCTGCAAAGCGAGCGATTTTAGCATTAAAGATGCGTATATTCATTGGGT
GAAACAGCGTCCGAAACAGGGCCTGGAATGGATTGGCTGGATTTATCCGGCGAAC
GGCTATACCAAATATAACGAAAAATTTCGTGGCAAAGCGACCCTGACCGCGGATA
AAAGCAGCAGCACCGCGTATatgCAGCTGAGCAGCCTGACCAGCGAAGATAGCGCG
GTGTATTTTTGCGTGCGTGAACCGCTGCCGCATAACTATGCGatgGATTATTGGGGC
CAGGGCACCAGCGTGACCGTGAGCAGC
Selecting six portions of dried meatThe serum of the interference is used as a false positive experiment detection sample (the sample does not contain tumor marker CA125 antigen), a pair of tumor marker CA125 antibodies are adopted to detect six serum samples in an enzyme-linked immunosorbent assay (ELISA), and due to the existence of the interference, the false positive phenomenon (OD) of different degrees exists in the six serum samples 450 The higher the reading, the more obvious the false positive is, and after 50 mug of passive blocker HBR-6 or 50 mug of blocker Block-5S disclosed by the invention is added into the false positive sample, the false positive phenomenon of six serum samples is eliminated to different degrees, and the data show that the capacity of eliminating an interfering substance by the Block-5S is obviously stronger than that of the passive blocker HBR-6, as shown in figure 1.
Example 2
The embodiment is a preparation method of an active type autophagy antibody blocker Block-5S, which comprises the following steps:
s1, artificially synthesizing a Block-L nucleotide sequence containing six Complementarity Determining Regions (CDRs), and cloning the Block-L nucleotide sequence onto a plasmid vector containing a CMV enhancer/promoter element conventionally;
s2, the constructed recombinant plasmid pCMV-Block-L is transformed into a conventional EcoliK-12 prokaryotic host cell, and 500 mug of endotoxin-free plasmid is extracted through the EcoliK-12 amplification plasmid;
s3, artificially synthesizing a Block-H nucleotide sequence containing six Complementarity Determining Regions (CDRs), and cloning the Block-H nucleotide sequence onto a plasmid vector containing a CMV enhancer/promoter element conventionally;
s4, the constructed recombinant plasmid pCMV-Block-H is transformed into a conventional EcoliK-12 prokaryotic host cell, and 500 mug of endotoxin-free plasmid is extracted through the EcoliK-12 amplification plasmid;
s5, culturing host expression cell CHO cells by using CHO culture medium, and increasing the cell density to 3X 10 6 Preparation of 2L of culture flask with a density of 3X 10 6 CHO cells in individual/mL;
s6, measuring 3mL of sterile transfection reagent PEI (1 mg/mL) to 10mL of DMEA culture medium, fully and uniformly mixing for 1min by using a vortex oscillator, standing for 5min, preparing 13mL of PEI mixed solution, and preparing two parts;
s7, respectively adding 500 mug recombinant plasmids pCMV-Block-L and pCMV-Block-H into the PEI mixed solution, fully and uniformly mixing for 1min by using a vortex oscillator, and standing for 15min to prepare the pCMV-Block-L and pCMV-Block-H plasmid mixed solution;
s8, respectively adding the mixed solution of the pCMV-Block-L and the pCMV-Block-H plasmids into 1L to increase the density to 3X 10 6 In the CHO cell liquid of the individual/mL, gently shake the culture flask while dripping, make the cell mix with transfected complex;
s9, placing the cells at 35 ℃ and 5% CO 2 Shaking culture at 150rpm/min, cell culture for 7 days, centrifuging at 3000 Xg for 5min, and collecting cell supernatant;
s10, purifying the supernatant by using an affinity column ProteinL and an ion exchange column SP-Q to obtain recombinant proteins Block-L and Block-H;
s11, mixing the Block-L, the Block-H and the like to obtain the product Block-5S.
Example 3
The embodiment is a preparation method of an active type autophagy antibody blocker Block-5S, which comprises the following steps:
s1, artificially synthesizing a Block-L nucleotide sequence containing six Complementarity Determining Regions (CDRs), and cloning the Block-L nucleotide sequence onto a plasmid vector containing a CMV enhancer/promoter element conventionally;
s2, the constructed recombinant plasmid pCMV-Block-L is transformed into a conventional EcoliK-12 prokaryotic host cell, and 500 mug of endotoxin-free plasmid is extracted through the EcoliK-12 amplification plasmid;
s3, artificially synthesizing a Block-H nucleotide sequence containing six Complementarity Determining Regions (CDRs), and cloning the Block-H nucleotide sequence onto a plasmid vector containing a CMV enhancer/promoter element conventionally;
s4, the constructed recombinant plasmid pCMV-Block-H is transformed into a conventional EcoliK-12 prokaryotic host cell, and 500 mug of endotoxin-free plasmid is extracted through the EcoliK-12 amplification plasmid;
s5, culturing host expression cell CHO cells by using CHO culture medium, and increasing the cell density to 3X 10 6 Preparation of 2L of culture flask with a density of 3X 10 6 CHO cells per m;
s6, measuring 6mL of sterile transfection reagent PEI (1 mg/mL) to 20mL of DMEA culture medium, fully and uniformly mixing for 1min by using a vortex oscillator, and standing for 5min to prepare 26mL of PEI mixed solution;
s7, mixing 500 mug of recombinant plasmids pCMV-Block-L and pCMV-Block-H, adding into PEI mixed solution, fully and uniformly mixing for 1min by using a vortex oscillator, and standing for 15min to prepare pCMV-Block-LH plasmid mixed solution;
s8, dropwise adding the pCMV-Block-LH plasmid mixed solution into 2L to increase the density to 3X 10 6 In the CHO cell liquid of the individual/mL, gently shake the culture flask while dripping, make the cell mix with transfected complex;
s9, placing the cells at 35 ℃ and 5% CO 2 Shaking culture at 150rpm/min, cell culture for 7 days, centrifuging at 3000 Xg for 5min, and collecting cell supernatant;
s10, purifying the supernatant by using an affinity column ProteinL to obtain a recombinant protein, namely a product Block-5S.
The foregoing is merely illustrative of the preferred embodiments of this invention, and it will be appreciated by those skilled in the art that variations and modifications may be made without departing from the principles of this invention, and such variations and modifications are to be regarded as being within the scope of this invention.
Claims (7)
1. An active heterophagic antibody blocker Block-5S, wherein the blocker Block-5S comprises a light chain Block-L and a heavy chain Block-H; wherein the CDR sequences of the 6 complementarity determining regions of the light chain Block-L are respectively:
CDR1：RASESISNNIH，
CDR2：RANQSIS，
CDR3：LQYDSWPLT，
CDR4：HASQNLSGFLS，
CDR5：AASTLD，
CDR6：LQYTSFPITFG；
the 6 complementarity determining region CDR sequences of heavy chain Block-H are:
CDR1：FSDAYMY，
CDR2：TIGNGGSNHYTIYVESV，
CDR3：AYYGNYGDSTRLAY，
CDR4：FSIKDAYIH，
CDR5：WIYPANGYTKYNEKFRG，
CDR6：EPLPHNYAMD。
2. the active isophagic antibody blocker Block-5S of claim 1, wherein the light chain Block-L is obtained by linking light chains Block-L1 and Block-L2 via a flexible short peptide NAAAANAAAA; block-H is obtained by connecting heavy chains of Block-H1 and Block-H2 through flexible short peptide NAAAANAAAA.
3. The active isophagy blocker Block-5S of claim 2 wherein the amino acid sequence of the light chain Block-L1 is shown in SEQ ID No. 13; the amino acid sequence of the light chain Block-L2 is shown as SEQ ID NO. 14.
4. The active isophagy blocker Block-5S of claim 2 wherein the amino acid sequence of heavy chain Block-H1 is shown in SEQ ID No. 15; the amino acid sequence of the heavy chain Block-H2 is shown as SEQ ID NO. 16.
5. The active isophagy blocker Block-5S of claim 1 wherein the light chain amino acid sequence is set forth in SEQ ID No. 17; the heavy chain amino acid sequence is shown as SEQ ID NO. 19.
6. The active isophagy blocker Block-5S of claim 1 wherein the light chain nucleotide sequence is set forth in SEQ ID No. 18; the heavy chain nucleotide sequence is shown as SEQ ID NO. 20.
7. The method for preparing the active type autophagy antibody blocker Block-5S according to any one of claims 1 to 6, comprising the following steps:
s1, artificially synthesizing a nucleotide sequence;
s2, cloning the nucleotide to a plasmid vector through a gene cloning technology to obtain a recombinant plasmid;
s3, transfecting the recombinant plasmid into CHO (Chinese CHO) expression cells, so that the nucleotide is transcribed and translated in the cells and the recombinant protein is expressed;
s4, expression and purification of the recombinant protein, and then mixing or mixing and expression before purification.
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