CN117466996A - Novel coronavirus detection kit and application - Google Patents
Novel coronavirus detection kit and application Download PDFInfo
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- CN117466996A CN117466996A CN202210911180.3A CN202210911180A CN117466996A CN 117466996 A CN117466996 A CN 117466996A CN 202210911180 A CN202210911180 A CN 202210911180A CN 117466996 A CN117466996 A CN 117466996A
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Classifications
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- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
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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
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- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
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- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
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- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
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- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
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- C07—ORGANIC CHEMISTRY
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- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/55—Fab or Fab'
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
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- C07K2317/565—Complementarity determining region [CDR]
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Abstract
The invention provides a novel coronavirus detection kit and application thereof. Wherein the novel coronavirus detection kit comprises a recombinant antibody, the recombinant antibody comprises a first recombinant antibody and/or a first recombinant antibody, the first recombinant antibody or the second recombinant antibody respectively and independently comprises a Fab fragment and an Fc fragment, the Fab fragment is derived from IgG, and the Fc fragment is derived from IgM. Can solve the problem of insufficient sensitivity of SARS-CoV-2 antigen detection in the prior art, and is suitable for the biotechnology field.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a novel coronavirus detection kit and application thereof.
Background
Coronavirus is a single-stranded positive-strand RNA virus of the envelope. Coronaviruses are divided into four genera based on their genetic evolution, serology and host-specific differences: alpha-coronavirus genus, beta-coronavirus genus, gamma-coronavirus genus, delta-coronavirus genus. Viruses of the genus β -coronavirus are divided into four groups A, B, C, D. The alpha and beta coronaviruses primarily infect mammals including humans, domestic animals, pets, and the gamma and delta coronaviruses primarily infect birds and mammals. The novel coronavirus (SARS-CoV-2) belongs to the genus beta-coronavirus, and the SARS-CoV-2 is adjacent to the class of SARS and SARS-like viruses as seen from the position of the evolution tree, and the evolutionarily common outer group of them is HKU9-1 coronavirus parasitic to fruit bats. SARS-CoV-2 is enveloped and the particles are round or oval, often polymorphic, with diameters of 50-200nm.
The nucleic acid of SARS-CoV-2 is a non-segmented single-stranded positive-strand RNA encoding two large overlapping open reading frames (ORF 1a and ORF1 b), 4 structural proteins (S, E, M and N proteins), and 9 accessory proteins. Among them, N protein (nucleocapsid protein) is a core component of a viral particle, which binds to viral genomic RNA, and packages the RNA into Ribonucleoprotein (RNP) complex. N protein is a phosphorylated protein, and phosphorylation can regulate the conformation of N protein and enhance the affinity with RNA. The N protein is relatively more conservative and is not easy to mutate; meanwhile, the N protein has higher expression abundance in the novel coronavirus. Thus, the N protein is often used as a target protein for novel coronavirus antigen detection reagents.
In clinical practice, the common in vitro diagnostic methods for novel coronaviruses include novel coronavirus nucleic acid detection, novel coronavirus antibody detection, and novel coronavirus antigen detection. Among them, nucleic acid detection has high sensitivity and has been used as a gold standard for novel coronavirus detection, but the application of nucleic acid detection is limited due to the long time consumption of nucleic acid detection, the severe detection environment, the need of specialized equipment and technicians, etc. The antigen detection has the advantages of high detection speed, simple and convenient operation and higher accuracy, and can be used as a complementary detection means or a substitute detection means for nucleic acid detection. In general, antigen detection reagents detect novel coronavirus N proteins in a sample. Development of a novel coronavirus antigen detection kit, the key raw material is an antibody aiming at N protein. However, after the antibody aiming at the N protein in the prior art is applied to a novel coronavirus antigen detection reagent, the detection sensitivity is insufficient, the specificity is low, and the clinical use requirement cannot be met. Therefore, development of an anti-N protein antibody with high sensitivity and high specificity for detection of novel coronavirus antigen is required.
Disclosure of Invention
The invention mainly aims to provide a novel coronavirus detection kit and application thereof, so as to solve the problem of insufficient sensitivity of SARS-CoV-2 antigen detection in the prior art.
In order to achieve the above object, according to a first aspect of the present invention, there is provided a novel coronavirus detection kit comprising a recombinant antibody comprising a first recombinant antibody and/or a second recombinant antibody, each of the first recombinant antibody or the second recombinant antibody independently comprising a Fab fragment and an Fc fragment, the Fab fragment being derived from IgG and the Fc fragment being derived from IgM.
Further, the Fab fragment comprises a SARS-CoV-2 nucleocapsid protein antibody variable region, the SARS-CoV-2 nucleocapsid protein antibody variable region comprises a SARS-CoV-2 nucleocapsid protein antibody light chain variable region and a SARS-CoV-2 nucleocapsid protein antibody heavy chain variable region, the SARS-CoV-2 nucleocapsid protein antibody light chain variable region of the first recombinant antibody comprises: SEQ ID NO:1: VL1-CDR1: SASQGIRNYLN; SEQ ID NO:2: VL1-CDR2: YTSTIHS; SEQ ID NO:3: VL1-CDR3: MQYFPSKTY; the SARS-CoV-2 nucleocapsid protein antibody heavy chain variable region of the first recombinant antibody comprises: SEQ ID NO:4: VH1-CDR1: DYNMG; SEQ ID NO:5: VH1-CDR2: ALIPNNGGTIYNQKFKG; SEQ ID NO:6: VH1-CDR3: EAYRDYDVKTWFAY; the SARS-CoV-2 nucleocapsid antibody light chain variable region of the second recombinant antibody comprises: SEQ ID NO:7: VL2-CDR1: QASESISNYLS; SEQ ID NO:8: VL2-CDR2: gassles; SEQ ID NO:9: VL2-CDR3: QGGYYSSGATFT; the SARS-CoV-2 nucleocapsid protein antibody heavy chain variable region of the second recombinant antibody comprises: SEQ ID NO:10: VH2-CDR1: SYAMS; SEQ ID NO:11: VH2-CDR2: ILSSDGNTYYASWAKG; SEQ ID NO:12: VH2-CDR3: FFYDDYDDLDIIFF.
Further, the amino acid sequence of the heavy chain variable region of the first recombinant antibody is as set forth in SEQ ID NO:13, as shown in: EVLLQQSGPELVNPGASVKIPCKASGYTFTDYNMGWVKQSHGKSLEWIGALIPNNGGTIYNQKFKGKATLTVDESSSTAYMELRSLTSEDTAVYYCAREAYRDYDVKTWFAYWGQGTLVTVSA; the amino acid sequence of the light chain variable region of the first recombinant antibody is shown in SEQ ID NO:14, as shown in: DIQMTQTTSSLSASLGDRVTLSCSASQGIRNYLNWYQQKPDGTVKLLIYYTSTIHSGVPSRFSGSGSGTDYSLTISNLEPEDIATYFCMQYFPSKTYFGGGTKLEIK; the amino acid sequence of the heavy chain variable region of the second recombinant antibody is as set forth in SEQ ID NO:15, as shown in: QSVEESGGRLVTPGTPLTLTCTVSGFSISSYAMSWVRQAPGKGLEYIGILSSDGNTYYASWAKGRFTLSKTSTTVDLKITSPTTEDTATYFCARFFYDDYDDLDIIFFWGQGTLVTVSS; the amino acid sequence of the light chain variable region of the second recombinant antibody is as shown in SEQ ID NO:16, as shown in: DPVMTQTPASVSGPVGGTVTINCQASESISNYLSWYQQKPGQPPKLLIYGASTLESGVPSRFSGSGSGTDFTLTISGVQCDDAATYYCQGGYYSSGATFTFGGGTEVVVK.
Further, the Fab fragment is murine; preferably, the amino acid sequence of the Fab fragment light chain portion of the first recombinant antibody is as set forth in SEQ ID NO:17, as shown in: DIQMTQTTSSLSASLGDRVTLSCSASQGIRNYLNWYQQKPDGTVKLLIYYTSTIHSGVPSRFSGSGSGTDYSLTISNLEPEDIATYFCMQYFPSKTYFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC; the amino acid sequence of the heavy chain part of the Fab fragment of the first recombinant antibody is shown in SEQ ID NO:18, as shown in: EVLLQQSGPELVNPGASVKIPCKASGYTFTDYNMGWVKQSHGKSLEWIGALIPNNGGTIYNQKFKGKATLTVDESSSTAYMELRSLTSEDTAVYYCAREAYRDYDVKTWFAYWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDC; the amino acid sequence of the Fab fragment light chain portion of the second recombinant antibody is set forth in SEQ ID NO:19, as shown in: DPVMTQTPASVSGPVGGTVTINCQASESISNYLSWYQQKPGQPPKLLIYGASTLESGVPSRFSGSGSGTDFTLTISGVQCDDAATYYCQGGYYSSGATFTFGGGTEVVVKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC; the amino acid sequence of the Fab fragment heavy chain portion of the second recombinant antibody is set forth in SEQ ID NO:20, as shown in: QSVEESGGRLVTPGTPLTLTCTVSGFSISSYAMSWVRQAPGKGLEYIGILSSDGNTYYASWAKGRFTLSKTSTTVDLKITSPTTEDTATYFCARFFYDDYDDLDIIFFWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC.
Further, the Fc fragment of the first recombinant antibody comprises A1-CH2, A1-CH3 and A1-CH4, and the amino acid sequences of A1-CH2 are shown in SEQ ID NO:21, as shown in: AVAEMNPNVNVFVPPRDGFSGPAPRKSKLICEATNFTPKPITVSWLKDGKLVESGFTTDPVTIENKGSTPQTYKVISTLTISEIDWLNLNVYTCRVDHRGLTFLKNVSSTCAA; the amino acid sequences of A1-CH3 are shown in SEQ ID NO:22, as shown in: SPSTDILTFTIPPSFADIFLSKSANLTCLVSNLATYETLNISWASQSGEPLETKIKIMESHPNGTFSAKGVASVCVEDWNNRKEFVCTVTHRDLPSPQKKFISKPN; the amino acid sequences of A1-CH4 are shown in SEQ ID NO:23, as shown in: EVHKHPPAVYLLPPAREQLNLRESATVTCLVKGFSPADISVQWLQRGQLLPQEKYVTSAPMPEPGAPGFYFTHSILTVTEEEWNSGETYTCVVGHEALPHLVTERTVDKSTGKPTLYNVSLIMSDTGGTCY.
Further, the amino acid sequence of the Fc fragment of the first recombinant antibody is as set forth in SEQ ID NO:24, as shown in: AVAEMNPNVNVFVPPRDGFSGPAPRKSKLICEATNFTPKPITVSWLKDGKLVESGFTTDPVTIENKGSTPQTYKVISTLTISEIDWLNLNVYTCRVDHRGLTFLKNVSSTCAASPSTDILTFTIPPSFADIFLSKSANLTCLVSNLATYETLNISWASQSGEPLETKIKIMESHPNGTFSAKGVASVCVEDWNNRKEFVCTVTHRDLPSPQKKFISKPNEVHKHPPAVYLLPPAREQLNLRESATVTCLVKGFSPADISVQWLQRGQLLPQEKYVTSAPMPEPGAPGFYFTHSILTVTEEEWNSGETYTCVVGHEALPHLVTERTVDKSTGKPTLYNVSLIMSDTGGTCY; preferably, the full-length amino acid sequence of the light chain of the first recombinant antibody is as set forth in SEQ ID NO:25, as shown in: METGLRWLLLVAVLKGVQCDIQMTQTTSSLSASLGDRVTLSCSASQGIRNYLNWYQQKPDGTVKLLIYYTSTIHSGVPSRFSGSGSGTDYSLTISNLEPEDIATYFCMQYFPSKTYFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC; preferably, the heavy chain full-length amino acid sequence of the first recombinant antibody is as set forth in SEQ ID NO:26, as shown in: METGLRWLLLVAVLKGVQCEVLLQQSGPELVNPGASVKIPCKASGYTFTDYNMGWVKQSHGKSLEWIGALIPNNGGTIYNQKFKGKATLTVDESSSTAYMELRSLTSEDTAVYYCAREAYRDYDVKTWFAYWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCPAVAEMNPNVNVFVPPRDGFSGPAPRKSKLICEATNFTPKPITVSWLKDGKLVESGFTTDPVTIENKGSTPQTYKVISTLTISEIDWLNLNVYTCRVDHRGLTFLKNVSSTCAASPSTDILTFTIPPSFADIFLSKSANLTCLVSNLATYETLNISWASQSGEPLETKIKIMESHPNGTFSAKGVASVCVEDWNNRKEFVCTVTHRDLPSPQKKFISKPNEVHKHPPAVYLLPPAREQLNLRESATVTCLVKGFSPADISVQWLQRGQLLPQEKYVTSAPMPEPGAPGFYFTHSILTVTEEEWNSGETYTCVVGHEALPHLVTERTVDKSTGKPTLYNVSLIMSDTGGTCY.
Further, the Fc fragment of the second recombinant antibody comprises B1-CH2, B1-CH3 and B1-CH4, and the amino acid sequences of B1-CH2 are shown in SEQ ID NO:27, as shown in: VIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLGQSMFTCRVDHRGLTFQQNASSMCVP; the amino acid sequence of B1-CH3 is shown in SEQ ID NO:28, as shown: DQDTAIRVFAIPPSFASIFLTKSTKLCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPK; the amino acid sequences of B1-CH4 are shown in SEQ ID NO: 29: GVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY.
Further, the amino acid sequence of the Fc fragment of the second recombinant antibody is as set forth in SEQ ID NO:30, as shown in: VIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLGQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY; preferably, the light chain full length amino acid sequence of the second recombinant antibody is as set forth in SEQ ID NO:31 shows: METGLRWLLLVAVLKGVQCDPVMTQTPASVSGPVGGTVTINCQASESISNYLSWYQQKPGQPPKLLIYGASTLESGVPSRFSGSGSGTDFTLTISGVQCDDAATYYCQGGYYSSGATFTFGGGTEVVVKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC; preferably, the heavy chain full length amino acid sequence of the second recombinant antibody is as set forth in SEQ ID NO:32, as shown in: METGLRWLLLVAVLKGVQCQSVEESGGRLVTPGTPLTLTCTVSGFSISSYAMSWVRQAPGKGLEYIGILSSDGNTYYASWAKGRFTLSKTSTTVDLKITSPTTEDTATYFCARFFYDDYDDLDIIFFWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCLPVIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLGQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY.
In order to achieve the above object, according to a second aspect of the present invention, there is provided a nucleocapsid protein recombinant antibody comprising the first recombinant antibody or the second recombinant antibody in the novel coronavirus detection kit described above.
In order to achieve the above object, according to a third aspect of the present invention, there is provided a recombinant antibody pair comprising: the first recombinant antibody or the second recombinant antibody in the novel coronavirus detection kit, and an antibody which is different from the first recombinant antibody or the second recombinant antibody and specifically binds to SARS-CoV-2; preferably, the recombinant antibody pair comprises a first recombinant antibody and a second recombinant antibody.
In order to achieve the above object, according to a fourth aspect of the present invention, there is provided a nucleic acid molecule encoding the above nucleocapsid protein recombinant antibody.
In order to achieve the above object, according to a fifth aspect of the present invention, there is provided a nucleic acid vector containing the above nucleic acid molecule, the nucleic acid vector comprising an expression cassette, a recombinant vector, a recombinant bacterium, or a recombinant virus.
In order to achieve the above object, according to a sixth aspect of the present invention, there is provided a novel coronavirus detection kit, or a nucleocapsid protein recombinant antibody, or an application of the recombinant antibody in preparing a SARS-CoV-2 virus antigen detection reagent or kit.
In order to achieve the above object, according to a seventh aspect of the present invention, there is provided a detection method for detecting a neocrown antigen in vitro, comprising the steps of: obtaining a biological sample from a subject, the biological sample selected from the group consisting of blood, plasma, serum, oral, throat and nose washes, bronchoalveolar lavage, sputum, saliva, stool or tissue, and the like. The biological sample of the subject is reacted with the first recombinant antibody and the second recombinant antibody simultaneously or separately, and antigen quantification/qualitative determination is achieved by detecting the signal forming the ternary complex of the first recombinant antibody-antigen-second recombinant antibody.
By applying the technical scheme of the invention, the first recombinant antibody and/or B is utilized 1 Compared with the recombinant antibodies aiming at SARS-CoV-2 virus in the prior art, the prepared novel coronavirus detection kit has better sensitivity and specificity, and can more accurately identify a sample to be detected.
Detailed Description
It should be noted that, in the case of no conflict, the embodiments and features in the embodiments may be combined with each other. The present invention will be described in detail with reference to examples.
As mentioned in the background art, the prior art has the problems of insufficient sensitivity and accuracy for detecting SARS-CoV-2 antigen. Thus, the inventors have tried to explore a novel coronavirus detection kit containing a novel recombinant antibody in the present application, thereby improving the sensitivity for SARS-CoV-2 antigen detection. A series of protection schemes of the present application are thus presented.
In a first exemplary embodiment of the present application, a novel coronavirus detection kit is provided, comprising a recombinant antibody comprising a first recombinant antibody and/or a second recombinant antibody, each independently comprising a Fab fragment and an Fc fragment, the Fab fragment being derived from IgG and the Fc fragment being derived from IgM.
The recombinant antibody formed by combining Fab derived from IgG and Fc derived from IgM has better detection sensitivity compared with the antibody of which Fab and Fc are derived from the same immunoglobulin. Multimeric antibody molecules can be formed using the "IgG fab+igm Fc" recombinant antibody protocol; the Fab region provides good affinity with the epitope, so that the antigen epitope binding valence of the antibody molecule is obviously increased after the multimer is formed, the binding of the antibody molecule and the antigen epitope is facilitated, and the detection sensitivity is improved.
The first recombinant antibody and the second recombinant antibody both have the capability of specifically binding SARS-CoV-2 antigen, and can respectively or jointly play a role in a novel coronavirus detection kit to improve the detection sensitivity of SARS-CoV-2 antigen.
The first recombinant antibody and the second recombinant antibody are recombinant antibodies, and are distinguished by "first", "second", and the like, and are not sequentially distinguished, but are merely for distinguishing the types of recombinant antibodies.
In a preferred embodiment, the Fab fragment comprises a SARS-CoV-2 nucleocapsid protein antibody variable region, the SARS-CoV-2 nucleocapsid protein antibody variable region comprises a SARS-CoV-2 nucleocapsid protein antibody light chain variable region and a SARS-CoV-2 nucleocapsid protein antibody heavy chain variable region, the SARS-CoV-2 nucleocapsid protein antibody light chain variable region of the first recombinant antibody comprises: SEQ ID NO:1: VL1-CDR1: SASQGIRNYLN; SEQ ID NO:2: VL1-CDR2: YTSTIHS; SEQ ID NO:3: VL1-CDR3: MQYFPSKTY; the SARS-CoV-2 nucleocapsid protein antibody heavy chain variable region of the first recombinant antibody comprises: SEQ ID NO:4: VH1-CDR1: DYNMG; SEQ ID NO:5: VH1-CDR2: ALIPNNGGTIYNQKFKG; SEQ ID NO:6: VH1-CDR3: EAYRDYDVKTWFAY; the SARS-CoV-2 nucleocapsid antibody light chain variable region of the second recombinant antibody comprises: SEQ ID NO:7: VL2-CDR1: QASESISNYLS; SEQ ID NO:8: VL2-CDR2: gassles; SEQ ID NO:9: VL2-CDR3: QGGYYSSGATFT; the SARS-CoV-2 nucleocapsid protein antibody heavy chain variable region of the second recombinant antibody comprises: SEQ ID NO:10: VH2-CDR1: SYAMS; SEQ ID NO:11: VH2-CDR2: ILSSDGNTYYASWAKG; SEQ ID NO:12: VH2-CDR3: FFYDDYDDLDIIFF.
The N-terminus of an antibody molecule has a region of widely varying amino acid sequences, known as the variable region, which includes the heavy chain variable region and the light chain variable region. Antibody heavy chain variable region (VH) and light chain variable region (VL) genes consist of 4 relatively conserved Framework Regions (FR) and 3 Complementarity Determining Regions (CDRs), respectively. There is a site in the variable region that can bind to an antigen, i.e., an antigen binding site. The non-CDR portion of the variable region is called the Framework Region (FR) which is beta-sheet structure and is conserved in base composition and arrangement sequence.
Thus mRNA can be extracted from hybridoma cells or spleen cells, reverse transcribed into cDNA, and synthetic primers amplify the light and heavy chain variable regions of the antibody molecule. The light chain signal peptide and the light chain VL-CL gene are recombined to form a light chain whole molecule gene, and the heavy chain signal peptide, the heavy chain VH-CH1 and the Fc gene are recombined to form a heavy chain whole molecule gene.
In a preferred embodiment, the amino acid sequence of the heavy chain variable region of the first recombinant antibody is as set forth in SEQ ID NO:13, as shown in: EVLLQQSGPELVNPGASVKIPCKASGYTFTDYNMGWVKQSHGKSLEWIGALIPNNGGTIYNQKFKGKATLTVDESSSTAYMELRSLTSEDTAVYYCAREAYRDYDVKTWFAYWGQGTLVTVSA; the amino acid sequence of the light chain variable region of the first recombinant antibody is shown in SEQ ID NO:14, as shown in: DIQMTQTTSSLSASLGDRVTLSCSASQGIRNYLNWYQQKPDGTVKLLIYYTSTIHSGVPSRFSGSGSGTDYSLTISNLEPEDIATYFCMQYFPSKTYFGGGTKLEIK; the amino acid sequence of the heavy chain variable region of the second recombinant antibody is as set forth in SEQ ID NO:15, as shown in: QSVEESGGRLVTPGTPLTLTCTVSGFSISSYAMSWVRQAPGKGLEYIGILSSDGNTYYASWAKGRFTLSKTSTTVDLKITSPTTEDTATYFCARFFYDDYDDLDIIFFWGQGTLVTVSS; the amino acid sequence of the light chain variable region of the second recombinant antibody is as shown in SEQ ID NO:16, as shown in: DPVMTQTPASVSGPVGGTVTINCQASESISNYLSWYQQKPGQPPKLLIYGASTLESGVPSRFSGSGSGTDFTLTISGVQCDDAATYYCQGGYYSSGATFTFGGGTEVVVK.
In a preferred embodiment, the Fab fragment of the recombinant antibody is murine; preferably, the amino acid sequence of the Fab fragment light chain portion of the first recombinant antibody is as set forth in SEQ ID NO:17, as shown in: DIQMTQTTSSLSASLGDRVTLSCSASQGIRNYLNWYQQKPDGTVKLLIYYTSTIHSGVPSRFSGSGSGTDYSLTISNLEPEDIATYFCMQYFPSKTYFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC; the amino acid sequence of the heavy chain part of the Fab fragment of the first recombinant antibody is shown in SEQ ID NO:18, as shown in: EVLLQQSGPELVNPGASVKIPCKASGYTFTDYNMGWVKQSHGKSLEWIGALIPNNGGTIYNQKFKGKATLTVDESSSTAYMELRSLTSEDTAVYYCAREAYRDYDVKTWFAYWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDC; the amino acid sequence of the Fab fragment light chain portion of the second recombinant antibody is set forth in SEQ ID NO:19, as shown in: DPVMTQTPASVSGPVGGTVTINCQASESISNYLSWYQQKPGQPPKLLIYGASTLESGVPSRFSGSGSGTDFTLTISGVQCDDAATYYCQGGYYSSGATFTFGGGTEVVVKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC; the amino acid sequence of the Fab fragment heavy chain portion of the second recombinant antibody is set forth in SEQ ID NO:20, as shown in: QSVEESGGRLVTPGTPLTLTCTVSGFSISSYAMSWVRQAPGKGLEYIGILSSDGNTYYASWAKGRFTLSKTSTTVDLKITSPTTEDTATYFCARFFYDDYDDLDIIFFWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC.
Fab fragments (Fragment of antigen binding), also known as antigen binding fragments, are regions of an antibody structure that can bind to antigen. Fab fragment consists of a complete light chain and heavy chain VH and CH1 domains with a molecular weight of about 5X 10 4 . Both the light chain and the heavy chain have a constant region and a variable region, and disulfide bonds are present between the light chain and the heavy chain. Thus the light chain portion of the Fab fragment is the light chain in the Fab fragment, including the light chain variable region (VL) and the light chain constant region (CL); the heavy chain portion of the Fab fragment is the heavy chain variable region (VH) and the heavy chain constant region CH1 of the heavy chain. The Fab fragment light chain portion and the Fab fragment heavy chain portion are disulfide linked to form a Fab fragment.
In a preferred embodiment, the Fc fragment of the first recombinant antibody comprises the amino acid sequences A1-CH2, A1-CH3 and A1-CH4, A1-CH2 having the amino acid sequences shown in SEQ ID NO:21, as shown in: AVAEMNPNVNVFVPPRDGFSGPAPRKSKLICEATNFTPKPITVSWLKDGKLVESGFTTDPVTIENKGSTPQTYKVISTLTISEIDWLNLNVYTCRVDHRGLTFLKNVSSTCAA; the amino acid sequences of A1-CH3 are shown in SEQ ID NO:22, as shown in: SPSTDILTFTIPPSFADIFLSKSANLTCLVSNLATYETLNISWASQSGEPLETKIKIMESHPNGTFSAKGVASVCVEDWNNRKEFVCTVTHRDLPSPQKKFISKPN; the amino acid sequences of A1-CH4 are shown in SEQ ID NO:23, as shown in: EVHKHPPAVYLLPPAREQLNLRESATVTCLVKGFSPADISVQWLQRGQLLPQEKYVTSAPMPEPGAPGFYFTHSILTVTEEEWNSGETYTCVVGHEALPHLVTERTVDKSTGKPTLYNVSLIMSDTGGTCY.
The Fc fragment is a crystallizable fragment (fragment crystallizable, fc) consisting of the CH2 and CH3 domains of Ig (immunoglobulin) (IgM and IgE also include a CH4 domain).
In a preferred embodiment, the Fc fragment of the first recombinant antibody has the amino acid sequence set forth in SEQ ID NO:24, as shown in: AVAEMNPNVNVFVPPRDGFSGPAPRKSKLICEATNFTPKPITVSWLKDGKLVESGFTTDPVTIENKGSTPQTYKVISTLTISEIDWLNLNVYTCRVDHRGLTFLKNVSSTCAASPSTDILTFTIPPSFADIFLSKSANLTCLVSNLATYETLNISWASQSGEPLETKIKIMESHPNGTFSAKGVASVCVEDWNNRKEFVCTVTHRDLPSPQKKFISKPNEVHKHPPAVYLLPPAREQLNLRESATVTCLVKGFSPADISVQWLQRGQLLPQEKYVTSAPMPEPGAPGFYFTHSILTVTEEEWNSGETYTCVVGHEALPHLVTERTVDKSTGKPTLYNVSLIMSDTGGTCY; preferably, the full-length amino acid sequence of the light chain of the first recombinant antibody is as set forth in SEQ ID NO:25, as shown in: METGLRWLLLVAVLKGVQCDIQMTQTTSSLSASLGDRVTLSCSASQGIRNYLNWYQQKPDGTVKLLIYYTSTIHSGVPSRFSGSGSGTDYSLTISNLEPEDIATYFCMQYFPSKTYFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC; preferably, the heavy chain full-length amino acid sequence of the first recombinant antibody is as set forth in SEQ ID NO:26, as shown in: METGLRWLLLVAVLKGVQCEVLLQQSGPELVNPGASVKIPCKASGYTFTDYNMGWVKQSHGKSLEWIGALIPNNGGTIYNQKFKGKATLTVDESSSTAYMELRSLTSEDTAVYYCAREAYRDYDVKTWFAYWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCPAVAEMNPNVNVFVPPRDGFSGPAPRKSKLICEATNFTPKPITVSWLKDGKLVESGFTTDPVTIENKGSTPQTYKVISTLTISEIDWLNLNVYTCRVDHRGLTFLKNVSSTCAASPSTDILTFTIPPSFADIFLSKSANLTCLVSNLATYETLNISWASQSGEPLETKIKIMESHPNGTFSAKGVASVCVEDWNNRKEFVCTVTHRDLPSPQKKFISKPNEVHKHPPAVYLLPPAREQLNLRESATVTCLVKGFSPADISVQWLQRGQLLPQEKYVTSAPMPEPGAPGFYFTHSILTVTEEEWNSGETYTCVVGHEALPHLVTERTVDKSTGKPTLYNVSLIMSDTGGTCY.
In a preferred embodiment, the Fc fragment of the second recombinant antibody comprises B1-CH2, B1-CH3 and B1-CH4, the amino acid sequences of B1-CH2 are as set forth in SEQ ID NO:27, as shown in: VIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLGQSMFTCRVDHRGLTFQQNASSMCVP; the amino acid sequence of B1-CH3 is shown in SEQ ID NO:28, as shown: DQDTAIRVFAIPPSFASIFLTKSTKLCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPK; the amino acid sequences of B1-CH4 are shown in SEQ ID NO: 29: GVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY.
In a preferred embodimentIn embodiments, the Fc fragment of the second recombinant antibody has the amino acid sequence set forth in SEQ ID NO:30, as shown in: VIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLGQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY; preferably, the light chain full length amino acid sequence of the second recombinant antibody is as set forth in SEQ ID NO:31 shows: METGLRWLLLVAVLKGVQCDPVMTQTPASVSGPVGGTVTINCQASESISNYLSWYQQKPGQPPKLLIYGASTLESGVPSRFSGSGSGTDFTLTISGVQCDDAATYYCQGGYYSSGATFTFGGGTEVVVKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC; preferably, the heavy chain full length amino acids of the second recombinant antibody: the sequence is shown in SEQ ID NO:32, as shown in: METGLRWLLLVAVLKGVQCQSVEESGGRLVTPGTPLTLTCTVSGFSISSYAMSWVRQAPGKGLEYIGILSSDGNTYYASWAKGRFTLSKTSTTVDLKITSPTTEDTATYFCARFFYDDYDDLDIIFFWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCLPVIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLGQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY. Preferably, the first recombinant antibody comprises recombinant antibody a 1 The second recombinant antibody comprises recombinant antibody B 1 。
In a second exemplary embodiment of the present application, a nucleocapsid protein recombinant antibody is provided, comprising the first recombinant antibody or the second recombinant antibody in the novel coronavirus assay kit described above. Preferably, the first recombinant antibody comprises recombinant antibody a 1 The second recombinant antibody comprises recombinant antibody B 1 。
Recombinant antibody A 1 And recombinant antibody B 1 Are all novel recombinant antibodies found in the present application.In the present application, the recombinant antibody scheme is adopted to recombine the Fab fragment of IgG and the Fc fragment of IgM to obtain the two antibodies with higher titers, which are suitable for detecting the neocrown antigen, including but not limited to the recombinant antibody A in the examples 1 And recombinant antibody B 1 . The sensitivity of the novel crown antigen detection reagent is greatly improved, and the two recombinant antibodies can be applied to other projects related to SARS-CoV-2.
In a third exemplary embodiment of the present application, there is provided a recombinant antibody pair comprising: the first recombinant antibody or the second recombinant antibody in the novel coronavirus detection kit, and an antibody which is different from the first recombinant antibody or the second recombinant antibody and specifically binds to SARS-CoV-2; preferably, the recombinant antibody pair comprises a first recombinant antibody and a second recombinant antibody. Preferably, the first recombinant antibody comprises recombinant antibody a 1 The second recombinant antibody comprises recombinant antibody B 1 。
Antibodies capable of binding to SARS-CoV-2, including the first recombinant antibody or the second recombinant antibody, as well as other antibodies capable of specifically recognizing SARS-CoV-2, are known in the art. Comprising a first recombinant antibody and/or B 1 Can be used in the fields of SARS-CoV-2 detection by a double antibody sandwich method, and the like, and improves the sensitivity and the specificity of SARS-CoV-2 detection.
In a fourth exemplary embodiment of the present application, a nucleic acid molecule encoding the above nucleocapsid protein recombinant antibody is provided.
In a fifth exemplary embodiment of the present application, a nucleic acid vector is provided comprising the nucleic acid molecule described above, including an expression cassette, a recombinant vector, a recombinant bacterium, or a recombinant virus.
Nucleic acid vectors in the present application, including nucleic acid-layered vectors, such as expression cassettes, recombinant vectors (e.g., plasmids), and the like; also included are organism-level vectors (hosts), such as host strains (recombinant bacteria) carrying the above-mentioned nucleic acid molecules, host viruses (recombinant viruses) carrying the above-mentioned nucleic acid molecules, and the like; also included are compound-layered vectors, such as nanoparticles for delivery of nucleic acid molecules, and the like.
In a sixth exemplary embodiment of the present application, a novel coronavirus detection kit, or a nucleocapsid protein recombinant antibody, or a recombinant antibody pair, or a nucleic acid molecule, or a nucleic acid vector, is provided for use in preparing a SARS-CoV-2 virus antigen detection reagent or kit.
In a seventh exemplary embodiment of the present application, there is provided a detection method for detecting a neocrown antigen in vitro, comprising the steps of: obtaining a biological sample from a subject, the biological sample selected from the group consisting of blood, plasma, serum, oral, throat and nose washes, bronchoalveolar lavage, sputum, saliva, stool or tissue, and the like. The biological sample of the subject is reacted with the first recombinant antibody and the second recombinant antibody simultaneously or separately, and antigen quantification/qualitative determination is achieved by detecting the signal forming the ternary complex of the first recombinant antibody-antigen-second recombinant antibody.
Unless otherwise specifically indicated, the various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or may be prepared by existing methods.
The practice of the present invention involves conventional virology, immunology, microbiology, molecular biology methods and DNA recombination techniques well known to those skilled in the art, some of which are described below for illustrative purposes, except where explicitly stated to the contrary. Other embodiments may be practiced in other embodiments, mutatis mutandis, of embodiments of the present description unless explicitly stated otherwise.
The advantageous effects of the present application will be explained in further detail below in connection with specific examples.
Example 1 antibody screening
The following antibody screens were performed using hybridoma fusion techniques. The principle of the hybridoma fusion technology is that the lymphocyte B cells of the spleen of an immunized mouse are subjected to fusion hybridization with myeloma cells of the mouse, so that the lymphocyte B cells can obtain unlimited proliferation capacity, the secretory antibodies in cell supernatants are subjected to ELISA detection, cell holes expressing specific antibodies are screened, and monoclonal antibodies are obtained through further subcloning. Through complete in vivo immune process, the animal can generate high affinity antibody, and the combined electrofusion ensures higher fusion rate and larger number of fusion, so that the target antibody with excellent performance can be screened.
The method comprises the following specific steps:
(1) immunized mice
Recombinant expression NP antigen (N protein, nucleocapsid protein) is taken as immunogen, SPF grade Balb/c mice with age of 6-8 weeks are fully emulsified by 200 mug/500 mug of NP protein-containing antigen and equal volume Freund's complete adjuvant, and injected subcutaneously in multiple points; antigen was emulsified 3 weeks apart with Freund's incomplete adjuvant, injected subcutaneously at multiple points, boosted 2 times, and finally injected intraperitoneally.
(2) Cell fusion and subclone screening
Taking spleen of immunized mice, grinding and separating to obtain dispersed single spleen cells, mixing the spleen cells and myeloma cells by using PEG, centrifuging after culture medium termination, suspending and spreading the mixture into a 96-well plate, changing liquid after one week, and taking supernatant for enzyme-free indirect detection. When the enzyme-immune method is used for detection, the coating antigen is recombinant NP antigen, and high-value positive holes of antibodies aiming at the coating antigen are selected. Subcloning the cells in the antibody high-value positive holes by a limiting dilution method, culturing for one week, continuing enzyme-free indirect detection, and selecting the antibody high-value holes for limiting dilution; repeating for 3-4 times until the detection hole is positive for the antibody and the cells in the hole are single colonies, and selecting the cell hole with the highest OD value for expansion culture to obtain a series of specific hybridoma cell strains, wherein the hybridoma cell strains comprise a hybridoma cell strain 3B8,4F5 aiming at NP antigen.
(3) Preparation and purification of monoclonal antibodies
Performing expansion culture on the single colony cells, and performing affinity purification by an SPA method to obtain the murine monoclonal antibody A 0 Murine monoclonal antibody B 0 。
(4) Affinity validation of murine monoclonal antibodies
Detection of 2 strains of murine monoclonal antibodies, murine monoclonal antibody A, by BIAcore technology 0 (clone number: 3B 8) and murine monoclonal antibody B 0 (clone number: 4F 5), and other hybridoma fines2 strains of mouse monoclonal antibody (mouse monoclonal antibody C) 0 Mouse monoclonal antibody D 0 ) Binding affinity to CoV-2Ag. The experimental procedure was performed according to biacore r 3000, biacore AB instructions. Specific affinity F (ab') fragments (Jackson lmmunoResearch) of the goat anti-murine lgG Fc region were immobilized on CM5 sensor chips by amine coupling chemistry. HBS-EP was selected as flow buffer at a flow rate of 10. Mu.L/min. After injection of 10. Mu.L of antibody (4. Mu.g/mL), different concentrations of CoV-2Ag were titrated at a flow rate of 30. Mu.L/min. Antibody affinity was determined at CoV-2Ag antigen concentrations of no more than 20 nM. The equilibrium dissociation constant Kd (M) represents the degree of dissociation of the antigen-antibody in the equilibrium state, and a smaller Kd (M) indicates a stronger affinity of the antigen-antibody.
BIAcore technology was used to detect murine monoclonal antibody affinity. The equilibrium dissociation constants Kd (M) of different murine mabs and CoV-2Ag are shown in Table 1.
TABLE 1
| Antibody numbering | Ka(M -1 s -1 ) | Kd(s -1 ) | Kd(M) |
| Murine monoclonal antibody A 0 | 4.25×10 5 | 6.25×10 -5 | 1.47×10 -10 |
| Murine monoclonal antibody B 0 | 3.99×10 5 | 6.01×10 -5 | 1.51×10 -10 |
| Mouse monoclonal antibody C 0 | 6.77×10 4 | 8.98×10 -5 | 1.33×10 -9 |
| Mouse monoclonal antibody D 0 | 7.29×104 | 10.1×10 -4 | 1.39×10 -9 |
(5) Murine monoclonal antibody pairing regimen screening
By enzyme-linked immunosorbent assay (ELISA), higher affinity murine monoclonal antibody A is obtained by screening 0 With mouse monoclonal antibody B 0 Coating and marking are respectively carried out, a novel coronavirus N antigen (CoV-2 Ag for short, manufacturer: new industrial organism) is detected, and an optimal pairing scheme is screened.
1) The murine monoclonal antibody A was diluted with 0.06M pH9.6 carbonate buffer solution, respectively 0 And mouse monoclonal antibody B 0 The final concentration was set to 6. Mu.g/mL. 96-well polystyrene plates were added, 0.1mL per well, and incubated at 37℃for 2 hours or overnight at 4 ℃. The next day, the wells were blocked with 0.02M PBS buffer, pH7.2, containing 10% calf serum (NBS), 0.15 mL/well, at 37℃for 2 hours.
2) 1mg HRP was dissolved in 0.1ml 0.1mol/L NaHCO 3 In the solution, the concentration is 10mg/mL; adding an equal volume of 10mmol/L NaIO 4 Mixing the solution, covering the bottle stopper, and keeping out light at room temperature for 2 hours; adding 2% (v/v) glycol solution with the same volume as the enzyme solution, mixing, and keeping out of the sun for 30 minutes at 4 ℃; 1mg of murine monoclonal antibody (murine monoclonal antibody A) 0 Or mouse monoclonal antibody B 0 ) Dialyzing against 50mmol/L CBS buffer solution with pH of 9.6 at 40deg.C overnight; finally, 40 mu L of dialyzate is added 5mg/mL NaBH 4 Mixing the solutions, light-shielding at 4deg.C for 2 hr, purifying with column, identifying enzyme binding substance, adding equal amount of glycerol, packaging, and preserving at-20deg.C.
3) CoV-2Ag was diluted in a gradient with 0.06M pH9.6 carbonate buffer, and the concentration after dilution was 20pg/mL, 100pg/mL, and 500pg/mL in this order.
4) The antigens after the gradient dilution of the different solutions are respectively dripped into a prepared 96-well microplate, and incubated for 30 minutes at 37 ℃.
5) After 3 washes with 10mM PBST, HRP-labeled murine antibodies were added, respectively, and incubated for 30 minutes at 37 ℃.
6) After 3 washes with 10mM PBST, 100. Mu.L of chromogenic substrate TMB was added to each well and incubated at 37℃for 15 minutes in the absence of light.
7) 50. Mu.L of dilute sulfuric acid solution was added to each well, and the absorbance at 450nm was measured.
And screening an optimal pairing scheme by adopting an enzyme-linked immunosorbent assay (ELISA) method. Experimental results murine monoclonal antibody A 0 As a capture antibody, murine monoclonal antibody B 0 The highest potency of the collocation scheme as a marker. See table 2 for experimental data.
TABLE 2
EXAMPLE 2 construction of expression plasmid
Expanding 3B8,4F5 hybridoma cell strain, collecting about 10 hybridoma cell strain in logarithmic growth phase 7 mRNA was extracted from each cell.
(1) Extraction of mRNA of New crown NP antibody
(a) The cell line was taken and mRNA was extracted using the kit.
(b) 200. Mu.L of ProteinaseK solution was used and mixed well.
(c) 200. Mu.L of buffer GB was added, mixed well by inversion, left at 70℃for 10Min, the solution was cleared, and the inner wall of the tube cap was centrifuged to remove water droplets.
(d) Adding 200 mu L absolute ethyl alcohol, fully shaking and uniformly mixing, and briefly centrifuging to remove water drops on the inner wall of the tube cover.
(e) And (3) adding the solution obtained in the last step into an adsorption column for centrifugation, and pouring out waste liquid.
(f) Buffer solution GD is added into the adsorption column, and the waste liquid is centrifugally poured out.
(g) Adding a rinsing liquid into the adsorption column, centrifuging to remove waste liquid, and washing twice.
(h) After centrifugation for two minutes, the mixture was left at room temperature for two minutes, and the residual rinse solution in the adsorption column was dried.
(i) Adding elution buffer solution, and centrifuging to obtain new crown NP mRNA.
(2) Reverse transcription
(a) In a 0.5mL microcentrifuge tube, 2. Mu.g of total RNA was added, and an appropriate amount of DEPC H2O was added to make the total volume 11. Mu.L. Add 10. Mu.M Oligo (dT) 15. Mu.L to the tube, mix gently and centrifuge.
(b) The tube was immediately inserted into an ice bath by heating at 70℃for 10 min.
A mixture of the following reagents was then added:
mix gently and centrifuge. Incubating at 42 ℃ for 2-5min.
(c) Superscript II 1. Mu.L was added and incubated in a 42℃water bath for 50min.
(d) The reaction was terminated by heating at 70℃for 15 min.
(e) The tube was inserted into ice, 1. Mu.L of RNase H was added, and incubated at 37℃for 20min to degrade the residual RNA. Preserving at-20 ℃ for standby.
(3) Amplifying light chain and heavy chain V region genes
Primers designed according to Orlandi et al (1989) amplify the variable region of the mouse variable region gene and the variable region of the antibody gene was amplified using the amplification product as a template. Heavy chain VH (about 360 bp) and light chain VL (about 330 bp) fragments were recovered, inserted into the Teasy vector and sequenced on 4 samples each. The sequenced sequences were classified and analyzed for homology in the GeneBank nucleic acid database. And designing a primer containing an enzyme cutting site according to the PCR product sequence, and amplifying again to ensure that the antibody variable region gene fragment has the enzyme cutting site matched with the vector, so that the antibody variable region gene fragment is convenient to insert into an expression vector, and the amplified product is recovered and purified for standby.
Sequencing results were as follows:
SEQ ID NO：14(VL1)：
DIQMTQTTSSLSASLGDRVTLSCSASQGIRNYLNWYQQKPDGTVKLLIYYTSTIHSGVPSRFSGSGSGTDYSLTISNLEPEDIATYFCMQYFPSKTYFGGGTKLEIK。
SEQ ID NO：13(VH1)：
EVLLQQSGPELVNPGASVKIPCKASGYTFTDYNMGWVKQSHGKSLEWIGALIPNNGGTIYNQKFKGKATLTVDESSSTAYMELRSLTSEDTAVYYCAREAYRDYDVKTWFAYWGQGTLVTVSA。
EQ ID NO：16(VL2)：
DPVMTQTPASVSGPVGGTVTINCQASESISNYLSWYQQKPGQPPKLLIYGASTLESGVPSRFSGSGSGTDFTLTISGVQCDDAATYYCQGGYYSSGATFTFGGGTEVVVK。
SEQ ID NO：15(VH2)：
QSVEESGGRLVTPGTPLTLTCTVSGFSISSYAMSWVRQAPGKGLEYIGILSSDGNTYYASWAKGRFTLSKTSTTVDLKITSPTTEDTATYFCARFFYDDYDDLDIIFFWGQGTLVTVSS。
(4) chimeric antibody vector construction
And (3) recombining the light chain signal peptide and the light chain VL+CL genes to form light chain full-length amino acids according to the antibody kappa, the mIgM and the human IgM obtained by the GeneBank search, and recombining the heavy chain signal peptide, the heavy chain VH-CH1 and the Fc genes to form heavy chain full-length amino acids. The entrusted aurey company performs gene synthesis based on insect cell (species) codon preference. Obtaining recombinant antibody A 1 Recombinant antibody B 1 Recombinant antibody A 0 ' and recombinant antibody B 0 ' light, heavy chain full-length nucleotide sequence.
Recombinant antibody A 1 The full-length amino acid sequence of the light chain is shown as SEQ ID NO:25, as shown in:
METGLRWLLLVAVLKGVQCDIQMTQTTSSLSASLGDRVTLSCSASQGIRNYLNWYQQKPDGTVKLLIYYTSTIHSGVPSRFSGSGSGTDYSLTISNLEPEDIATYFCMQYFPSKTYFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC。
recombinant antibody A 1 The full-length nucleotide sequence of the light chain is shown as SEQ ID NO: shown at 35.
atggaaactggtcttcgatggttgttgttagtggcggtgctaaagggggtacagtgtgatatccaaatgacccaaacgacctcatcgttatcagcgtccctcggtgatagggtaacactgtcctgttccgcgtcgcaaggcattcgcaattatctgaactggtatcagcagaaaccagatggcacagttaaactccttatctactatacgtcaaccatacattcgggagtaccaagtcgtttcagcgggagtggaagtggaacagactacagcctgaccatttccaacctagaacccgaggacattgctacttatttttgcatgcagtacttcccgagtaaaacgtatttcggtggcggtacaaaactggagataaaacgggctgacgccgcacctactgtttctatctttcctccgagtagcgaacaattaacttcagggggagcatcggtcgtgtgcttcctaaataatttttacccgaaggatataaacgttaagtggaagattgacgggtctgaaagacaaaacggcgtccttaacagctggacggatcaggacagcaaggattcgacatactcaatgtcttctactctcacattgaccaaggacgagtacgagagacacaatagctatacgtgcgaggccacgcacaagacttctacctctcccatcgtcaaatcctttaatcgaaacgaatgt。
Recombinant antibody A 1 The heavy chain of (2) is 'mouse IgG VH+mouse IgG CH1+mouse IgM CH 2+mouse IgM CH 3+mouse IgM CH 4', and the full-length amino acid sequence of the heavy chain is shown as SEQ ID NO:26, as shown in:
METGLRWLLLVAVLKGVQCEVLLQQSGPELVNPGASVKIPCKASGYTFTDYNMGWVKQSHGKSLEWIGALIPNNGGTIYNQKFKGKATLTVDESSSTAYMELRSLTSEDTAVYYCAREAYRDYDVKTWFAYWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCPAVAEMNPNVNVFVPPRDGFSGPAPRKSKLICEATNFTPKPITVSWLKDGKLVESGFTTDPVTIENKGSTPQTYKVISTLTISEIDWLNLNVYTCRVDHRGLTFLKNVSSTCAASPSTDILTFTIPPSFADIFLSKSANLTCLVSNLATYETLNISWASQSGEPLETKIKIMESHPNGTFSAKGVASVCVEDWNNRKEFVCTVTHRDLPSPQKKFISKPNEVHKHPPAVYLLPPAREQLNLRESATVTCLVKGFSPADISVQWLQRGQLLPQEKYVTSAPMPEPGAPGFYFTHSILTVTEEEWNSGETYTCVVGHEALPHLVTERTVDKSTGKPTLYNVSLIMSDTGGTCY。
recombinant antibody A 1 The full-length nucleotide sequence of the heavy chain is shown as SEQ ID NO: shown at 36.
atggagaccggtctccggtggcttttgttagttgcagtgttgaagggtgtgcagtgtgaagttcttctgcaacagagtgggcccgaattggtaaatcctggagcgtccgtgaagattccatgcaaggcttccggatataccttcacggactataacatgggttgggtaaagcaatcacatggcaagagcctggaatggatcggggctctcattcctaataacgggggcacgatttacaatcagaagtttaaaggcaaagctacattaaccgtagatgagtcttcctcgaccgcatacatggagcttcgcagtttaacatccgaagacaccgccgtgtattactgtgcgcgagaggcgtacagagactatgacgttaaaacatggttcgcgtactggggccaaggtacgttggtcactgtctcagctgcaaagacgacgcctccctcagtataccctctcgccccgggatctgccgcccaaacgaattccatggtcactcttgggtgtctagtgaagggctattttcctgaaccggtcacggtgacttggaactctggatctttgagttcgggagtgcatacatttcctgccgtactacagtccgatttgtatacactctcgtcgtcggttacggtaccatcatctacatggccgtctgagaccgttacctgtaatgtcgctcatccggcatcaagtactaaagtggacaagaagatcgtgccacgagactgcccggctgttgctgaaatgaacccgaacgtcaacgttttcgtgccgcctagggatgggtttagcggtccggcgcctcgcaaatctaagctcatctgcgaagccacaaactttactcccaaacccattacggttagctggctaaaagacggtaagctcgtagaatcgggcttcactacggatccagtaacaattgagaataaaggatccactccacagacctataaagtcataagtacgctcaccataagcgaaatagattggctgaatctgaacgtgtatacttgtagggtagatcaccgtgggcttacattcctgaaaaatgtatcgagtacgtgcgcagcatcgccctctacagatatactcacatttactattccaccatcatttgctgatatcttcctatcaaaaagtgccaacttgacatgtttagtatcaaatttagcaacctacgagactctaaacatctcgtgggcctcgcaaagtggtgagccgcttgaaaccaaaattaagatcatggagagtcatcccaatggcactttttctgcgaaaggtgttgcctcagtttgcgtcgaagactggaacaatagaaaagaattcgtttgcacagtcactcaccgtgatttaccatccccacagaagaaatttatcagtaagccgaatgaagtccacaaacacccccccgcagtatacctcctgccacccgcgcgagagcaattgaacttacgcgagagtgcaactgtgacatgtctagtcaagggcttcagcccagcggacatatcagttcaatggcttcagagaggacagctgctgccacaagagaaatatgtcaccagcgcgcctatgcccgaaccgggagcacctgggttttacttcacccattcaatactaacggtgactgaagaggagtggaattccggggagacgtacacttgcgtagtgggccacgaggccctacctcaccttgtaaccgaacggacggttgacaaaagcaccggtaagcccactttatataacgtgagcttaataatgagcgatacaggagggacgtgctac。
Recombinant antibody B 1 The full-length amino acid sequence of the light chain is shown as SEQ ID NO:31 shows:
METGLRWLLLVAVLKGVQCDPVMTQTPASVSGPVGGTVTINCQASESISNYLSWYQQKPGQPPKLLIYGASTLESGVPSRFSGSGSGTDFTLTISGVQCDDAATYYCQGGYYSSGATFTFGGGTEVVVKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。
recombinant antibody B 1 The full-length nucleotide sequence of the light chain is shown as SEQ ID NO: shown at 37.
atggagacaggtttgcgctggctcctactggtggccgttctcaaaggcgttcagtgtgaccctgtaatgactcagacgccggcttccgtttcaggcccagtgggaggtaccgtcaccatcaactgccaggcatcggaaagtatatctaactatctgtcatggtatcaacaaaagccgggacagcccccaaaattacttatttatggcgcaagcacgctagagagtggggtgccctcccgtttctcggggtccggtagtggaaccgatttcacattaacgattagtggagtgcagtgtgatgatgccgcgacttactactgccaagggggttactactcgagcggagcgacattcacatttggtggcggcaccgaggtcgtagtcaagcgaacggttgctgctccttccgtttttatattcccaccgtctgacgagcaattgaagtctggtactgcatcggtcgtatgtctgctcaataacttttaccctcgggaagccaaggtccagtggaaagtagacaacgcgttacaaagtgggaatagccaagaatcagtgaccgagcaggatagtaaagatagcacgtattccctatcatctactcttaccctttctaaagccgactatgagaaacacaaggtttatgcgtgcgaagtaactcatcaaggcttgagctcgcccgtaacaaagtcatttaataggggggaatgt。
Recombinant antibody B 1 The heavy chain of (2) is 'mouse IgG VH+mouse IgG CH1+human IgM CH 2+human IgM CH 3+human IgM CH 4', and the full-length amino acid sequence of the heavy chain is shown as SEQ ID NO:32, as shown in:
METGLRWLLLVAVLKGVQCQSVEESGGRLVTPGTPLTLTCTVSGFSISSYAMSWVRQAPGKGLEYIGILSSDGNTYYASWAKGRFTLSKTSTTVDLKITSPTTEDTATYFCARFFYDDYDDLDIIFFWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCLPVIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLGQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY。
Recombinant antibody B 1 The full-length nucleotide sequence of the heavy chain is shown as SEQ ID NO: shown at 38.
atggaaaccggccttagatggttgttgctcgttgcagttctcaagggcgtccagtgccagtcagtcgaagagtcagggggtaggctagtcacacccggtacgccactgaccctgacttgcacagtttcggggttttcaattagcagctacgccatgtcctgggtgcgacaggcccccgggaaaggtcttgagtatatcggaatactctcgagcgacggcaatacatattatgcaagttgggctaaggggcgttttacactgagtaaaaccagtacgaccgttgatttaaagatcacatcacccacgacggaggacaccgcaacatacttttgcgcacggttcttttacgatgattacgacgacctagacattatattcttttggggtcaaggcaccctagtaacagtctcttcagcatcgactaagggcccgtcggtttttccactcgcgccttctagtaagtccacaagcggaggcacagctgccctggggtgtttagttaaggattatttccccgagccagtgacggtatcatggaactcgggagcgctaacctcgggagtacacactttcccggctgtgctgcaatctagtggattgtactcactgtcctctgtagtgaccgtgcccagctcctcgctcgggacccaaacttatatatgtaacgtaaatcacaagccatcgaacacaaaggtagacaaaaaggttgaaccaaaaagctgtttacccgttattgcagagctgccgccgaaagtctcggtgttcgttcctcctagagatggattctttggaaatccacgtaagtcaaagctcatatgtcaagctactggattttctcctagacaaattcaagtatcttggttacgggagggtaagcaggtaggttccggggtaacgactgatcaggttcaggccgaggcaaaagaaagcggtcctacgacgtataaggtcacgtctacgttaaccataaaagagagtgactggttaggtcaatccatgttcacttgcagggtcgaccatcgtggtctaactttccaacagaatgcctcttcgatgtgcgtcccggatcaggacacggcgattcgcgtcttcgcaatcccgcccagtttcgcgtcaattttcttgacgaaaagtacaaaattgtgtttggttactgatttgactacatatgactcagtaaccatatcctggactaggcagaatggcgaggcggttaaaacccatacgaacatctccgaatcacacccaaacgcgacattctccgcggttggggaagctagcatctgcgaagatgattggaacagcggtgagcgctttacctgcaccgtaactcatactgatcttccttctcctctcaaacagacgatctctaggcctaaaggagtcgcgttacaccggccagacgtatacctacttccgccggcgcgtgaacagctaaaccttagagaaagcgctacaataacttgcttagtgactggattttctcctgccgatgtgtttgtgcaatggatgcaacgaggccagcctctatcaccagagaaatacgtaacgagtgccccaatgcccgagccgcaagcacccgggcggtattttgctcattctatccttacggtctcggaagaagagtggaatacaggcgaaacatacacttgtgtcgtggctcatgaggcccttccaaaccgagtcaccgaacgcactgtggacaaaagcacagggaagccgacactctacaatgtcagtcttgtgatgtccgataccgctggcacctgttat。
Recombinant antibody A 0 ' light chain full-length amino acid sequence, and recombinant antibody A 1 The full-length amino acid sequences of the light chains are identical, as shown in SEQ ID NO: 25.
Recombinant antibody A 0 The heavy chain is 'mouse IgG VH+mouse IgM CH 1+mouse IgM CH 2+mouse IgM CH 3+mouse IgM CH 4', and the full-length amino acid sequence of the heavy chain is shown in SEQ ID NO:33, as shown in:
METGLRWLLLVAVLKGVQCEVLLQQSGPELVNPGASVKIPCKASGYTFTDYNMGWVKQSHGKSLEWIGALIPNNGGTIYNQKFKGKATLTVDESSSTAYMELRSLTSEDTAVYYCAREAYRDYDVKTWFAYWGQGTLVTVSAESQSFPNVFPLVSCESPLSDKNLVAMGCLARDFLPSTISFTWNYQNNTEVIQGIRTFPTLRTGGKYLATSQVLLSPKSILEGSDEYLVCKIHYGGKNKDLHVPIPAVAEMNPNVNVFVPPRDGFSGPAPRKSKLICEATNFTPKPITVSWLKDGKLVESGFTTDPVTIENKGSTPQTYKVISTLTISEIDWLNLNVYTCRVDHRGLTFLKNVSSTCAASPSTDILTFTIPPSFADIFLSKSANLTCLVSNLATYETLNISWASQSGEPLETKIKIMESHPNGTFSAKGVASVCVEDWNNRKEFVCTVTHRDLPSPQKKFISKPNEVHKHPPAVYLLPPAREQLNLRESATVTCLVKGFSPADISVQWLQRGQLLPQEKYVTSAPMPEPGAPGFYFTHSILTVTEEEWNSGETYTCVVGHEALPHLVTERTVDKSTGKPTLYNVSLIMSDTGGTCY。
recombinant antibody A 0 The full-length nucleotide sequence of the heavy chain is shown in SEQ ID NO: 39.
atggaaacaggattgcggtggttacttctggtggccgtgctgaaaggagtccagtgtgaggtcctcctgcagcagtcaggcccggaactagttaatccaggtgcgagtgtgaaaatcccgtgcaaggctagcggatacacctttaccgattacaacatgggatgggtaaaacagtcgcatggcaagtccctagagtggataggtgctctcataccaaacaacggaggtacaatttataatcaaaagttcaaagggaaagcgacgctaacggtagatgagtcatcatcgacggcctatatggaattgcgcagcctaacaagcgaggatacggcagtgtactactgcgcgcgagaggcctacagggactatgatgtcaagacctggttcgcttactggggtcaaggcacgttggtgactgtgagtgcagaatctcaatcatttccaaatgtctttcctttggtgagctgtgagagtccattgtcagacaagaatctagtggcaatgggttgtctagctcgcgattttttgccatcgacaatttcattcacctggaactatcaaaacaacactgaggtaatccaagggatacgtactttccccaccttacgaacaggtggaaagtatttggcaacatcgcaagtgctgcttagccctaagagcatcctcgagggtagtgatgaatatttagtttgtaagatccattacggcggtaaaaacaaggacctacatgtaccgattccggcagtcgccgagatgaaccctaatgtaaacgtcttcgttccgccacgtgacgggttttctggcccagctccgaggaaaagcaaactcatttgtgaagccacgaactttactcctaaacccatcacagtctcttggcttaaggatggcaagttagtagagtccggattcaccactgacccagtaaccatcgagaataaggggtccactccgcaaacttacaaagttatcagcaccctaactatttcggagatagactggttaaatttaaatgtatacacttgcagagtcgaccaccgaggcttgaccttcctcaagaacgtcagttctacatgtgccgcttcgcctagcactgacattttaacttttaccattccgcctagttttgcagacatcttcttgtcgaaatccgccaatctgacgtgcctggtatcgaatcttgcaacctatgaaactcttaatataagttgggcgtcgcagagtggagaaccgctcgaaacgaaaataaagatcatggaatcacatccgaacgggacattctctgccaagggcgttgcttctgtttgtgtggaggattggaataatcggaaagaattcgtctgcaccgttacgcaccgcgatctcccttccccacagaaaaaattcatttccaaaccaaacgaagtacacaagcacccgcctgcggtatacctcctccctcctgctagggaacaactgaatttgagagagtctgcgacggtcacatgtctggttaagggtttttcacccgccgacatttctgttcaatggcttcagagaggccagcttctaccccaggaaaaatatgttacatccgcgcccatgcccgaacccggggcgcccggtttttattttactcactccatacttacggtaacggaggaagagtggaactctggagaaacttatacatgcgttgttgggcacgaagcattaccacatcttgtgacggagcgtacagtcgataagtctacggggaaacccacactttataatgtgtcactgataatgtccgataccggagggacctgctac。
Recombinant antibody B 0 ' light chain full-length amino acid sequence, and recombinant antibody B 1 The full-length amino acid sequences of the light chains are identical, as shown in SEQ ID NO: shown at 31.
Recombinant antibody B 0 The heavy chain is 'mouse IgG VH+mouse IgM CH 1+mouse IgM CH 2+mouse IgM CH 3+mouse IgM CH 4', and the full-length amino acid sequence of the heavy chain is shown in SEQ ID NO:34, as shown in:
METGLRWLLLVAVLKGVQCQSVEESGGRLVTPGTPLTLTCTVSGFSISSYAMSWVRQAPGKGLEYIGILSSDGNTYYASWAKGRFTLSKTSTTVDLKITSPTTEDTATYFCARFFYDDYDDLDIIFFWGQGTLVTVSSESQSFPNVFPLVSCESPLSDKNLVAMGCLARDFLPSTISFTWNYQNNTEVIQGIRTFPTLRTGGKYLATSQVLLSPKSILEGSDEYLVCKIHYGGKNKDLHVPIPAVAEMNPNVNVFVPPRDGFSGPAPRKSKLICEATNFTPKPITVSWLKDGKLVESGFTTDPVTIENKGSTPQTYKVISTLTISEIDWLNLNVYTCRVDHRGLTFLKNVSSTCAASPSTDILTFTIPPSFADIFLSKSANLTCLVSNLATYETLNISWASQSGEPLETKIKIMESHPNGTFSAKGVASVCVEDWNNRKEFVCTVTHRDLPSPQKKFISKPNEVHKHPPAVYLLPPAREQLNLRESATVTCLVKGFSPADISVQWLQRGQLLPQEKYVTSAPMPEPGAPGFYFTHSILTVTEEEWNSGETYTCVVGHEALPHLVTERTVDKSTGKPTLYNVSLIMSDTGGTCY。
recombinant antibody B 0 The full-length nucleotide sequence of the heavy chain is shown in SEQ ID NO: 40.
atggaaacaggtctacgttggcttctattggtcgctgttctcaaaggtgtccagtgccagtccgtggaagaaagtggaggccgactcgtaactcctggaactccgttgactttaacctgtacagtaagtggtttctctatctcgtcttatgcgatgtcttgggtaaggcaggctccaggtaaagggctggagtacatcggcattctcagctccgacggcaatacttactacgcatcttgggctaaggggcgcttcaccctatccaaaacatcaacaaccgtagatttgaaaatcactagccccacaactgaagacactgcaacgtatttttgtgcaagatttttttacgatgattatgacgatcttgatattatatttttttggggacaaggcacgctcgtgacggttagtagtgagagtcagtcctttccgaacgtgttccccctagtatcttgtgaatccccattaagtgacaagaatctagtcgccatgggctgtctggcacgagatttcttaccaagcacgatttctttcacttggaattatcaaaacaataccgaggttatccaggggatccgtacgttccccaccctacgtaccgggggtaaatacctggccacctcccaagtactcctttctccgaaatctattcttgagggttcagatgaatatttggtttgtaaaattcactacggcgggaagaataaagatctacacgtccccatacccgcggttgccgagatgaacccaaatgtcaacgtcttcgttccccccagagacggattttcaggcccagccccgcgcaagtcaaagcttatatgcgaagccacaaactttacgccaaagccgataacagtgtcatggctgaaggacggcaaattagttgaaagcggtttcaccacggaccctgtcactatagagaacaagggctcgacgcctcagacctacaaggtcatcagcaccctgacaattagcgagattgattggcttaacttaaatgtgtacacatgcagggtggatcaccgagggctgacctttctaaagaacgtgagctccacgtgcgcggcttcaccgtcaaccgacatacttactttcacgatcccccctagtttcgcggatatatttttatccaaatcggccaatttaacttgcttagtctcgaatttggcaacctatgaaaccctaaacatatcatgggcatcgcagtctggggagcctctcgagacgaaaattaaaatcatggagtcgcatccgaatgggacttttagcgccaagggagttgcttccgtgtgtgtagaagactggaacaaccggaaggagtttgtttgtaccgtaacacaccgggacctcccatcgcctcagaaaaaatttatatcgaagccaaatgaggtgcataaacatcctccggcggtctacttgttaccacctgcgagagagcaattgaatctccgcgaatcggctactgtaacttgtctggttaaagggttctcaccagcagatatatcggttcaatggttgcaaaggggtcaacttttgcctcaagaaaagtatgtaacttctgcaccgatgcctgaacccggtgctccgggattctatttcacgcacagtatcctcacagtaacggaggaagaatggaacagcggtgagacatacacctgcgtggtaggacatgaagcgctgccacatctcgtgacagagcggacggtcgacaagagcacgggaaagcccacactgtataatgtgtcccttattatgtccgacacaggaggcacgtgctat。
(5) Preparation of high titer antibodies
(i.) design primers the sequences were constructed onto pFastbac Dual vectors.
(ii) transferring the recombinant expression vector to DH10bac by chemical transformation.
(a) At each transformation, one MAX EfficiencyR DH Bac branch was thawed on ice TM Competent cells.
(b) At each transformation, gently mix and transfer 100. Mu.L DH10Bac TM Cells were placed into a pre-chilled 1.5mL polypropylene tube.
(c) An appropriate amount of plasmid DNA was added to the cells and gently mixed. It is not necessary to blow up and down and mix them uniformly.
pFastBac TM Recombinant: 1ng
pFastBac TM Control plasmid: 1ng
pUC19 control: 50pg of
(d) Cells were incubated on ice for 30 min.
(e) The cells were heat-shocked at 42℃for 45 seconds and were never shaken.
(f) The tubes were immediately transferred to ice and cooled for 2 minutes.
(g) 900. Mu.L of room temperature SOC medium was added.
(h)pFastBac TM Conversion: the centrifuge tube was shaken at 225rpm for 4 hours at 37 ℃. pUC19 conversion: the centrifuge tube was shaken at 225rpm for 1 hour at 37 ℃.
(i) Each time pFastBac TM Conversion: 10-fold serial dilutions of cells were prepared using SOC medium (10 -1 、10 -2 、10 -3 ). mu.L of each dilution of cells was inoculated on LB agar plates containing 50. Mu.g/mL kanamycin, 7. Mu.g/mL gentamycin, 10. Mu.g/mL tetracycline, 100. Mu.g/mL Bluo-gal (halogenated indole-. Beta. -galactoside) and 40. Mu.g/mL IPTG (isopropylthiogalactoside).
pUC19 conversion: cells were diluted 1:100 with SOC medium. mu.L of the dilution was inoculated on LB agar plates containing 100. Mu.g/mL ampicillin.
(j) The agar plates were incubated at 37℃for 48 hours and white spot colony assays were selected.
(iii) screening recombinant bacmid by blue-white
(a) 10 white colonies were picked and re-plated on freshly prepared LB agar plates containing 50. Mu.g/mL kanamycin, 7. Mu.g/mL gentamycin, 10. Mu.g/mL tetracycline, 100. Mu.g/mL Bluo-gal and 40. Mu.g/mL IPTG. The agar plates were incubated overnight at 37 ℃.
(b) The monoclonal clone confirmed to have a white phenotype was picked from the recoated agar plates containing Bluo-gal and IPTG and inoculated into liquid medium containing 50. Mu.g/mL kanamycin, 7. Mu.g/mL gentamicin and 10. Mu.g/mL tetracycline.
(iv.) PCR validation of recombinant bacmid
Recombinant bacmid DNA was analyzed by PCR.
The method comprises the following specific steps: using pUC/M13 forward or reverse primers, specific primersTaq polymerase, the recombinant bacmid DNA was amplified using the following procedure. pUC/M13 forward or reverse primer and gene specific primer mixtures were used to verify that recombinant bacmid construction was successful. For each sample, a 50 μl PCR reaction was established in a 0.5mL microcentrifuge tube as follows:
(v) Amplification was performed using the following cycling parameters:
from the reaction, 5-10. Mu.L was taken and analyzed by agarose gel electrophoresis.
(vi.) transfection of recombinant bacmid into insect cells
(a) Confirmation that Sf9 cells are in log phase (1.5-2.5X10) 6 Individual cells/mL), and the survival rate is higher than 95%.
(b) If the cell density is 1.5X10 6 –2.5×10 6 Within the individual cells/mL range, and no antibiotics in the culture, step b1 is continued. If the cell density is outside this range or if the cell culture contains antibiotics, steps b2-b3 are performed.
(b1) 2mL of Grace insect cell culture medium without additives (without antibiotics and serum) was added to each well. Inoculating 8×10 in each well 5 Sf9 cells as described in step a. The medium is not changed or the cells are rinsed. The residual medium will increase transfection efficiency. Cells were allowed to adhere to the wall for 15 minutes at room temperature in a fume hood. Continuing to step c.
(b2) 1.5mL of Grace insect cell culture medium (without antibiotics) supplemented with 10% Fetal Bovine Serum (FBS) was mixed with 8.5mL of Grace insect cell culture medium without additives (without FBS or antibiotics) to prepare 10mL of plate culture medium.
(b3) Inoculating 8×10 in each well 5 Sf9 cells as described in step a. Cells were allowed to adhere to the wall for 15 minutes at room temperature in a fume hood. The medium was aspirated. The plate medium described in step b2 was added to each well. Continuing to step c.
(c) For each transfected sample, complexes were prepared as follows:
(c1) Mix Cellfectin before use R II Reagent (cat. No. 10362-100), 8. Mu.L was diluted in 100. Mu.L of Grace medium without additives (without antibiotics or serum). Vortex briefly and mix well.
Note that: the mixture may be left at room temperature for up to 30 minutes.
(c2) Mu.g of baculovirus DNA was diluted in 100. Mu.L of Grace medium without additives (without antibiotics or serum). Mix gently.
(c3) The diluted DNA was mixed with diluted Cellfectin II (total volume 210. Mu.L). Mix gently and incubate at room temperature for 15-30min.
(d) 210. Mu.L of the DNA liposome mixture or transfection mixture (step c 3) was added dropwise to the cells obtained in step b. Cells were incubated at 27℃for 3-5 hours.
(e) The transfection mixture was aspirated and 2mL of complete medium (e.g., grace insect cell medium with additives and 10% fetal bovine serum) was added. Antibiotics may be optionally used.
(f) Cells were incubated at 27℃for 72 hours until signs of viral infection were observed.
(vii) harvesting the baculovirus. Preparation of P1 Virus stock
(a) Once transfected cells showed signs of post infection (e.g., 72 hours post-transfection), each well of virus-containing medium (2 mL) was collected and transferred to a sterile 15mL centrifuge tube. Centrifugation at 500 Xg for 5 min removed cells and larger debris.
(b) The clarified supernatant was transferred to a new 15mL centrifuge tube. This is a stock solution of P1 virus. Stored at 4deg.C in dark. And (3) injection: if it is desired to concentrate the virus stock to obtain higher titers, the virus supernatant may be filtered after a low speed centrifugation step using a 0.2 μm low protein binding filter.
(viii.) re-infection of insect cells Sf9
(a) On the day of infection, sf9 cell suspensions were prepared at 2X 10 6 Cell/well density. Cells were allowed to adhere by incubation at room temperature for 1 hour.
(b) After 1 hour, the cells were examined by an inverted microscope to confirm that they had adhered.
(c) An appropriate amount of P1 virus stock was added to each well.
(d) The cells were incubated in a humidified incubator at 27℃for 48 hours.
(e) 48 hours after transfection, 2mL of virus-containing medium per well was collected and transferred to a sterile 15mL centrifuge tube. Centrifugation at 500 Xg for 5 min removed cells and larger debris to give a clarified stock solution of viral bacilli. And (3) injection: viruses may be collected later (e.g., 72 hours) after infection. The optimal collection time is not determined and should be determined for each baculovirus recombinant. Note that the activity of the culture gradually decreases due to the constant lysis of the cells.
(f) The supernatant was transferred to a new 15mL centrifuge tube. This is a stock solution of P2 virus. Stored at 4deg.C in dark. If long-term storage is required, P2 stock solution can be packaged and stored at-80deg.C in dark place.
(ix.) harvesting of recombinant proteins
(a) Sf9 cells were seeded in 24-well plates, 6X 10 per well 5 And each. Cells were allowed to adhere for at least 30 minutes.
(b) The medium was removed and the cells were rinsed once with fresh medium. Replaced with 300 μl fresh medium.
(c) pFastBac at the appropriate multiplicity of infection (MOI) was added to each well TM Baculovirus stock solution. Including appropriate controls (e.g., empty control (uninfected) cells, pFastBac TM Positive control baculovirus, recombinant baculovirus with confirmed effect).
(d) Cells were incubated in a humidified incubator at 27 ℃.
(e) Cells (or medium if the recombinant protein is secreted) are harvested at the appropriate time (i.e., 24, 48, 72, 96 hours post-transfection). If cells are collected, the medium is removed and the cells are rinsed once with serum-free medium.
(x.) purification of recombinant proteins
Purifying the expression supernatant by corresponding column according to the operation of GE AKTA Pure protein separation and purification system instruction to obtain purified recombinant antibody, namely recombinant antibody A 0 ', recombinant antibody A 1 Recombinant antibody B 0 ' and recombinant antibody B 1 。
EXAMPLE 3 binding potency validation of recombinant antibodies
By ELISA, purified murine monoclonal antibody A 0 Competing with the corresponding recombinant antibody to bind to the novel coronavirus N antigen (CoV-2 Ag for short, manufacturer: new industrial organism), and comparing the difference of the antibody binding titers of the control group and the experimental group. The following are specific test protocols:
1) Dilution of murine monoclonal antibody A with 0.06M pH9.6 carbonate buffer 0 The final concentration was set to 6. Mu.g/mL. 96-well polystyrene plates were added, 0.1mL per well, and incubated at 37℃for 2 hours or overnight at 4 ℃. The next day, the wells were blocked with 0.02M PBS buffer, pH7.2, containing 10% calf serum (NBS), 0.15 mL/well, at 37℃for 2 hours.
2) With 0.06M pH9.6 carbonate buffer solution and murine monoclonal antibody A respectively 0 (concentration of 6. Mu.g/mL) solution, recombinant antibody A 0 ' (at a concentration of 6. Mu.g/mL) solution and recombinant antibody A 1 The (concentration is 6 mu g/mL) solution is used for gradient dilution of CoV-2Ag, and the concentration of the diluted CoV-2Ag is 20pg/mL, 100pg/mL and 500pg/mL in sequence.
3) The antigens after the gradient dilution of the different solutions are dripped into a pre-prepared 96-well microplate and incubated for 30 minutes at 37 ℃.
4) After washing 3 times with 10mM PBST (phosphate Tween buffer), HRP (horseradish peroxidase) -labeled murine anti-HA-tagged monoclonal antibody was added and incubated for 30 minutes at 37 ℃.
5) After 3 washes with 10mM PBST, 100. Mu.L of chromogenic substrate TMB was added to each well and incubated at 37℃for 15 minutes in the absence of light.
6) 50. Mu.L of dilute sulfuric acid solution was added to each well, and the absorbance at 450nm was measured.
By enzyme-linked immunosorbent assay (ELISA) of purified murine monoclonal antibody B 0 Competing with the corresponding recombinant antibody to bind to the novel coronavirus NP antigen (CoV-2 Ag for short, manufacturer: new production organism), and comparing the difference of the antibody binding titers of the control group and the experimental group. The verification evaluation method is the same as the above, and the difference is that: murine monoclonal antibody B 0 Recombinant antibody B corresponding thereto 0 ' and recombinant antibody B 1 The murine monoclonal antibody A in the experimental scheme is respectively replaced 0 Recombinant antibody A corresponding thereto 0 ' and recombinant antibody A 1 。
The experimental results show that the active recombinant antibodies are successfully expressed, and the binding titers of the recombinant antibodies (mouse Fab+mouse IgM Fc/human IgM Fc) and the recombinant antibodies (mouse IgG variable region+mouse IgM constant region) and the antigen are not very different. Table 3 shows the results of the specific experiments.
TABLE 3 Table 3
Example 4 preparation of magnetic microparticle chemiluminescent kit
The specificity of the antibodies was evaluated using a magnetic particle chemiluminescence platform from Shenzhen New Protect biomedical engineering Co., ltd. Purifying the obtained anti-SARS CoV-2 antibody (mouse monoclonal antibody A 0 Recombinant antibody A 1 And recombinant antibody A 0 ') coating magnetic microspheres respectively, purifying to obtain anti-SARS CoV-2 antibody (mouse monoclonal antibody B) 0 Recombinant antibody B 1 And recombinant antibody B 0 ') separately labeling luminophores (ABEI, N- (4-aminobutyl) -N-ethyl isoluminolNo), a novel coronavirus antigen magnetic particle chemiluminescence kit is prepared, and the detection principle of the kit is a double-antibody sandwich method.
1) Antibody coated magnetic microsphere
Taking 1mg of magnetic microspheres (manufacturer: new industrial organism, product number: B001) in a beaker, and removing the magnetic microsphere protective liquid by an externally applied magnetic field; adding carbonate buffer solution (pH 9.5) with the concentration of 0.1mol/L which is five times the coating volume, stirring for 2-3 minutes while ultrasonic, removing the supernatant after magnetic separation, and repeating for 3 times; the magnetic microsphere is suspended to 20mg/mL by the buffer solution for cleaning, then 10mg/mL of carbodiimide is added for activation, after being stirred uniformly, 6 mug of anti-SARS-CoV-2 antibody is added, the temperature is kept at 37 ℃ for 2 hours, finally the coated magnetic microsphere is cleaned and resuspended to 20mg/mL by 0.01mol/L PBS buffer solution (pH 7.4), and the intermediate of the magnetic microsphere component is obtained.
2) Luminescent agent labeled antibody
1mg of anti-SARS-CoV-2 antibody is taken in a dialysis bag, 0.01mol/L PBS buffer solution (pH 7.4) is added to adjust the concentration of the antibody to 1.0mg/mL for dialysis, after the dialysis of the raw material is finished, the dialyzed raw material is transferred into a sample bottle, 25 mug of ABEI activated ester is added, the reaction is carried out for 2 hours at 25 ℃ in a constant temperature water bath shaker, and after the reaction is finished, the binding substance of the ABEI activated ester is subjected to gel column purification and sample collection to obtain a luminescent marker component intermediate product.
3) Preparing new coronavirus antigen detection kit (magnetic particle chemiluminescence method)
(1) The intermediate of the magnetic microsphere component is diluted by PBS solution, so that the concentration of the magnetic microsphere in the working solution is 1mg/mL and the concentration of the antibody coated on the magnetic microsphere is 6 mug/mL.
(2) The luminescent marker component intermediate was diluted with PBS to give an ABEI concentration of 12.5ng/mL and an ABEI-labeled antibody concentration of 0.5. Mu.g/mL in the working solution.
(3) Preparing Tris-HCl solution containing surfactant, which is the buffer component of the kit.
(4) A low-point calibrator/calibrator comprising a solution of novel coronavirus N protein at a concentration of 1AU/mL.
(5) High-point calibration/calibrator containing solutions of novel coronavirus N protein at a concentration of 16AU/mL.
(6) The negative quality control material is PBS solution.
(7) The positive quality control material is a solution containing novel coronavirus N protein, and the concentration of the novel coronavirus N protein is 4AU/mL.
Example 5 sensitivity verification of different antibody pairing modes in the kit
(1) And respectively diluting the two intermediate magnetic microsphere components by using PBS (phosphate buffer solution) to ensure that the concentration of the magnetic microsphere in the working solution is 1mg/mL and the concentration of the antibody coated on the magnetic microsphere is 6 mug/mL.
(2) The two luminescent marker component intermediates were diluted with PBS solution to give an ABEI concentration of 12.5ng/mL and an ABEI-labeled antibody concentration of 0.5. Mu.g/mL in the working solution.
(3) The 2 magnetic ball working solutions and the 1 ABEI working solution prepared above were mixed in pairs, and the diluted solution (with the number Z) and the gradient diluted CoV-2Ag (with the concentrations of 20pg/mL (with the number L) and 500pg/mL (with the number H)) were detected. The sample/calibrator/quality control material, buffer, magnetic microsphere and luminescent marker are incubated together, the novel coronavirus antigen in the sample/calibrator/quality control material is combined with the novel coronavirus N protein antibody coated by the magnetic microsphere and the novel coronavirus N protein antibody marked by ABEI, and after incubation, unbound substances are removed by magnetic separation and washing. Adding substrate solution (NaOH, H) for full-automatic immune inspection system 2 O 2 ) A chemiluminescent reaction is initiated, producing an optical signal. The relative light intensity (RLU) measured by the photomultiplier is then calculated as { H (RLU) -Z (RLU) } and { L (RLU) -Z (RLU) }, respectively. In the study, we used the value of { H (RLU) -Z (RLU) } as the index of the paired titers, the ratio of { L (RLU) -Z (RLU) } as the index of the measurement sensitivity, and the optimal antibody paired regimen was selected by the sensitivity and titers obtained by the calculation of the detection target substances of different collocation schemes.
Experimental results show that recombinant antibody A 1 And recombinant antibody B 1 When used alone, the kit has certain sensitivity and potencyAnd (3) lifting, and optimizing the matching scheme when the two are matched. Table 4 shows the results of the specific experiments.
TABLE 4 Table 4
Continuing table 4:
example 6 verification of the Capacity of different novel coronavirus strains
Samples of different novel coronavirus strains (inactivated virus cultures) were diluted to 5.0TCID with 0.05mol/L Tris-HCl buffer (pH 8.6) respectively 50 and/mL for studying the ability of recombinant antibodies to detect different novel coronavirus strains. The information for the different novel coronavirus strains is shown in table 5 below.
TABLE 5
Selection of "Capture antibody as recombinant antibody A 1 Detection antibody is recombinant antibody B 1 The scheme prepares the novel coronavirus antigen magnetic particle chemiluminescence kit. The sample/calibrator/quality control material, buffer, magnetic microsphere and luminescent marker are incubated together, novel coronavirus N protein in the sample/calibrator/quality control material is combined with novel coronavirus N protein antibody coated by the magnetic microsphere and novel coronavirus N protein antibody marked by ABEI, and unbound substances are removed by magnetic separation and washing after incubation. Adding substrate solution (NaOH, H) for full-automatic immune inspection system 2 O 2 ) A chemiluminescent reaction is initiated, producing an optical signal. The relative light intensity (RLU) measured by the photomultiplier is proportional to the concentration of the novel coronavirus antigen in the sample. Detection result<1.0AU/mL, the novel coronavirus antigen was judged negative; judging when the detection result is more than or equal to 1.0AU/mLIs positive to the novel coronavirus antigen.
The detection result shows that the concentration is 5.0TCID 50 The sample of each strain can be detected as positive result by the novel coronavirus antigen kit, which shows that the kit has good detection capability for different novel coronavirus strains. The specific results are shown in Table 6.
TABLE 6
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Example 7 analytical specificity verification
Normal human samples and negative samples for dilution of potential cross-over material were derived from hospitals. Commercial pathogen inactivated cultures of potentially cross-reactive materials were selected for analysis specificity studies, and the potentially cross-reactive materials in table 7 were diluted to the corresponding concentrations in the following table using negative mix samples, respectively, to obtain samples for analysis specificity studies.
TABLE 7
| Potential cross numbering | Virus type | Concentration of virus |
| C1 | HKU1 | 1.5×10 5 TCID50/mL |
| C2 | OC43 | 1.5×10 5 TCID50/mL |
| C3 | NL63 | 1.5×10 5 TCID50/mL |
| C4 | 229E | 1.5×10 5 TCID50/mL |
| C5 | MERS coronavirus | 1.5×10 5 TCID50/mL |
| C6 | SARS coronavirus | 1.5×10 5 TCID50/mL |
| C7 | H1N1 (seasonal) | 1.5×10 5 TCID50/mL |
| C8 | Influenza b Yamagata | 3.8×10 5 TCID50/mL |
| C9 | Respiratory syncytial virus A | 1.5×10 5 TCID50/mL |
| C10 | Respiratory syncytial virus B | 1.5×10 5 TCID50/mL |
| C11 | Mycoplasma pneumoniae | 2.70×10 6 CCU/mL |
| C12 | Chlamydia pneumoniae | 1.70×10 6 IFU/mL |
Selection of "Capture antibody as recombinant antibody A 1 Detection antibody is recombinant antibody B 1 The scheme prepares the novel coronavirus antigen magnetic particle chemiluminescence kit. And (3) detecting the samples by using the kit respectively, and repeating the test for 3 times for each sample to judge the detection condition of the novel coronavirus antigen. The detection results showed negative results, indicating that these potential cross-over substances did not affect the detection of the novel coronavirus antigen. The specific detection results are shown in table 8:
TABLE 8
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Example 8 clinical Performance verification
The positive and negative samples detected by the novel coronavirus 2019-nCoV nucleic acid detection kit (fluorescence PCR method) (manufacturer: jiangsu Shuo Shi Biotechnology Co., ltd.) are used as detection samples. Based on the capture antibody is murine monoclonal antibody A 0 The detection antibody is murine monoclonal antibody B 0 The detection kit of the scheme is used as a control group kit, and is based on the fact that the capture antibody is a recombinant antibody A1 and the detection antibody is a recombinant antibodyThe detection kit of the B1' scheme is used as an experimental group kit for respectively detecting 189 PCR positive samples and 94 PCR negative samples, counting the coincidence degree of the PCR result and evaluating the clinical sensitivity and specificity.
The results show that compared with the PCR results, the clinical sensitivity of the experimental group kit is 80.42 percent and the clinical specificity is 97.87 percent; the clinical sensitivity of the control kit was 62.43% and the clinical specificity 96.81%. The results show that when the recombinant antibody A1 and the recombinant antibody B1 are adopted, the diagnostic sensitivity and the specificity of the novel coronavirus antigen are obviously improved.
The results of the fluorescence PCR method are summarized in Table 9, wherein Ct is less than or equal to 37, which is a positive sample.
TABLE 9
The results of the fluorescence PCR method for negative samples with Ct >37 and the relevant antigen detection results are summarized in Table 10.
Table 10
From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: recombinant antibody A as described above 1 And B 1 For the novel recombinant antibodies obtained in the present application, recombinant antibody A was used 1 And/or B 1 Compared with the recombinant antibodies against SARS-CoV-2 virus in the prior art, the prepared kit has better sensitivity and specificity, and can identify the sample to be detected more accurately.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (13)
1. A novel coronavirus detection kit is characterized in that the novel coronavirus detection kit comprises a recombinant antibody, the recombinant antibody comprises a first recombinant antibody and/or a second recombinant antibody,
the first recombinant antibody or the second recombinant antibody each independently comprises a Fab fragment and an Fc fragment,
the Fab fragment is derived from an IgG,
the Fc fragment is derived from IgM.
2. The novel coronavirus detection kit of claim 1, wherein the Fab fragment comprises a SARS-CoV-2 nucleocapsid protein antibody variable region,
the SARS-CoV-2 nucleocapsid protein antibody variable region comprises a SARS-CoV-2 nucleocapsid protein antibody light chain variable region and a SARS-CoV-2 nucleocapsid protein antibody heavy chain variable region,
the SARS-CoV-2 nucleocapsid antibody light chain variable region of said first recombinant antibody comprises:
SEQ ID NO：1：VL1-CDR1：SASQGIRNYLN；
SEQ ID NO：2：VL1-CDR2：YTSTIHS；
SEQ ID NO：3：VL1-CDR3：MQYFPSKTY；
the SARS-CoV-2 nucleocapsid protein antibody heavy chain variable region of said first recombinant antibody comprises:
SEQ ID NO：4：VH1-CDR1：DYNMG；
SEQ ID NO：5：VH1-CDR2：ALIPNNGGTIYNQKFKG；
SEQ ID NO：6：VH1-CDR3：EAYRDYDVKTWFAY；
the SARS-CoV-2 nucleocapsid antibody light chain variable region of said second recombinant antibody comprises:
SEQ ID NO：7：VL2-CDR1：QASESISNYLS；
SEQ ID NO：8：VL2-CDR2：GASTLES；
SEQ ID NO：9：VL2-CDR3：QGGYYSSGATFT；
the heavy chain variable region of the SARS-CoV-2 nucleocapsid protein antibody of the second recombinant antibody comprises:
SEQ ID NO：10：VH2-CDR1：SYAMS；
SEQ ID NO：11：VH2-CDR2：ILSSDGNTYYASWAKG；
SEQ ID NO：12：VH2-CDR3：FFYDDYDDLDIIFF。
3. the novel coronavirus detection kit as claimed in claim 2, wherein,
The amino acid sequence of the heavy chain variable region of the first recombinant antibody is shown in SEQ ID NO:13, as shown in:
EVLLQQSGPELVNPGASVKIPCKASGYTFTDYNMGWVKQSHGKSLEWIGALIPNNGGTIYNQKFKGKATLTVDESSSTAYMELRSLTSEDTAVYYCAREAYRDYDVKTWFAYWGQGTLVTVSA；
the amino acid sequence of the light chain variable region of the first recombinant antibody is shown as SEQ ID NO:14, as shown in:
DIQMTQTTSSLSASLGDRVTLSCSASQGIRNYLNWYQQKPDGTVKLLIYYTSTIHSGVPSRFSGSGSGTDYSLTISNLEPEDIATYFCMQYFPSKTYFGGGTKLEIK；
the amino acid sequence of the heavy chain variable region of the second recombinant antibody is shown in SEQ ID NO:15, as shown in:
QSVEESGGRLVTPGTPLTLTCTVSGFSISSYAMSWVRQAPGKGLEYIGILSSDGNTYYASWAKGRFTLSKTSTTVDLKITSPTTEDTATYFCARFFYDDYDDLDIIFFWGQGTLVTVSS；
the amino acid sequence of the light chain variable region of the second recombinant antibody is shown in SEQ ID NO:16, as shown in:
DPVMTQTPASVSGPVGGTVTINCQASESISNYLSWYQQKPGQPPKLLIYGASTLESGVPSRFSGSGSGTDFTLTISGVQCDDAATYYCQGGYYSSGATFTFGGGTEVVVK。
4. the novel coronavirus detection kit as claimed in claim 1, wherein,
the amino acid sequence of the Fab fragment light chain part of the first recombinant antibody is shown as SEQ ID NO:17, as shown in:
DIQMTQTTSSLSASLGDRVTLSCSASQGIRNYLNWYQQKPDGTVKLLIYYTSTIHSGVPSRFSGSGSGTDYSLTISNLEPEDIATYFCMQYFPSKTYFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC；
the amino acid sequence of the heavy chain part of the Fab fragment of the first recombinant antibody is shown as SEQ ID NO:18, as shown in:
EVLLQQSGPELVNPGASVKIPCKASGYTFTDYNMGWVKQSHGKSLEWIGALIPNNGGTIYNQKFKGKATLTVDESSSTAYMELRSLTSEDTAVYYCAREAYRDYDVKTWFAYWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDC；
the amino acid sequence of the Fab fragment light chain part of the second recombinant antibody is shown in SEQ ID NO:19, as shown in:
DPVMTQTPASVSGPVGGTVTINCQASESISNYLSWYQQKPGQPPKLLIYGASTLESGVPSRFSGSGSGTDFTLTISGVQCDDAATYYCQGGYYSSGATFTFGGGTEVVVKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC；
the amino acid sequence of the heavy chain part of the Fab fragment of the second recombinant antibody is shown in SEQ ID NO:20, as shown in:
QSVEESGGRLVTPGTPLTLTCTVSGFSISSYAMSWVRQAPGKGLEYIGILSSDGNTYYASWAKGRFTLSKTSTTVDLKITSPTTEDTATYFCARFFYDDYDDLDIIFFWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC。
5. the novel coronavirus detection kit of claim 1, wherein the Fc fragment of the first recombinant antibody comprises A1-CH2, A1-CH3 and A1-CH4,
the amino acid sequence of the A1-CH2 is shown in SEQ ID NO:21, as shown in:
AVAEMNPNVNVFVPPRDGFSGPAPRKSKLICEATNFTPKPITVSWLKDGKLVESGFTTDPVTIENKGSTPQTYKVISTLTISEIDWLNLNVYTCRVDHRGLTFLKNVSSTCAA；
the amino acid sequence of A1-CH3 is shown in SEQ ID NO:22, as shown in:
SPSTDILTFTIPPSFADIFLSKSANLTCLVSNLATYETLNISWASQSGEPLETKIKIMESHPNGTFSAKGVASVCVEDWNNRKEFVCTVTHRDLPSPQKKFISKPN；
The amino acid sequence of A1-CH4 is shown in SEQ ID NO:23, as shown in:
EVHKHPPAVYLLPPAREQLNLRESATVTCLVKGFSPADISVQWLQRGQLLPQEKYVTSAPMPEPGAPGFYFTHSILTVTEEEWNSGETYTCVVGHEALPHLVTERTVDKSTGKPTLYNVSLIMSDTGGTCY。
6. the novel coronavirus detection kit of claim 5, wherein the Fc fragment of the first recombinant antibody has an amino acid sequence as set forth in SEQ ID NO:24, as shown in:
AVAEMNPNVNVFVPPRDGFSGPAPRKSKLICEATNFTPKPITVSWLKDGKLVESGFTTDPVTIENKGSTPQTYKVISTLTISEIDWLNLNVYTCRVDHRGLTFLKNVSSTCAASPSTDILTFTIPPSFADIFLSKSANLTCLVSNLATYETLNISWASQSGEPLETKIKIMESHPNGTFSAKGVASVCVEDWNNRKEFVCTVTHRDLPSPQKKFISKPNEVHKHPPAVYLLPPAREQLNLRESATVTCLVKGFSPADISVQWLQRGQLLPQEKYVTSAPMPEPGAPGFYFTHSILTVTEEEWNSGETYTCVVGHEALPHLVTERTVDKSTGKPTLYNVSLIMSDTGGTCY；
preferably, the full-length amino acid sequence of the light chain of the first recombinant antibody is as shown in SEQ ID NO:25, as shown in:
METGLRWLLLVAVLKGVQCDIQMTQTTSSLSASLGDRVTLSCSASQGIRNYLNWYQQKPDGTVKLLIYYTSTIHSGVPSRFSGSGSGTDYSLTISNLEPEDIATYFCMQYFPSKTYFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC; preferably, the heavy chain full-length amino acid sequence of the first recombinant antibody is as shown in SEQ ID NO:26, as shown in:
METGLRWLLLVAVLKGVQCEVLLQQSGPELVNPGASVKIPCKASGYTFTDYNMGWVKQSHGKSLEWIGALIPNNGGTIYNQKFKGKATLTVDESSSTAYMELRSLTSEDTAVYYCAREAYRDYDVKTWFAYWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCPAVAEMNPNVNVFVPPRDGFSGPAPRKSKLICEATNFTPKPITVSWLKDGKLVESGFTTDPVTIENKGSTPQTYKVISTLTISEIDWLNLNVYTCRVDHRGLTFLKNVSSTCAASPSTDILTFTIPPSFADIFLSKSANLTCLVSNLATYETLNISWASQSGEPLETKIKIMESHPNGTFSAKGVASVCVEDWNNRKEFVCTVTHRDLPSPQKKFISKPNEVHKHPPAVYLLPPAREQLNLRESATVTCLVKGFSPADISVQWLQRGQLLPQEKYVTSAPMPEPGAPGFYFTHSILTVTEEEWNSGETYTCVVGHEALPHLVTERTVDKSTGKPTLYNVSLIMSDTGGTCY。
7. the novel coronavirus detection kit of claim 5, wherein the Fc fragment of the second recombinant antibody comprises B1-CH2, B1-CH3 and B1-CH4,
the amino acid sequence of the B1-CH2 is shown in SEQ ID NO:27, as shown in:
VIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLGQSMFTCRVDHRGLTFQQNASSMCVP; the amino acid sequence of the B1-CH3 is shown in SEQ ID NO:28, as shown:
DQDTAIRVFAIPPSFASIFLTKSTKLCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPK；
The amino acid sequence of the B1-CH4 is shown in SEQ ID NO: 29:
GVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY。
8. the novel coronavirus detection kit of claim 7, wherein the Fc fragment of the second recombinant antibody has an amino acid sequence as set forth in SEQ ID NO:30, as shown in:
VIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLGQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY；
preferably, the full-length amino acid sequence of the light chain of the second recombinant antibody is as set forth in SEQ ID NO:31 shows:
METGLRWLLLVAVLKGVQCDPVMTQTPASVSGPVGGTVTINCQASESISNYLSWYQQKPGQPPKLLIYGASTLESGVPSRFSGSGSGTDFTLTISGVQCDDAATYYCQGGYYSSGATFTFGGGTEVVVKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC；
preferably, the heavy chain full-length amino acid sequence of the second recombinant antibody is as set forth in SEQ ID NO:32, as shown in:
METGLRWLLLVAVLKGVQCQSVEESGGRLVTPGTPLTLTCTVSGFSISSYAMSWVRQAPGKGLEYIGILSSDGNTYYASWAKGRFTLSKTSTTVDLKITSPTTEDTATYFCARFFYDDYDDLDIIFFWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCLPVIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLGQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY。
9. a nucleocapsid protein recombinant antibody, comprising a first recombinant antibody or a second recombinant antibody in the novel coronavirus detection kit of any one of claims 1 to 8.
10. A pair of recombinant antibodies, wherein the pair of recombinant antibodies comprises:
the first recombinant antibody or the second recombinant antibody in the novel coronavirus detection kit of any one of claims 1 to 8, and an antibody that is different from the first recombinant antibody or the second recombinant antibody and specifically binds to SARS-CoV-2;
preferably, the recombinant antibody pair comprises the first recombinant antibody and the second recombinant antibody.
11. A nucleic acid molecule encoding the recombinant nucleocapsid protein antibody of claim 9.
12. A nucleic acid vector comprising the nucleic acid molecule of claim 11, said nucleic acid vector comprising an expression cassette, a recombinant vector, a recombinant bacterium, or a recombinant virus.
13. Use of the novel coronavirus detection kit of any one of claims 1 to 8, or the recombinant nucleocapsid protein antibody of claim 9, or the recombinant antibody pair of claim 10, in the preparation of a SARS-CoV-2 virus antigen detection reagent or kit.
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