WO2017107131A1 - Tp recombinant antigen, and preparation method and application thereof - Google Patents

Tp recombinant antigen, and preparation method and application thereof Download PDF

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Publication number
WO2017107131A1
WO2017107131A1 PCT/CN2015/098666 CN2015098666W WO2017107131A1 WO 2017107131 A1 WO2017107131 A1 WO 2017107131A1 CN 2015098666 W CN2015098666 W CN 2015098666W WO 2017107131 A1 WO2017107131 A1 WO 2017107131A1
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polynucleotide
polypeptide
seq
antigen
nucleotide sequence
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PCT/CN2015/098666
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French (fr)
Chinese (zh)
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刘莉莉
李瑞净
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菲鹏生物股份有限公司
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Publication of WO2017107131A1 publication Critical patent/WO2017107131A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/18—Ion-exchange chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/30—Extraction; Separation; Purification by precipitation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • 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
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/571—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses for venereal disease, e.g. syphilis, gonorrhoea

Definitions

  • the invention relates to the field of immunoassay, in particular to a TP recombinant antigen and a preparation method and application thereof.
  • Syphilis is a sexually transmitted disease, the pathogen of which is Treponema pallidum, also known as Treponema pallidum (TP), which is mainly transmitted through sexual contact and blood, and produces a variety of symptoms and signs, and the time is hidden, the course of disease It lasts for a long time and can almost invade all organs of the body. It is a sexually transmitted disease that is second only to AIDS and seriously endangers the health of our people. In recent years, there has been an upward trend in China. According to the 2014 report on the major syphilis epidemic, since 2009, syphilis has been ranked third in the number of reported cases of Class B infectious diseases. In particular, the number of syphilis-positive pregnant women screened in prenatal clinics has increased significantly, bringing new problems to public health in China.
  • Treponema pallidum also known as Treponema pallidum (TP)
  • TP Treponema pallidum
  • the fluorescent trehalose antibody absorption test and the treponema hemagglutination test have greatly improved the specificity of the detection, but a large number of pathogens need to be prepared, and the quality is difficult to control, which is not conducive to large-scale blood screening work. Therefore, the development of rapid, simple, sensitive and specific diagnostic reagents is currently the main task.
  • the widely used treponema pallidum serum detection method is enzyme-linked immunosorbent assay (ELISA) and rapid detection of colloidal gold or latex. This method is based on the specific recognition of antigen-antibody. Enzyme-linked immunosorbent assay (ELISA) has the advantages of high sensitivity and good specificity, and has become a syphilis test. The mainstream method of measurement.
  • the colloidal gold or latex method has the advantages of quickness and convenience, and has been applied more and more widely.
  • Immunochromatography colloidal gold technology is a new type of diagnostic technology, which has been widely used.
  • the basic principle is as follows: labeling an antigen or antibody with colloidal gold, coating the corresponding paired antigen on the nitrocellulose membrane of the test strip or The antibody, when the sample contains the corresponding specific antibody or antigen, the colloidal gold labeled particles and the ligand in the sample combine to form a complex, and then chromatographed on the nitrocellulose membrane, and then coated antigen or antibody Capture, form a visible line (T line) visible to the naked eye, and determine the result by detecting the presence or absence of the line.
  • T line visible line
  • the label is a small label such as an enzyme, a luminescent substance or a radioactive substance
  • the labeling antigen is encapsulated by the label, so that the antigen epitope is embedded. Lead to a decrease in antigenic activity
  • the theoretically optimal sensitivity state is that the lower the labeling ratio, the higher the sensitivity, and the sensitivity is 1:1.
  • Treponema pallidum In the early 1980s, with the development of molecular biology technology, especially the emergence of genetic engineering (DNA recombination) technology, the study of Treponema pallidum entered a new stage, and the entire genomic DNA sequence of Treponema pallidum has been resolved. Through the cloning of recombinant Treponema pallidum DNA and its expression in E. coli, a variety of recombinant TP antigens were prepared, which provided a new way for the study of syphilis. However, prokaryotic expression lacks post-translational modification, and sometimes requires the expression of a special protein partner and further renaturation or preservation of the buffer to make its protein activity better.
  • a TP recombinant antigen comprising a chimeric expression polypeptide linked in turn and a flexible linker peptide which facilitates soluble expression, the chimeric expression polypeptide being a TPN15-TPN17-TPN47 chimeric expression polypeptide.
  • a GST tag is further included, the GST tag, the chimeric expression polypeptide, and the flexible linker peptide are linked in turn.
  • the TPN15-TPN17-TPN47 chimeric expression antigen is (a) a polypeptide encoded by a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID No. 1, (b), and a polynucleotide comprising a nucleotide sequence represented by SEQ ID No. 1 having a polynucleotide encoding at least 98% homology; or (c) a nucleotide represented by SEQ ID No. 1.
  • the flexible linker peptide is (a) a polypeptide encoded by a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID No. 2; (b), and SEQ ID No. 2 a polynucleotide having a nucleotide sequence consisting of a polynucleotide having a polynucleotide having at least 98% homology; or (c) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID No. a polypeptide in which one or more bases are deleted, substituted or increased by the resulting polynucleotide.
  • a method for preparing a TP recombinant antigen comprises the following steps:
  • Step 1 Providing a gene expression vector for expressing a TP recombinant antigen, the TP recombinant antigen comprising a chimeric expression polypeptide and a flexible link peptide capable of soluble expression, the chimeric expression polypeptide is TPN15- TPN17-TPN47 chimeric expression polypeptide;
  • Step two transforming the gene expression vector into a host cell, wherein the host cell is a prokaryotic cell;
  • Step 3 inducing expression of the host cell transformed with the gene expression vector, and separating the expression liquid;
  • Step 4 performing gradient analysis of saturated ammonium sulfate on the expression solution to determine sedimentation protein and sedimentation
  • the ammonium sulfate concentration of the target protein is added to the corresponding final concentration of ammonium sulfate, and the supernatant is collected after sufficient rest, and then the final concentration of the saturated ammonium sulfate protein is added to the supernatant, and the precipitate is collected after being completely rested.
  • the precipitate is redissolved to give a crude product;
  • Step 5 purifying the crude product by using an affinity column, and then performing affinity column and ion exchange purification to obtain the TP recombinant antigen.
  • the TP recombinant antigen further comprises a GST tag, the GST tag, the chimeric expression polypeptide, and the flexible linker peptide are linked in turn.
  • the TPN15-TPN17-TPN47 chimeric expression antigen is (a) a polypeptide encoded by a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID No. 1, (b), and a polynucleotide comprising a nucleotide sequence represented by SEQ ID No. 1 having a polynucleotide encoding at least 98% homology; or (c) a nucleotide represented by SEQ ID No. 1.
  • the flexible linker peptide is (a) a polypeptide encoded by a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID No. 2; (b), and SEQ ID No. 2 a polynucleotide having a nucleotide sequence consisting of a polynucleotide having a polynucleotide having at least 98% homology; or (c) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID No. a polypeptide in which one or more bases are deleted, substituted or increased by the resulting polynucleotide.
  • a syphilis detecting reagent wherein the syphilis detecting reagent contains a solution of the above TP recombinant antigen
  • the solution of the TP recombinant antigen contains Tween 20 in a mass percentage concentration of 0.1% to 0.3% or Triton X-100 in a mass percentage concentration of 0.1% to 0.3%.
  • the solution of the TP recombinant antigen contains 0.15% by weight of Tween 20 or 0.15% by weight of Triton X-100.
  • a syphilis test strip comprising a coating antigen, a labeled antigen, a GST monoclonal antibody and a label;
  • the labeled antigen comprises a GST tag, a chimeric expression polypeptide, and a flexible linker peptide that facilitates soluble expression, which is a TPN15-TPN17-TPN47 chimeric expression.
  • the coating antigen includes the chimeric expression polypeptide and the flexible link peptide
  • the label is indirectly bound to the labeled antigen by the anti-GST monoclonal antibody.
  • the TPN15-TPN17-TPN47 chimeric expression antigen is (a) a polypeptide encoded by a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID No. 1, (b), and a polynucleotide comprising a nucleotide sequence represented by SEQ ID No. 1 having a polynucleotide encoding at least 98% homology; or (c) a nucleotide represented by SEQ ID No. 1.
  • the flexible linker peptide is (a) a polypeptide encoded by a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID No. 2; (b), and SEQ ID No. 2 a polynucleotide having a nucleotide sequence consisting of a polynucleotide having a polynucleotide having at least 98% homology; or (c) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID No. a polypeptide in which one or more bases are deleted, substituted or increased by the resulting polynucleotide.
  • a syphilis detection kit comprising the above syphilis detection reagent.
  • a syphilis detection kit comprising the above syphilis test strip.
  • This TP recombinant antigen modifies the TPN15-TPN17-TPN47 chimeric expression polypeptide by introducing a flexible link peptide which contributes to soluble expression, and the TP recombinant antigen has a better protein activity than the conventional TP antigen.
  • FIG. 1 is a flow chart showing a method for preparing a TP recombinant antigen according to an embodiment
  • FIG. 2 is a front elevational view of a syphilis test strip of an embodiment
  • Figure 3 is a schematic cross-sectional view of the syphilis test strip shown in Figure 2;
  • FIG. 4 is a schematic structural view of a syphilis detection kit according to an embodiment
  • Figure 5 is a chromatogram of the purity of SDS-PAGE gel coated with antigen and labeled antigen in Example 1.
  • the TP (P. pallidum) recombinant antigen of one embodiment comprises a chimeric expression polypeptide and a flexible linker peptide which facilitates soluble expression, and the chimeric expression polypeptide is a TPN15-TPN17-TPN47 chimeric expression polypeptide.
  • This TP recombinant antigen can be used as a coating antigen and a labeled antigen, and is applied to the field of syphilis detection.
  • the TP recombinant antigen also includes a GST tag, and the GST tag, the chimeric expression polypeptide, and the flexible linker peptide are sequentially linked.
  • the TPN15-TPN17-TPN47 chimeric expression antigen is: (a) a polypeptide encoded by a polynucleotide consisting of the nucleotide sequence shown in SEQ ID No. 1, (b), and SEQ ID No. a polynucleotide comprising a nucleotide sequence of 1 having a polynucleotide encoding at least 98% homology; or (c) a multinuclear comprising the nucleotide sequence of SEQ ID No. 1.
  • the flexible link peptide is: (a) a polypeptide encoded by a polynucleotide consisting of the nucleotide sequence shown in SEQ ID No. 2; (b) and a nucleoside represented by SEQ ID No. 2. a polynucleotide having a nucleic acid sequence consisting of a polynucleotide having at least 98% homology; or (c) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID No. 2, wherein one or A polypeptide obtained by deleting, substituting or increasing the obtained polynucleotide by a plurality of bases.
  • the GST tag is a polypeptide encoded by a polynucleotide consisting of the nucleotide sequence shown by SEQ ID No. 3.
  • the naturally occurring protein may be genetically altered, the base in the coding sequence is deleted, replaced or increased, or the amino acid is deleted, inserted, substituted or otherwise mutated, resulting in the amino acid sequence of the protein.
  • One or more amino acids are deleted, replaced or added. Therefore, there are some proteins whose physiological and biological activities are substantially equivalent to those of non-mutant proteins. These structures differ from the corresponding proteins, but polypeptides or proteins that have no apparent functional difference from the protein are referred to as functionally equivalent variants.
  • Functionally equivalent variants are equally applicable to polypeptides obtained by introducing one or more codons by artificial means such as deletions, insertions, and mutations to introduce such mutations into the amino acid sequence of a protein. Although more variants of this form can be obtained in this way, the resulting variant is a functionally equivalent variant with the physiological activity essentially equivalent to that of the original non-mutated protein.
  • the coding sequence of a functionally equivalent variant is homologous and, therefore, is altered by at least one (eg, a deletion, insertion or substitution of one or more bases in the coding sequence of the protein or one of the amino acid sequences of the protein or
  • a polypeptide or protein obtained by deleting, inserting or substituting a plurality of amino acids generally has an activity functionally equivalent to the protein, and therefore, a polypeptide encoded by the above nucleotide sequence or a polypeptide consisting of the above amino acid sequence, if Escherichia coli Periplasmic proteins have no significant functional differences and are included within the scope of the invention.
  • This TP recombinant antigen modifies the TPN15-TPN17-TPN47 chimeric expression polypeptide by introducing a flexible link peptide which contributes to soluble expression, and the TP recombinant antigen has a better protein activity than the conventional TP antigen.
  • the TP recombinant antigen can be used in the field of preparing syphilis detection reagents or preparing syphilis detection equipment.
  • the method for preparing the above TP recombinant antigen as shown in FIG. 1 comprises the following steps:
  • the gene expression vector is for expressing a TP recombinant antigen
  • the TP recombinant antigen comprises a chimeric expression polypeptide and a flexible link peptide which contributes to soluble expression
  • the chimeric expression polypeptide is a TPN15-TPN17-TPN47 chimeric expression polypeptide.
  • the TP recombinant antigen further comprises a GST tag as a labeled antigen, and the GST tag, the chimeric expression polypeptide and the flexible link peptide are sequentially linked.
  • the TPN15-TPN17-TPN47 chimeric expression antigen is: (a) a polypeptide encoded by a polynucleotide consisting of the nucleotide sequence shown in SEQ ID No. 1, (b), and SEQ ID No. a polynucleotide comprising a nucleotide sequence of 1 having a polynucleotide encoding at least 98% homology; or (c) a multinuclear comprising the nucleotide sequence of SEQ ID No. 1.
  • the flexible link peptide is: (a) a polypeptide encoded by a polynucleotide consisting of the nucleotide sequence shown in SEQ ID No. 2; (b) and a nucleoside represented by SEQ ID No. 2. a polynucleotide having a nucleic acid sequence consisting of a polynucleotide having at least 98% homology; or (c) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID No. 2, wherein one or A polypeptide obtained by deleting, substituting or increasing the obtained polynucleotide by a plurality of bases.
  • the gene expression vector can be constructed by selecting a gene fragment of a helper protein, designing a primer, enzymatic cleavage site on the primer, PCR amplification of the accessory protein gene fragment, and ligation to the expression after digestion with the corresponding enzyme.
  • a recombinant plasmid is obtained.
  • a gene fragment of syphilis detection antigen was selected, primers were designed, and the primers were ligated with restriction enzyme sites.
  • the genomic detection antigen gene fragment was amplified by PCR and ligated into the recombinant plasmid with the corresponding enzyme to obtain the gene expressing the fusion protein.
  • Expression vector is described by selecting a gene fragment of a helper protein, designing a primer, enzymatic cleavage site on the primer, PCR amplification of the accessory protein gene fragment, and ligation to the expression after digestion with the corresponding enzyme.
  • the gene expression vector can select a plasmid for bicistronic expression, for example, pET-24a (Novagen, Cat. No. 69864-3), pET-30a, and the like. Wherein, the expression sequence of the flexible linker peptide serves as a second cistron.
  • the host cell can be a prokaryotic cell, such as E. coli.
  • the gene expression vector transformation operation is carried out by the method recommended by the kit manufacturer.
  • the host cell is a competent cell, such as an E. coli competent cell, and the constructed gene expression vector is added to the competent cell, and the heat shock treatment is performed to make the feeling The cell membrane structure of the cells is disturbed, and a gap appears on the cell membrane to allow the gene expression vector to enter the cell, and then cultured at a constant temperature to resuscitate the host cell.
  • Tween 20 or Triton X-100 is also included in S40.
  • the mass concentration of Tween 20 or Triton X-100 is 0.1% to 0.3%.
  • the Tween 20 or Triton X-100 has a mass percent concentration of 0.15%.
  • Prokaryotic expression of TP recombinant antigen often contains more polymer structure, and a certain amount of Tween 20 or Triton X-100 is better solved in the purification process.
  • the crude product obtained by purifying S40 is purified by an affinity column, and then subjected to affinity column and ion exchange purification to obtain a TP recombinant antigen.
  • a syphilis detection reagent contains a solution of the above TP recombinant antigen.
  • the solution of the TP recombinant antigen contains Tween 20 or Triton X-100.
  • the mass concentration of Tween 20 or Triton X-100 is 0.1% to 0.3%.
  • the Tween 20 or Triton X-100 has a mass percent concentration of 0.15%.
  • Prokaryotic expression of TP recombinant antigen often contains more polymer structure, and the addition of a certain amount of Tween 20 or Triton X-100 during use is better to solve the problem.
  • the syphilis test strip of one embodiment includes a coating antigen, a labeled antigen, a GST monoclonal antibody, and a label.
  • the labeled antigen comprises a GST tag, a chimeric expression polypeptide, and a flexible linker peptide that facilitates soluble expression, and the chimeric expression polypeptide is a TPN15-TPN17-TPN47 chimeric expression polypeptide.
  • the coating antigen includes the above chimeric expression polypeptide linked in sequence and the above flexible link peptide;
  • the label binds indirectly to the labeled antigen by an anti-GST monoclonal antibody.
  • the label may be larger nanoparticles such as colloidal gold, colloidal selenium, colloidal silver or latex.
  • the syphilis detection kit of one embodiment includes the syphilis detection reagent described above or the syphilis test strip described above.
  • the syphilis detection kit of one embodiment shown in FIGS. 2, 3, and 4 includes a syphilis test strip 100, a housing 200, and other detection reagents.
  • the syphilis test strip 100 includes a support sheet 110, a sample pad 120, a gold standard pad 130, a nitrocellulose membrane 140, an absorbent pad 150, a test line 160, and a quality control line 170.
  • the sample pad 120, the gold standard pad 130, the nitrocellulose membrane 140, and the absorption pad 150 are sequentially disposed on the support sheet 110 from one end to the other end of the support sheet 110.
  • the sample pad 120 partially overlaps the gold standard pad 130
  • the gold standard pad 130 partially overlaps the nitrocellulose membrane 140
  • the nitrocellulose membrane 140 partially overlaps the absorption pad 150.
  • the detection line 160 and the quality control line 170 are disposed on the nitrocellulose membrane, and the detection line 160 is disposed at one end of the gold standard pad 130, and the quality control line 170 is disposed at one end of the absorption pad 150.
  • the support sheet 110 is made of a material that does not absorb water.
  • Sample pad 120 is used for sample spotting.
  • the anti-GST monoclonal monomer is coated with colloidal gold particles and coupled to the labeled antigen and uniformly coated on the gold pad 130.
  • the coating antigen is coated on the nitrocellulose membrane 140.
  • the labeled antigen comprises a GST tag, a chimeric expression polypeptide, and a flexible linker peptide that facilitates soluble expression, and the chimeric expression polypeptide is a TPN15-TPN17-TPN47 chimeric expression polypeptide.
  • the coating antigen includes the above chimeric expression polypeptides ligated in sequence as well as the above flexible link peptide.
  • Detection line 160 is an affinity purified anti-syphilis antigen and quality control line 170 is a goat anti-mouse IgG antibody.
  • the mark on the gold standard pad 130 is colloidal gold. It can be understood that in other embodiments, the mark may also be larger nanoparticles such as colloidal selenium, colloidal silver or latex.
  • the anti-syphilis antigen used is a Treponema pallidum antigen.
  • test strip 100 can be placed within housing 200 of the test kit.
  • a sample insertion hole 210 and a observation window 220 are opened in the housing 200.
  • the sample well 210 corresponds to the position of the sample pad 120.
  • the detection line 160 and the quality control line 170 are exposed in the observation window 220 for convenient observation.
  • detection reagents can be prepared directly in the laboratory as needed.
  • the above detection kit detects the presence or absence of a Treponema pallidum antibody in the test material by a double antigen sandwich method. At the time of detection, all the Treponema pallidum antibodies in the sample are first bound to the labeled antigen indirectly labeled by the colloidal gold, and the reaction complex migrates forward along the coating membrane due to capillary action. If the sample contains treponema pallidum antibody and reaches the detection line 160, Upon encountering the coated antigen coated on the nitrocellulose membrane 140, a coated antigen-tested Treponema pallidum antibody-labeled antigen-marker complex is formed, thereby enriching on the detection line 160 to form a red precipitation line.
  • the syphilis antigen is captured by the goat anti-mouse monoclonal antibody through the detection line 160, and is enriched on the quality control line 170 to form a red precipitation line. A positive result was obtained when the red line was simultaneously present on the test line 160 and the quality control line 170. If the sample does not contain the Treponema pallidum antibody, when the reaction complex reaches the detection line 160, the coated antigen will not form the coated antigen-tested Treponema pallidum antibody-labeled antigen-label complex, and the reaction complex passes the detection. Line 160, enriched only on the quality control line 170 to form a red precipitate line, at which point a negative result was judged.
  • the experimental methods without specific conditions are usually in accordance with conventional conditions, for example, see Sambrook, EF Frych, T Manny Artis, etc. (Jin Dongyan, Li Mengfeng, etc.)
  • the conditions described in the molecular cloning experimental guide [M] (Beijing: Science Press, 1992) or the method recommended by the manufacturer of the kit are implemented. All procedures are carried out in accordance with standard procedures in the art, and the reagents or carriers used are conventional reagents or conventional carriers.
  • coating antigens by coating, labeling antigens, immunogen sources, and hybridoma cells.
  • TpN17 GeneBank No: M74825
  • TpN15 GeneBank No: U73115.1
  • TpN47 GeneBank No: NC_000919.1 gene dominant epitopes
  • design overlapping PCR primers to synthesize TpN17 The active segments corresponding to 23aa-106aa), TpN15 (31aa-109aa), TpN47 (185aa-410aa) were amplified by bridge PCR and introduced into the flexible link peptide TRX, which facilitates soluble expression, to obtain a chimeric gene.
  • the gene was ligated with pMD18-T vector, transformed into E.
  • coli picked for monoclonal, and the correct positive plasmid was identified by PCR, and the plasmid was extracted and identified.
  • the plasmids identified by PCR identification and restriction enzyme digestion were sequenced, and the results were identical to the designed sequences.
  • the correctly sequenced plasmid was digested with the corresponding enzyme designed at both ends of the restriction enzyme, and the excised target sequence was ligated into the expression vector pET-24a.
  • the recombinant plasmid carrying the antigen is pET-24a-TPAG-TRX (including HIS tag), the plasmid is a bicistronic expression plasmid, wherein TRX is a second cistron; the recombinant plasmid labeled with the antigen is PET30a-GST-TPAG-TRX (containing HIS tag and GST peptide), the plasmid is a bicistronic expression plasmid, wherein TRX is a second cistron.
  • TPAG is the expression sequence of the TPN15-TPN17-TPN47 chimeric expression antigen, and the sequence is shown in SEQ ID No. 1;
  • TRX is the expression sequence of the chimeric expression polypeptide, and the sequence is shown in SEQ ID No. 2.
  • Immunogen and hybridoma cell selection antigens PGEX-2T, PGEX-6P-1, PGEX-5X-1, and pET-41a plasmids were used to transform the expression host ER2566.
  • the appropriate amount of transformed host bacteria was coated on resistant LB solid culture plates, and cultured overnight at 37 ° C. On the next day, 7 colonies capable of growing on resistant culture plates were picked and inoculated into 3 mL resistant strains.
  • the LB liquid medium was cultured at 37 ° C for 5 hours, and the host strain transformed with the empty vector was taken as a control. IPTG was added to a final concentration of 0.25 mM, and culture was induced for 6 h at 30 ° C and 180 rpm.
  • the cells were collected by centrifugation, and the cells were resuspended in 40 ⁇ L of 20 mM PBS buffer, and 20 ⁇ L of 3 ⁇ loading Buffer was added thereto, boiled in boiling water for 10 minutes, and then subjected to SDS-PAGE electrophoresis. Coomassie brilliant blue staining, the strain transformed into the recombinant plasmid had a distinct protein expression band at the predicted molecular weight position, whereas the strain transformed only into the empty plasmid did not have the band.
  • One microliter of the transformed host strain was inoculated into 500 mL of resistant LB medium, cultured overnight at 37 ° C, 200 rpm, and IPTG was added the next morning to a final concentration of 0.25 mM, 30 ° C, and induction at 180 rpm for 4 h.
  • the cells were collected by centrifugation, and an appropriate amount of lysis buffer was added thereto. After ultrasonication, the supernatant was centrifuged.
  • the ammonium sulfate gradient analysis was carried out separately, and the coated antigen was analyzed by using 10% saturated ammonium sulfate for the protein, and the protein was precipitated with 20% ammonium sulfate; the labeled antigen was directly precipitated with the target protein by 35%; the immunogen and the hybridoma cells were screened for the antigen.
  • the protein was precipitated with 15% saturated ammonium sulfate and the protein was precipitated with 30% ammonium sulfate.
  • the crude antigen was obtained after treatment with ammonium sulfate.
  • the coated antigen was purified by NI affinity medium and SP ion exchange chromatography to obtain the target protein and ensured that 0.15% Tween 20 or TritonX-100 was contained in the balance solution and elution buffer; the labeled antigen was purified by NI affinity medium and glutathione
  • the glycopeptide affinity medium was purified to obtain the target protein; the final purity of both antigens was over 90% (see Figure 5, coated antigen and labeled antigen SDS-PAGE gel purity electrophoresis map: lane 1 protein Marker; lane 2 purified and coated Antigen; labeled with antigen after washing in lane 3), stored at -20 ° C until use.
  • the immunogen and hybridoma cell screening antigens were purified by glutathione affinity medium to obtain the target protein, and stored at -20 ° C until use.
  • the recombinant GST protein solution obtained above was dialyzed against PBS, diluted to 1.0 mg/mL with PBS, mixed with an equal volume of Freund's complete adjuvant, and fully emulsified to obtain an oily emulsion.
  • the emulsion was administered subcutaneously to the back site of 6-week-old female BALB/c mice at a dose of 0.2 mL.
  • the abdominal cavity was boosted, that is, the same amount of antigen was mixed with the Freund's incomplete adjuvant, and the immunization was increased to four needles.
  • the tail blood was collected, the serum was separated, and the titer was determined by indirect ELISA. It can be used for fusion at 1:10000.
  • Three days before the fusion the same dose of antigen was mixed with an equal volume of 0.9% sodium chloride injection for intraperitoneal injection of booster immunization.
  • the immunization method was the same as above.
  • BALB/c mouse peritoneal macrophages were used as feeder cells.
  • the BALB/c mice were sacrificed by neck-stretching, 75% alcohol was immersed in the whole body, and the abdominal skin was cut with scissors under aseptic operation.
  • the peritoneum was exposed, and 5 mL of RPMI 1640 basic culture solution was injected into the abdominal cavity with a syringe. Rinse repeatedly, recover the rinse solution, centrifuge at 1000 rpm for 5 minutes, leave a precipitate, resuspend with RPMI 1640 screening medium (in RPMI 1640 complete medium containing HAT), adjust the cell concentration to 1 ⁇ 10 5 /mL, and add 96 wells. Plates, 150 ⁇ L/well, cultured overnight at 37 ° C, 5% CO 2 .
  • mice Three days after the last immunization of the mice, the spleens were taken out under aseptic conditions, placed in a dish, rinsed once with RPMI 1640 base medium, and placed in a nylon beaker on a small beaker to be filtered to prepare a cell suspension. After centrifugation, the supernatant was discarded, and the RPMI 1640 base medium was resuspended, and this was repeated three times and counted.
  • Mouse myeloma cells Sp2/0 were screened by 8-azaguanine and cultured to logarithmic growth phase. Two large bottles were prepared to make cell suspension, centrifuged, and the supernatant was discarded and resuspended in RPMI 1640 base medium. Repeat three times and count.
  • the myeloma cells and the immune spleen cells were mixed at a ratio of 1:10, and washed once with a RPMI 1640 base culture solution in a 50 mL plastic centrifuge tube, and centrifuged at 1,200 rpm for 8 minutes. The supernatant was discarded, the cells were mixed, 1 mL of 50% PEG1500 fusion was slowly added, and after 1 minute of fusion, 15 mL of RPMI1640 basal medium was added to terminate the cell fusion. Centrifuge for 5 minutes at 1,000 rpm. The supernatant was discarded and gently incubated with 50 mL of RPMI 1640 screening medium and aliquoted into 10 96-well plates (fed cells). Incubate at 50 ⁇ L/well, 37 ° C, 5% CO 2 . The culture was continued until the sixth day, and the HT medium (HTMI-containing RPMI1640 complete medium) was changed twice.
  • HT medium HTMI-containing RPMI1640 complete
  • Different GST recombinant proteins were diluted with 0.06 M pH 9.6 carbonate buffer solution (PGEX-2T, PGEX-6P-1, PGEX-5X-1, pET-41a, PET30a-GST-TPAG-TRX, respectively), while pET -24a-TPAG was used as a negative control to a final concentration of 1 ⁇ g/mL.
  • 0.1 mL per well was added to a 96-well polystyrene plate at 37 ° C for 2 hours or 4 ° C overnight. The next day, it was blocked with 0.02 M pH 7.2 PBS containing 10% calf serum or 1% skim milk powder at 0.15 mL/well for 2 hours at 37 ° C for detection.
  • mice Six to eight weeks of robust BALB/c mice were selected, and each mouse was intraperitoneally injected with 0.5 mL of pristane; after 10 days, 1 x 10 6 hybridoma cells were intraperitoneally injected. After inoculation of cells for 7 to 10 days, ascites can be produced, and the animal's health and ascites signs should be closely observed. As much as possible of ascites, while the mice are killed before death, the mice are sacrificed and the ascites is inhaled into the test tube with a dropper. The mice can obtain 5-10 mL of ascites. Ascites was collected, centrifuged, and stored in a refrigerator at -20 °C.
  • PET30a-GST-TPAG-TRX was added to the 100 ⁇ l colloidal gold-labeled GST monoclonal antibody complex, fully mixed and stored at 4 ° C. .
  • the indirect labeled gold standard complex was named PET30a-GST-TPAG-TRX-GSTAb-AU.
  • the gold standard compound was diluted 10 times with a colloidal gold dilution solution, and the glass fiber was immersed and lyophilized to prepare a gold standard pad.
  • 1mL colloidal gold plus 15 ⁇ L of 0.2M K2CO3 labeled 10 ⁇ g, GSTAb258 monoclonal antibody the product has the highest sensitivity and specificity.
  • T line detection line
  • the T line is close to the gold standard pad end, about 5 mm from the gold standard pad end; diluted with the same dilution liquid to prepare the goat anti-mouse monoclonal antibody to 0.5 mg/mL
  • the C line is close to the absorption pad and is about 3 mm away from the absorption pad. The distance between the two lines is 5 ⁇ 8mm, and it is dried at 37°C, and the package is ready for use.
  • the coated nitrocellulose membrane, the gold standard pad, the absorbent pad, the sample pad, and the polyester plate were sequentially laminated as shown in the figure, and cut into strips of 3 mm width. For every 10 strips, add a desiccant and vacuum package. Store at 4 to 30 °C.
  • the indirect labeled double antigen sandwich method gold label detection system of the above Example 3 was used for detecting TP antibodies, and the sensitivity, specificity, repeatability, stability, precision and the like were superior to the existing commercial kits.
  • the sensitivity of the kit A was 98.4%
  • the sensitivity of the kit B was 100%
  • the specific kit A was 99.75%
  • the kit B was 99.9%. It is indicated that the kit of the present embodiment is superior to the existing products in both sensitivity and specificity, and can be completely used for rapid diagnosis of conventional syphilis.
  • the finished kit of this example has good stability.

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Abstract

Provided are a TP recombinant antigen, and a preparation method and an application thereof. The TP recombinant antigen comprises sequentially connected chimeric expression polypeptides and a flexible linker which facilitates soluble expression. The chimeric expression polypeptides are TPN15-TPN17-TPN47 chimeric-expression polypeptides.

Description

TP重组抗原及其制备方法和应用TP recombinant antigen and preparation method and application thereof 技术领域：Technical field:
本发明涉及免疫检测领域，特别是涉及一种TP重组抗原及其制备方法和应用。The invention relates to the field of immunoassay, in particular to a TP recombinant antigen and a preparation method and application thereof.
背景技术：Background technique:
梅毒是一种性传播疾病，其病原体是***，也叫***(Treponema pallidum，TP)，主要通过性接触和血液传播，并产生多种多样的症状和体征，且时隐时显，病程可持续很长，几乎可侵犯全身各器官。是仅次于艾滋病的严重危害我国人民身体健康的性传播疾病，近年来在我国有上升的趋势，据2014年梅毒重点疫情报告，2009年以来梅毒位于乙类传染病报告发病数的第三位；尤其是在产前门诊筛查出的梅毒抗体阳性的孕妇显著增加，给我国的公共卫生带来新的问题。Syphilis is a sexually transmitted disease, the pathogen of which is Treponema pallidum, also known as Treponema pallidum (TP), which is mainly transmitted through sexual contact and blood, and produces a variety of symptoms and signs, and the time is hidden, the course of disease It lasts for a long time and can almost invade all organs of the body. It is a sexually transmitted disease that is second only to AIDS and seriously endangers the health of our people. In recent years, there has been an upward trend in China. According to the 2014 report on the major syphilis epidemic, since 2009, syphilis has been ranked third in the number of reported cases of Class B infectious diseases. In particular, the number of syphilis-positive pregnant women screened in prenatal clinics has increased significantly, bringing new problems to public health in China.
国内外对梅毒的发病机制和诊断方式进行了大量的研究，梅毒的诊断和治疗后效果评价一直是利用体液免疫学指标，目前国内对血源筛选还有部分采用RPR和TRUST方法，其主要缺点是敏感性和特异性不高，许多非梅毒性疾病，包括类风湿性关节炎、***性红斑狼疮、慢性迁延性肝炎等均可呈阳性反应。而荧光***抗体吸收试验和***血凝试验虽大大改善了检测的特异性，但是需制备大量病原体，且质量难以控制，也不利于大规模的血液筛查工作。因此，研制快速、简便、灵敏且特异的诊断试剂是当前的主要任务。A large number of studies have been carried out on the pathogenesis and diagnosis of syphilis at home and abroad. The diagnosis and treatment evaluation of syphilis has always been the use of humoral immunological indicators. At present, there are some RPR and TRUST methods for blood source screening in China. Sensitivity and specificity are not high, and many non-syphilitic diseases, including rheumatoid arthritis, systemic lupus erythematosus, and chronic persistent hepatitis, can be positive. The fluorescent trehalose antibody absorption test and the treponema hemagglutination test have greatly improved the specificity of the detection, but a large number of pathogens need to be prepared, and the quality is difficult to control, which is not conducive to large-scale blood screening work. Therefore, the development of rapid, simple, sensitive and specific diagnostic reagents is currently the main task.
目前应用比较广泛的***血清检测方法为酶联免疫吸附试验(ELISA)和胶体金或乳胶快速检测。此方法以抗原-抗体的特异性识别为基础。酶联免疫吸附试验(ELISA)具有灵敏度高、特异性好的优势，已经成为梅毒检 测的主流方法。而胶体金或乳胶法具有快捷、方便的优势，已经应用得越来越广泛。At present, the widely used treponema pallidum serum detection method is enzyme-linked immunosorbent assay (ELISA) and rapid detection of colloidal gold or latex. This method is based on the specific recognition of antigen-antibody. Enzyme-linked immunosorbent assay (ELISA) has the advantages of high sensitivity and good specificity, and has become a syphilis test. The mainstream method of measurement. The colloidal gold or latex method has the advantages of quickness and convenience, and has been applied more and more widely.
免疫层析胶体金技术是新型的诊断技术，已得到较为广泛的应用，基本原理如下：利用胶体金标记一种抗原或抗体，在试纸条的硝酸纤维素膜上包被相应的配对抗原或抗体，检测时当样品中含有相应的特异性抗体或抗原时，胶体金标记颗粒和样品中配体相结合形成复合物，然后在硝酸纤维素膜上层析，再被包被的抗原或抗体捕获，形成肉眼可见的检测线(T线)，通过检测线的有无实现对结果的判定。Immunochromatography colloidal gold technology is a new type of diagnostic technology, which has been widely used. The basic principle is as follows: labeling an antigen or antibody with colloidal gold, coating the corresponding paired antigen on the nitrocellulose membrane of the test strip or The antibody, when the sample contains the corresponding specific antibody or antigen, the colloidal gold labeled particles and the ligand in the sample combine to form a complex, and then chromatographed on the nitrocellulose membrane, and then coated antigen or antibody Capture, form a visible line (T line) visible to the naked eye, and determine the result by detecting the presence or absence of the line.
从方法学上讲，目前梅毒ELISA和胶体金检测用的都是标记物和标记抗原直接标记的双抗原夹心法，其存在的固有缺陷如下：Methodologically, the current syphilis ELISA and colloidal gold detection are both double-antibody sandwich methods for direct labeling of labeled and labeled antigens, and their inherent defects are as follows:
1、当标记物为酶、发光物质、放射性物质等小标记物时，为了提高灵敏度，需要提高标记比例，但是比例过高会使标记抗原被标记物所包裹，使得抗原表位被包埋而导致抗原活性降低；1. When the label is a small label such as an enzyme, a luminescent substance or a radioactive substance, in order to increase the sensitivity, it is necessary to increase the labeling ratio, but if the ratio is too high, the labeling antigen is encapsulated by the label, so that the antigen epitope is embedded. Lead to a decrease in antigenic activity;
2、当标记物为较大的纳米颗粒如使用胶体金、乳胶或其他纳米颗粒类标记物时，理论上最佳的灵敏度状态是标记比例越低灵敏度越高，摩尔比为1：1时灵敏度最高，但由于直接标记过程中，标记抗原用量过低会导致标记沉淀等不易标记因素而使标记比例无法降得过低，从而导致灵敏度偏低，同时标记抗原使用量的提高也会导致特异性降低。因此，急需在现有梅毒双抗原夹心法试剂盒的上面做出进一步的改进，提高试剂盒的灵敏度的同时改善特异性。2. When the label is a large nanoparticle such as colloidal gold, latex or other nanoparticle label, the theoretically optimal sensitivity state is that the lower the labeling ratio, the higher the sensitivity, and the sensitivity is 1:1. The highest, but due to the low labeling antigen in the direct labeling process, it will lead to labeling and other difficult labeling factors, so that the labeling ratio cannot be lowered too low, resulting in low sensitivity, and the increase in the amount of labeled antigen will also lead to specificity. reduce. Therefore, there is an urgent need to make further improvements on the existing syphilis double antigen sandwich kit to improve the sensitivity of the kit while improving specificity.
80年代初，随着分子生物学技术的发展，特别是基因工程(DNA重组)技术的出现使***的研究进入一个新的阶段，***的全部基因组DNA序列己经被解析。通过重组***DNA的克隆以及在大肠杆菌中的表达，制备出多种重组TP抗原，为梅毒的研究提供了一条新的途径。但原核表达缺乏翻译后修饰功能，有时需要特殊蛋白伴侣一起表达并进一步的进行复性处理或保存缓冲液的摸索才能使其蛋白活性较好的表现出来。 In the early 1980s, with the development of molecular biology technology, especially the emergence of genetic engineering (DNA recombination) technology, the study of Treponema pallidum entered a new stage, and the entire genomic DNA sequence of Treponema pallidum has been resolved. Through the cloning of recombinant Treponema pallidum DNA and its expression in E. coli, a variety of recombinant TP antigens were prepared, which provided a new way for the study of syphilis. However, prokaryotic expression lacks post-translational modification, and sometimes requires the expression of a special protein partner and further renaturation or preservation of the buffer to make its protein activity better.
发明内容：Summary of the invention:
基于此，有必要提供一种蛋白活性较好的TP重组抗原及其制备方法和应用。Based on this, it is necessary to provide a TP recombinant antigen with better protein activity, a preparation method and application thereof.
一种TP重组抗原，包括依次连接的嵌合表达多肽以及有助于可溶性表达的柔性链接肽，所述嵌合表达多肽为TPN15-TPN17-TPN47嵌合表达多肽。A TP recombinant antigen comprising a chimeric expression polypeptide linked in turn and a flexible linker peptide which facilitates soluble expression, the chimeric expression polypeptide being a TPN15-TPN17-TPN47 chimeric expression polypeptide.
在一个实施例中，还包括GST标签，所述GST标签、所述嵌合表达多肽以及所述柔性链接肽依次连接。In one embodiment, a GST tag is further included, the GST tag, the chimeric expression polypeptide, and the flexible linker peptide are linked in turn.
在一个实施例中，所述TPN15-TPN17-TPN47嵌合表达抗原为(a)、由SEQ ID No.1所示的核苷酸序列组成的多核苷酸编码得到的多肽；(b)、与SEQ ID No.1所示的核苷酸序列组成的多核苷酸具有至少98％同源性的多核苷酸编码得到的多肽；或(c)、由SEQ ID No.1所示的核苷酸序列组成的多核苷酸，其中一个或多个碱基被缺失、替代或增加得到的多核苷酸编码得到的多肽。In one embodiment, the TPN15-TPN17-TPN47 chimeric expression antigen is (a) a polypeptide encoded by a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID No. 1, (b), and a polynucleotide comprising a nucleotide sequence represented by SEQ ID No. 1 having a polynucleotide encoding at least 98% homology; or (c) a nucleotide represented by SEQ ID No. 1. A polynucleotide consisting of a sequence in which one or more bases are deleted, replaced or increased by a polypeptide encoded by the resulting polynucleotide.
在一个实施例中，所述柔性链接肽为(a)、由SEQ ID No.2所示的核苷酸序列组成的多核苷酸编码得到的多肽；(b)、与SEQ ID No.2所示的核苷酸序列组成的多核苷酸具有至少98％同源性的多核苷酸编码得到的多肽；或(c)、由SEQ ID No.2所示的核苷酸序列组成的多核苷酸，其中一个或多个碱基被缺失、替代或增加得到的多核苷酸编码得到的多肽。In one embodiment, the flexible linker peptide is (a) a polypeptide encoded by a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID No. 2; (b), and SEQ ID No. 2 a polynucleotide having a nucleotide sequence consisting of a polynucleotide having a polynucleotide having at least 98% homology; or (c) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID No. a polypeptide in which one or more bases are deleted, substituted or increased by the resulting polynucleotide.
一种TP重组抗原的制备方法，包括如下步骤：A method for preparing a TP recombinant antigen comprises the following steps:
步骤一、提供基因表达载体，所述基因表达载体用于表达TP重组抗原，所述TP重组抗原包括嵌合表达多肽以及有助于可溶性表达的柔性链接肽，所述嵌合表达多肽为TPN15-TPN17-TPN47嵌合表达多肽； Step 1. Providing a gene expression vector for expressing a TP recombinant antigen, the TP recombinant antigen comprising a chimeric expression polypeptide and a flexible link peptide capable of soluble expression, the chimeric expression polypeptide is TPN15- TPN17-TPN47 chimeric expression polypeptide;
步骤二、将所述基因表达载体转化到宿主细胞中，所述宿主细胞为原核细胞；Step two, transforming the gene expression vector into a host cell, wherein the host cell is a prokaryotic cell;
步骤三、对转化了所述基因表达载体的所述宿主细胞进行诱导表达，分离得到表达液；Step 3: inducing expression of the host cell transformed with the gene expression vector, and separating the expression liquid;
步骤四、对所述表达液中进行饱和硫酸铵梯度分析，确定沉杂蛋白和沉 目的蛋白的硫酸铵浓度，加入相应的沉杂的硫酸铵终浓度，充分静止后收集上清，接着在所述上清中加入饱和硫酸铵沉目的蛋白的终浓度，充分静止后收集沉淀，将所述沉淀重新溶解，得到粗产物；以及Step 4: performing gradient analysis of saturated ammonium sulfate on the expression solution to determine sedimentation protein and sedimentation The ammonium sulfate concentration of the target protein is added to the corresponding final concentration of ammonium sulfate, and the supernatant is collected after sufficient rest, and then the final concentration of the saturated ammonium sulfate protein is added to the supernatant, and the precipitate is collected after being completely rested. The precipitate is redissolved to give a crude product;
步骤五、利用亲和柱纯化所述粗产物，再进行亲和柱及离子交换纯化，得到所述TP重组抗原。Step 5: purifying the crude product by using an affinity column, and then performing affinity column and ion exchange purification to obtain the TP recombinant antigen.
在一个实施例中，所述TP重组抗原还包括GST标签，所述GST标签、所述嵌合表达多肽以及所述柔性链接肽依次连接。In one embodiment, the TP recombinant antigen further comprises a GST tag, the GST tag, the chimeric expression polypeptide, and the flexible linker peptide are linked in turn.
在一个实施例中，所述TPN15-TPN17-TPN47嵌合表达抗原为(a)、由SEQ ID No.1所示的核苷酸序列组成的多核苷酸编码得到的多肽；(b)、与SEQ ID No.1所示的核苷酸序列组成的多核苷酸具有至少98％同源性的多核苷酸编码得到的多肽；或(c)、由SEQ ID No.1所示的核苷酸序列组成的多核苷酸，其中一个或多个碱基被缺失、替代或增加得到的多核苷酸编码得到的多肽。In one embodiment, the TPN15-TPN17-TPN47 chimeric expression antigen is (a) a polypeptide encoded by a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID No. 1, (b), and a polynucleotide comprising a nucleotide sequence represented by SEQ ID No. 1 having a polynucleotide encoding at least 98% homology; or (c) a nucleotide represented by SEQ ID No. 1. A polynucleotide consisting of a sequence in which one or more bases are deleted, replaced or increased by a polypeptide encoded by the resulting polynucleotide.
在一个实施例中，所述柔性链接肽为(a)、由SEQ ID No.2所示的核苷酸序列组成的多核苷酸编码得到的多肽；(b)、与SEQ ID No.2所示的核苷酸序列组成的多核苷酸具有至少98％同源性的多核苷酸编码得到的多肽；或(c)、由SEQ ID No.2所示的核苷酸序列组成的多核苷酸，其中一个或多个碱基被缺失、替代或增加得到的多核苷酸编码得到的多肽。In one embodiment, the flexible linker peptide is (a) a polypeptide encoded by a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID No. 2; (b), and SEQ ID No. 2 a polynucleotide having a nucleotide sequence consisting of a polynucleotide having a polynucleotide having at least 98% homology; or (c) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID No. a polypeptide in which one or more bases are deleted, substituted or increased by the resulting polynucleotide.
一种梅毒检测试剂，所述梅毒检测试剂含有上述的TP重组抗原的溶液；A syphilis detecting reagent, wherein the syphilis detecting reagent contains a solution of the above TP recombinant antigen;
所述TP重组抗原的溶液中含有质量百分浓度为0.1％～0.3％的吐温20或质量百分浓度为0.1％～0.3％的Triton X-100。The solution of the TP recombinant antigen contains Tween 20 in a mass percentage concentration of 0.1% to 0.3% or Triton X-100 in a mass percentage concentration of 0.1% to 0.3%.
在一个实施例中，所述TP重组抗原的溶液中含有质量百分浓度为0.15％的吐温20或质量百分浓度为0.15％的Triton X-100。In one embodiment, the solution of the TP recombinant antigen contains 0.15% by weight of Tween 20 or 0.15% by weight of Triton X-100.
一种梅毒检测试纸，包括包被抗原、标记抗原、GST单克隆抗体及标记物；A syphilis test strip comprising a coating antigen, a labeled antigen, a GST monoclonal antibody and a label;
所述标记抗原包括依次连接的GST标签、嵌合表达多肽以及有助于可溶性表达的柔性链接肽，所述嵌合表达多肽为TPN15-TPN17-TPN47嵌合表达 多肽；The labeled antigen comprises a GST tag, a chimeric expression polypeptide, and a flexible linker peptide that facilitates soluble expression, which is a TPN15-TPN17-TPN47 chimeric expression. Polypeptide
所述包被抗原包括依次连接的所述嵌合表达多肽以及所述柔性链接肽；The coating antigen includes the chimeric expression polypeptide and the flexible link peptide;
所述标记物通过所述抗GST单克隆抗体与所述标记抗原间接结合。The label is indirectly bound to the labeled antigen by the anti-GST monoclonal antibody.
在一个实施例中，所述TPN15-TPN17-TPN47嵌合表达抗原为(a)、由SEQ ID No.1所示的核苷酸序列组成的多核苷酸编码得到的多肽；(b)、与SEQ ID No.1所示的核苷酸序列组成的多核苷酸具有至少98％同源性的多核苷酸编码得到的多肽；或(c)、由SEQ ID No.1所示的核苷酸序列组成的多核苷酸，其中一个或多个碱基被缺失、替代或增加得到的多核苷酸编码得到的多肽。In one embodiment, the TPN15-TPN17-TPN47 chimeric expression antigen is (a) a polypeptide encoded by a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID No. 1, (b), and a polynucleotide comprising a nucleotide sequence represented by SEQ ID No. 1 having a polynucleotide encoding at least 98% homology; or (c) a nucleotide represented by SEQ ID No. 1. A polynucleotide consisting of a sequence in which one or more bases are deleted, replaced or increased by a polypeptide encoded by the resulting polynucleotide.
在一个实施例中，所述柔性链接肽为(a)、由SEQ ID No.2所示的核苷酸序列组成的多核苷酸编码得到的多肽；(b)、与SEQ ID No.2所示的核苷酸序列组成的多核苷酸具有至少98％同源性的多核苷酸编码得到的多肽；或(c)、由SEQ ID No.2所示的核苷酸序列组成的多核苷酸，其中一个或多个碱基被缺失、替代或增加得到的多核苷酸编码得到的多肽。In one embodiment, the flexible linker peptide is (a) a polypeptide encoded by a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID No. 2; (b), and SEQ ID No. 2 a polynucleotide having a nucleotide sequence consisting of a polynucleotide having a polynucleotide having at least 98% homology; or (c) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID No. a polypeptide in which one or more bases are deleted, substituted or increased by the resulting polynucleotide.
一种梅毒检测试剂盒，包括上述的梅毒检测试剂。A syphilis detection kit comprising the above syphilis detection reagent.
一种梅毒检测试剂盒，包括上述的梅毒检测试纸。A syphilis detection kit comprising the above syphilis test strip.
这种TP重组抗原通过引入有助于可溶性表达的柔性链接肽对TPN15-TPN17-TPN47嵌合表达多肽进行修饰，相对于传统的TP抗原，这种TP重组抗原的蛋白活性较好。This TP recombinant antigen modifies the TPN15-TPN17-TPN47 chimeric expression polypeptide by introducing a flexible link peptide which contributes to soluble expression, and the TP recombinant antigen has a better protein activity than the conventional TP antigen.
附图说明DRAWINGS
图1为一实施方式的TP重组抗原的制备方法的流程图；1 is a flow chart showing a method for preparing a TP recombinant antigen according to an embodiment;
图2为一实施方式的梅毒检测试纸的正面示意图；2 is a front elevational view of a syphilis test strip of an embodiment;
图3为如图2所示的梅毒检测试纸的截面示意图；Figure 3 is a schematic cross-sectional view of the syphilis test strip shown in Figure 2;
图4为一实施方式的梅毒检测试剂盒的结构示意图；4 is a schematic structural view of a syphilis detection kit according to an embodiment;
图5为实施例1中包被抗原和标记抗原的SDS-PAGE胶纯度电泳图。 Figure 5 is a chromatogram of the purity of SDS-PAGE gel coated with antigen and labeled antigen in Example 1.
具体实施方式detailed description
下面主要结合附图及具体实施例对TP重组抗原及其制备方法和应用做进一步的解释说明。The TP recombinant antigen and its preparation method and application are further explained below mainly in conjunction with the accompanying drawings and specific examples.
一实施方式的TP(***)重组抗原，包括嵌合表达多肽以及有助于可溶性表达的柔性链接肽，嵌合表达多肽为TPN15-TPN17-TPN47嵌合表达多肽。The TP (P. pallidum) recombinant antigen of one embodiment comprises a chimeric expression polypeptide and a flexible linker peptide which facilitates soluble expression, and the chimeric expression polypeptide is a TPN15-TPN17-TPN47 chimeric expression polypeptide.
这种TP重组抗原可以用作包被抗原和标记抗原，应用到梅毒检测领域。This TP recombinant antigen can be used as a coating antigen and a labeled antigen, and is applied to the field of syphilis detection.
作为标记抗原时，这种TP重组抗原还包括GST标签，GST标签、嵌合表达多肽以及柔性链接肽依次连接。As a marker antigen, the TP recombinant antigen also includes a GST tag, and the GST tag, the chimeric expression polypeptide, and the flexible linker peptide are sequentially linked.
具体的，TPN15-TPN17-TPN47嵌合表达抗原为：(a)、由SEQ ID No.1所示的核苷酸序列组成的多核苷酸编码得到的多肽；(b)、与SEQ ID No.1所示的核苷酸序列组成的多核苷酸具有至少98％同源性的多核苷酸编码得到的多肽；或(c)、由SEQ ID No.1所示的核苷酸序列组成的多核苷酸，其中一个或多个碱基被缺失、替代或增加得到的多核苷酸编码得到的多肽。Specifically, the TPN15-TPN17-TPN47 chimeric expression antigen is: (a) a polypeptide encoded by a polynucleotide consisting of the nucleotide sequence shown in SEQ ID No. 1, (b), and SEQ ID No. a polynucleotide comprising a nucleotide sequence of 1 having a polynucleotide encoding at least 98% homology; or (c) a multinuclear comprising the nucleotide sequence of SEQ ID No. 1. A glycoside acid in which one or more bases are deleted, substituted or increased by the resulting polynucleotide encoding the resulting polypeptide.
具体的，柔性链接肽为：(a)、由SEQ ID No.2所示的核苷酸序列组成的多核苷酸编码得到的多肽；(b)、与SEQ ID No.2所示的核苷酸序列组成的多核苷酸具有至少98％同源性的多核苷酸编码得到的多肽；或(c)、由SEQ ID No.2所示的核苷酸序列组成的多核苷酸，其中一个或多个碱基被缺失、替代或增加得到的多核苷酸编码得到的多肽。Specifically, the flexible link peptide is: (a) a polypeptide encoded by a polynucleotide consisting of the nucleotide sequence shown in SEQ ID No. 2; (b) and a nucleoside represented by SEQ ID No. 2. a polynucleotide having a nucleic acid sequence consisting of a polynucleotide having at least 98% homology; or (c) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID No. 2, wherein one or A polypeptide obtained by deleting, substituting or increasing the obtained polynucleotide by a plurality of bases.
GST标签为由SEQ ID No.3所示的核苷酸序列组成的多核苷酸编码得到的多肽。The GST tag is a polypeptide encoded by a polynucleotide consisting of the nucleotide sequence shown by SEQ ID No. 3.
由于蛋白编码序列的多态性及变异，天然存在的蛋白质会出现基因突变，编码序列中碱基被缺失、替代或增加，或氨基酸的缺失、***、取代或其它变异，从而导致蛋白质的氨基酸序列出现一个或多个氨基酸被缺失、替代或增加。因此，存在着一些生理和生物活性上基本等同于无变异蛋白质的蛋白。这些结构不同于相应的蛋白质，但与该蛋白质没有明显的功能差异的多肽或蛋白称为功能等同变异体。 Due to the polymorphism and variation of the protein coding sequence, the naturally occurring protein may be genetically altered, the base in the coding sequence is deleted, replaced or increased, or the amino acid is deleted, inserted, substituted or otherwise mutated, resulting in the amino acid sequence of the protein. One or more amino acids are deleted, replaced or added. Therefore, there are some proteins whose physiological and biological activities are substantially equivalent to those of non-mutant proteins. These structures differ from the corresponding proteins, but polypeptides or proteins that have no apparent functional difference from the protein are referred to as functionally equivalent variants.
功能等同的变异体同样适用于通过缺失、***和突变等人工手段改变一个或多个密码子，从而向一种蛋白质的氨基酸序列中导入这类变异而制成的多肽。尽管这样能获得更多不同形式的变异体，但所得的变异体作为功能等同变异体的前提是其生理活性基本等同于原始无变异蛋白质的活性。Functionally equivalent variants are equally applicable to polypeptides obtained by introducing one or more codons by artificial means such as deletions, insertions, and mutations to introduce such mutations into the amino acid sequence of a protein. Although more variants of this form can be obtained in this way, the resulting variant is a functionally equivalent variant with the physiological activity essentially equivalent to that of the original non-mutated protein.
一般的，功能等同变异体的编码序列是同源的，因此，由至少一种改变(如蛋白质的编码序列中一个或多个碱基的缺失、***或取代或者蛋白质的氨基酸序列中有一个或多个氨基酸缺失、***或取代)所得的多肽或蛋白一般具有功能上等同于所述蛋白质的活性，因此，由上述核苷酸序列编码的到的蛋白或上述氨基酸序列组成的多肽，如果大肠杆菌周质蛋白没有明显的功能差异，也包括在本发明的范围内。In general, the coding sequence of a functionally equivalent variant is homologous and, therefore, is altered by at least one (eg, a deletion, insertion or substitution of one or more bases in the coding sequence of the protein or one of the amino acid sequences of the protein or A polypeptide or protein obtained by deleting, inserting or substituting a plurality of amino acids generally has an activity functionally equivalent to the protein, and therefore, a polypeptide encoded by the above nucleotide sequence or a polypeptide consisting of the above amino acid sequence, if Escherichia coli Periplasmic proteins have no significant functional differences and are included within the scope of the invention.
这种TP重组抗原通过引入有助于可溶性表达的柔性链接肽对TPN15-TPN17-TPN47嵌合表达多肽进行修饰，相对于传统的TP抗原，这种TP重组抗原的蛋白活性较好。This TP recombinant antigen modifies the TPN15-TPN17-TPN47 chimeric expression polypeptide by introducing a flexible link peptide which contributes to soluble expression, and the TP recombinant antigen has a better protein activity than the conventional TP antigen.
这种TP重组抗原可以应用于制备梅毒检测试剂领域或制备梅毒检测设备领域。The TP recombinant antigen can be used in the field of preparing syphilis detection reagents or preparing syphilis detection equipment.
如图1所示的上述的TP重组抗原的制备方法，包括如下步骤：The method for preparing the above TP recombinant antigen as shown in FIG. 1 comprises the following steps:
S10、提供基因表达载体。S10. Providing a gene expression vector.
基因表达载体用于表达TP重组抗原，TP重组抗原包括嵌合表达多肽以及有助于可溶性表达的柔性链接肽，嵌合表达多肽为TPN15-TPN17-TPN47嵌合表达多肽。The gene expression vector is for expressing a TP recombinant antigen, and the TP recombinant antigen comprises a chimeric expression polypeptide and a flexible link peptide which contributes to soluble expression, and the chimeric expression polypeptide is a TPN15-TPN17-TPN47 chimeric expression polypeptide.
优选的，这种TP重组抗原作为标记抗原时还包括GST标签，GST标签、嵌合表达多肽以及柔性链接肽依次连接。Preferably, the TP recombinant antigen further comprises a GST tag as a labeled antigen, and the GST tag, the chimeric expression polypeptide and the flexible link peptide are sequentially linked.
具体的，TPN15-TPN17-TPN47嵌合表达抗原为：(a)、由SEQ ID No.1所示的核苷酸序列组成的多核苷酸编码得到的多肽；(b)、与SEQ ID No.1所示的核苷酸序列组成的多核苷酸具有至少98％同源性的多核苷酸编码得到的多肽；或(c)、由SEQ ID No.1所示的核苷酸序列组成的多核苷酸，其中一个或多个碱基被缺失、替代或增加得到的多核苷酸编码得到的多肽。 Specifically, the TPN15-TPN17-TPN47 chimeric expression antigen is: (a) a polypeptide encoded by a polynucleotide consisting of the nucleotide sequence shown in SEQ ID No. 1, (b), and SEQ ID No. a polynucleotide comprising a nucleotide sequence of 1 having a polynucleotide encoding at least 98% homology; or (c) a multinuclear comprising the nucleotide sequence of SEQ ID No. 1. A glycoside acid in which one or more bases are deleted, substituted or increased by the resulting polynucleotide encoding the resulting polypeptide.
具体的，柔性链接肽为：(a)、由SEQ ID No.2所示的核苷酸序列组成的多核苷酸编码得到的多肽；(b)、与SEQ ID No.2所示的核苷酸序列组成的多核苷酸具有至少98％同源性的多核苷酸编码得到的多肽；或(c)、由SEQ ID No.2所示的核苷酸序列组成的多核苷酸，其中一个或多个碱基被缺失、替代或增加得到的多核苷酸编码得到的多肽。Specifically, the flexible link peptide is: (a) a polypeptide encoded by a polynucleotide consisting of the nucleotide sequence shown in SEQ ID No. 2; (b) and a nucleoside represented by SEQ ID No. 2. a polynucleotide having a nucleic acid sequence consisting of a polynucleotide having at least 98% homology; or (c) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID No. 2, wherein one or A polypeptide obtained by deleting, substituting or increasing the obtained polynucleotide by a plurality of bases.
一般的，基因表达载体的构建过程可以为：选取一段辅助蛋白的基因片段，设计引物，引物上带有酶切位点，PCR扩增辅助蛋白基因片段，连接到用相应酶切处理后的表达载体中，得到重组质粒。选取一段梅毒检测抗原的基因片段，设计引物，引物上带有酶切位点，PCR扩增梅毒检测抗原基因片段，连入到用相应酶切好的重组质粒中，得到可表达融合蛋白的基因表达载体。In general, the gene expression vector can be constructed by selecting a gene fragment of a helper protein, designing a primer, enzymatic cleavage site on the primer, PCR amplification of the accessory protein gene fragment, and ligation to the expression after digestion with the corresponding enzyme. In the vector, a recombinant plasmid is obtained. A gene fragment of syphilis detection antigen was selected, primers were designed, and the primers were ligated with restriction enzyme sites. The genomic detection antigen gene fragment was amplified by PCR and ligated into the recombinant plasmid with the corresponding enzyme to obtain the gene expressing the fusion protein. Expression vector.
基因表达载体可以选择双顺反子表达的质粒，例如，pET-24a(Novagen公司，货号：69864-3)、pET-30a，等。其中，柔性链接肽的表达序列作为第二顺反子。The gene expression vector can select a plasmid for bicistronic expression, for example, pET-24a (Novagen, Cat. No. 69864-3), pET-30a, and the like. Wherein, the expression sequence of the flexible linker peptide serves as a second cistron.
S20、将S10得到的基因表达载体转化到宿主细胞中。S20, transforming the gene expression vector obtained by S10 into a host cell.
宿主细胞可以为原核细胞，例如：大肠杆菌。The host cell can be a prokaryotic cell, such as E. coli.
一般的，基因表达载体转化操作参见试剂盒生产厂家推荐的方法实现，宿主细胞为感受态细胞，例如大肠杆菌感受态细胞，将构建好的基因表达载体加入感受态细胞，热激处理，使感受态细胞的细胞膜结构扰动，细胞膜上出现空隙以便基因表达载体进入细胞，之后恒温培养，使宿主细胞复苏。In general, the gene expression vector transformation operation is carried out by the method recommended by the kit manufacturer. The host cell is a competent cell, such as an E. coli competent cell, and the constructed gene expression vector is added to the competent cell, and the heat shock treatment is performed to make the feeling The cell membrane structure of the cells is disturbed, and a gap appears on the cell membrane to allow the gene expression vector to enter the cell, and then cultured at a constant temperature to resuscitate the host cell.
一般的，基因表达载体转化到宿主细胞后，还需要对转化后的宿主细胞进行抗性筛选，如在培养基中加入氨苄西林钠或卡那霉素。In general, after transformation of the gene expression vector into the host cell, it is also necessary to perform resistance screening on the transformed host cell, such as adding ampicillin sodium or kanamycin to the culture medium.
S30、对S20得到的转化了基因表达载体的宿主细胞进行诱导表达，分离得到表达液。S30, inducing expression of the host cell transformed with the gene expression vector obtained by S20, and isolating the expression liquid.
S40、对S30得到的表达液中进行饱和硫酸铵梯度分析，确定沉杂蛋白和沉目的蛋白的硫酸铵浓度，加入相应的沉杂的硫酸铵终浓度，充分静止后收集上清，接着在上清中加入饱和硫酸铵沉目的蛋白的终浓度，充分静止后收 集沉淀，将沉淀重新溶解，得到粗产物。S40, performing a gradient analysis of saturated ammonium sulfate in the expression liquid obtained in S30, determining the ammonium sulfate concentration of the precipitated protein and the sinking protein, adding the corresponding final concentration of ammonium sulfate, collecting the supernatant after being completely still, and then on the upper layer. Adding the final concentration of saturated ammonium sulfate to the target protein in the middle of the Qing Dynasty The precipitate was collected and the precipitate was redissolved to give a crude product.
S40中还包括向表达液中加入吐温20或Triton X-100的操作。吐温20或Triton X-100的质量百分浓度为0.1％～0.3％。优选的，吐温20或Triton X-100的质量百分浓度为0.15％。Also included in S40 is the addition of Tween 20 or Triton X-100 to the expression solution. The mass concentration of Tween 20 or Triton X-100 is 0.1% to 0.3%. Preferably, the Tween 20 or Triton X-100 has a mass percent concentration of 0.15%.
原核表达的TP重组抗原中往往含有较多的聚体结构，在纯化过程中加入一定量的吐温20或Triton X-100较好的解决了该问题。Prokaryotic expression of TP recombinant antigen often contains more polymer structure, and a certain amount of Tween 20 or Triton X-100 is better solved in the purification process.
S50、利用亲和柱纯化S40得到的粗产物，再进行亲和柱及离子交换纯化，得到TP重组抗原。S50, the crude product obtained by purifying S40 is purified by an affinity column, and then subjected to affinity column and ion exchange purification to obtain a TP recombinant antigen.
一实施方式的梅毒检测试剂，含有上述的TP重组抗原的溶液。A syphilis detection reagent according to one embodiment contains a solution of the above TP recombinant antigen.
TP重组抗原的溶液中含有吐温20或Triton X-100。吐温20或Triton X-100的质量百分浓度为0.1％～0.3％。优选的，吐温20或Triton X-100的质量百分浓度为0.15％。The solution of the TP recombinant antigen contains Tween 20 or Triton X-100. The mass concentration of Tween 20 or Triton X-100 is 0.1% to 0.3%. Preferably, the Tween 20 or Triton X-100 has a mass percent concentration of 0.15%.
原核表达的TP重组抗原中往往含有较多的聚体结构，在使用过程中加入一定量的吐温20或Triton X-100较好的解决了该问题。Prokaryotic expression of TP recombinant antigen often contains more polymer structure, and the addition of a certain amount of Tween 20 or Triton X-100 during use is better to solve the problem.
一实施方式的梅毒检测试纸，包括包被抗原、标记抗原、GST单克隆抗体及标记物。The syphilis test strip of one embodiment includes a coating antigen, a labeled antigen, a GST monoclonal antibody, and a label.
标记抗原包括依次连接的GST标签、嵌合表达多肽以及有助于可溶性表达的柔性链接肽，嵌合表达多肽为TPN15-TPN17-TPN47嵌合表达多肽。The labeled antigen comprises a GST tag, a chimeric expression polypeptide, and a flexible linker peptide that facilitates soluble expression, and the chimeric expression polypeptide is a TPN15-TPN17-TPN47 chimeric expression polypeptide.
包被抗原包括依次连接的上述嵌合表达多肽以及上述柔性链接肽；The coating antigen includes the above chimeric expression polypeptide linked in sequence and the above flexible link peptide;
标记物通过抗GST单克隆抗体与标记抗原间接结合。The label binds indirectly to the labeled antigen by an anti-GST monoclonal antibody.
嵌合表达多肽以及柔性链接肽的序列前文已经给出，在此不在赘述。The sequences of chimeric expression polypeptides as well as flexible link peptides have been previously provided and will not be described herein.
优选的，标记物可以为胶体金、胶体硒、胶体银或乳胶等较大的纳米颗粒。Preferably, the label may be larger nanoparticles such as colloidal gold, colloidal selenium, colloidal silver or latex.
一实施方式的梅毒检测试剂盒，包括上述的梅毒检测试剂，或者包括上述的梅毒检测试纸。The syphilis detection kit of one embodiment includes the syphilis detection reagent described above or the syphilis test strip described above.
具体的，如图2、图3和图4所示的一实施方式的梅毒检测试剂盒，包括梅毒检测试纸100、壳体200以及其他检测试剂。 Specifically, the syphilis detection kit of one embodiment shown in FIGS. 2, 3, and 4 includes a syphilis test strip 100, a housing 200, and other detection reagents.
梅毒检测试纸100包括支撑薄片110、样品垫120、金标垫130、硝酸纤维素膜140、吸收垫150、检测线160及质控线170。样品垫120、金标垫130、硝酸纤维素膜140及吸收垫150从支撑薄片110的一端向另一端依次设置在支撑薄片110上。样品垫120与金标垫130部分重叠，金标垫130与硝酸纤维素膜140部分重叠，硝酸纤维素膜140与吸收垫150部分重叠。检测线160及质控线170设在硝酸纤维素膜上，且检测线160设在靠近金标垫130的一端，质控线170设在靠近吸收垫150的一端。支撑薄片110采用不吸水的材料制作。样品垫120用于样品点样。抗GST单克隆单体包附胶体金颗粒并与标记抗原偶联后均匀涂覆在金标垫130上。包被抗原包被在硝酸纤维素膜140上。The syphilis test strip 100 includes a support sheet 110, a sample pad 120, a gold standard pad 130, a nitrocellulose membrane 140, an absorbent pad 150, a test line 160, and a quality control line 170. The sample pad 120, the gold standard pad 130, the nitrocellulose membrane 140, and the absorption pad 150 are sequentially disposed on the support sheet 110 from one end to the other end of the support sheet 110. The sample pad 120 partially overlaps the gold standard pad 130, the gold standard pad 130 partially overlaps the nitrocellulose membrane 140, and the nitrocellulose membrane 140 partially overlaps the absorption pad 150. The detection line 160 and the quality control line 170 are disposed on the nitrocellulose membrane, and the detection line 160 is disposed at one end of the gold standard pad 130, and the quality control line 170 is disposed at one end of the absorption pad 150. The support sheet 110 is made of a material that does not absorb water. Sample pad 120 is used for sample spotting. The anti-GST monoclonal monomer is coated with colloidal gold particles and coupled to the labeled antigen and uniformly coated on the gold pad 130. The coating antigen is coated on the nitrocellulose membrane 140.
标记抗原包括依次连接的GST标签、嵌合表达多肽以及有助于可溶性表达的柔性链接肽，嵌合表达多肽为TPN15-TPN17-TPN47嵌合表达多肽。包被抗原包括依次连接的上述嵌合表达多肽以及上述柔性链接肽。检测线160为亲和纯化的抗梅毒抗原，质控线170为羊抗鼠IgG抗体。The labeled antigen comprises a GST tag, a chimeric expression polypeptide, and a flexible linker peptide that facilitates soluble expression, and the chimeric expression polypeptide is a TPN15-TPN17-TPN47 chimeric expression polypeptide. The coating antigen includes the above chimeric expression polypeptides ligated in sequence as well as the above flexible link peptide. Detection line 160 is an affinity purified anti-syphilis antigen and quality control line 170 is a goat anti-mouse IgG antibody.
本实施方式中，金标垫130上的标记物为胶体金，可以理解，在其他实施方式中，标记物还可以为胶体硒、胶体银或乳胶等较大的纳米颗粒。本实施方式中，所用到的抗梅毒抗原为***抗原。In the present embodiment, the mark on the gold standard pad 130 is colloidal gold. It can be understood that in other embodiments, the mark may also be larger nanoparticles such as colloidal selenium, colloidal silver or latex. In the present embodiment, the anti-syphilis antigen used is a Treponema pallidum antigen.
结合图4，检测试纸100可置于检测试剂盒的壳体200内。壳体200上开设有加样孔210及观察窗220。加样孔210对应样品垫120的位置。检测线160及质控线170裸露于观察窗220中，方便观察。In conjunction with Figure 4, test strip 100 can be placed within housing 200 of the test kit. A sample insertion hole 210 and a observation window 220 are opened in the housing 200. The sample well 210 corresponds to the position of the sample pad 120. The detection line 160 and the quality control line 170 are exposed in the observation window 220 for convenient observation.
其他检测试剂可以根据需要直接在实验室制备。Other detection reagents can be prepared directly in the laboratory as needed.
上述检测试剂盒利用双抗原夹心法来检测被检材料中的是否含有***抗体。检测时，样品中所有的***抗体先和胶体金间接标记的标记抗原结合，由于毛细管作用，反应复合物沿包被膜向前泳动，若样品中有***抗体，到达检测线160时，遇到包被在硝酸纤维素膜140上的包被抗原，就会形成包被抗原-待测***抗体-标记抗原-标记物复合物，从而富集在检测线160上，形成红色沉淀线；未结合***抗体的金标 记梅毒抗原则通过检测线160，被羊抗鼠单抗捕获，富集在质控线170上，形成红色沉淀线。当检测线160与质控线170上同时有红色沉淀线时判为阳性结果。若样品中不含有***抗体，反应复合物到达检测线160时，遇到包被抗原就不会形成包被抗原-待测***抗体-标记抗原-标记物复合物，反应复合物通过检测线160，仅富集在质控线170上形成红色沉淀线，此时判为阴性结果。The above detection kit detects the presence or absence of a Treponema pallidum antibody in the test material by a double antigen sandwich method. At the time of detection, all the Treponema pallidum antibodies in the sample are first bound to the labeled antigen indirectly labeled by the colloidal gold, and the reaction complex migrates forward along the coating membrane due to capillary action. If the sample contains treponema pallidum antibody and reaches the detection line 160, Upon encountering the coated antigen coated on the nitrocellulose membrane 140, a coated antigen-tested Treponema pallidum antibody-labeled antigen-marker complex is formed, thereby enriching on the detection line 160 to form a red precipitation line. Gold standard that does not bind to Treponema pallidum antibodies The syphilis antigen is captured by the goat anti-mouse monoclonal antibody through the detection line 160, and is enriched on the quality control line 170 to form a red precipitation line. A positive result was obtained when the red line was simultaneously present on the test line 160 and the quality control line 170. If the sample does not contain the Treponema pallidum antibody, when the reaction complex reaches the detection line 160, the coated antigen will not form the coated antigen-tested Treponema pallidum antibody-labeled antigen-label complex, and the reaction complex passes the detection. Line 160, enriched only on the quality control line 170 to form a red precipitate line, at which point a negative result was judged.
下面为具体实施例部分。The following is part of the specific embodiment.
以下实施例中，如无特别说明，未注明具体条件的实验方法，通常按照常规条件，例如参见萨姆布鲁克、EF弗里奇、T曼尼阿蒂斯等(金冬雁，黎孟枫等译)所著的的分子克隆实验指南[M](北京：科学出版社，1992)中所述的条件或者试剂盒生产厂家推荐的方法实现。所有操作均采用本领域标准操作过程，所采用的试剂或载体等均为常规试剂或常规载体。In the following examples, unless otherwise specified, the experimental methods without specific conditions are usually in accordance with conventional conditions, for example, see Sambrook, EF Frych, T Manny Artis, etc. (Jin Dongyan, Li Mengfeng, etc.) The conditions described in the molecular cloning experimental guide [M] (Beijing: Science Press, 1992) or the method recommended by the manufacturer of the kit are implemented. All procedures are carried out in accordance with standard procedures in the art, and the reagents or carriers used are conventional reagents or conventional carriers.
实施例1Example 1
包被、标记抗原、免疫源和杂交瘤细胞筛选抗原的制备。Preparation of coating antigens by coating, labeling antigens, immunogen sources, and hybridoma cells.
通过文献查阅及生物信息学分析***TpN17(GeneBank No：M74825)、TpN15(GeneBank No：U73115.1)、TpN47(GeneBank No：NC_000919.1)基因优势表位，设计重叠PCR引物人工合成TpN17(23aa-106aa)、TpN15(31aa-109aa)、TpN47(185aa-410aa)所对应活性区段，通过桥式PCR扩增并引入有助于可溶性表达的柔性链接肽TRX，获得嵌合基因，将该基因和pMD18-T载体连接，转化大肠杆菌，挑取单克隆，PCR鉴定正确的阳性质粒，提取质粒酶切鉴定。将PCR鉴定和酶切鉴定都正确的质粒送测序，结果和设计序列完全一致。将测序正确的质粒用设计在其两端的酶切位点相应的酶进行酶切，切出的目的序列连入表达载体pET-24a中。包被抗原的重组质粒为pET-24a-TPAG-TRX(含HIS tag)，该质粒为双顺反子表达的质粒，其中TRX为第二顺反子；标记抗原的重组质粒为 PET30a-GST-TPAG-TRX(含HIS tag及GST peptide)，该质粒为双顺反子表达的质粒，其中TRX为第二顺反子。Through literature review and bioinformatics analysis of TpN17 (GeneBank No: M74825), TpN15 (GeneBank No: U73115.1), TpN47 (GeneBank No: NC_000919.1) gene dominant epitopes, design overlapping PCR primers to synthesize TpN17 ( The active segments corresponding to 23aa-106aa), TpN15 (31aa-109aa), TpN47 (185aa-410aa) were amplified by bridge PCR and introduced into the flexible link peptide TRX, which facilitates soluble expression, to obtain a chimeric gene. The gene was ligated with pMD18-T vector, transformed into E. coli, picked for monoclonal, and the correct positive plasmid was identified by PCR, and the plasmid was extracted and identified. The plasmids identified by PCR identification and restriction enzyme digestion were sequenced, and the results were identical to the designed sequences. The correctly sequenced plasmid was digested with the corresponding enzyme designed at both ends of the restriction enzyme, and the excised target sequence was ligated into the expression vector pET-24a. The recombinant plasmid carrying the antigen is pET-24a-TPAG-TRX (including HIS tag), the plasmid is a bicistronic expression plasmid, wherein TRX is a second cistron; the recombinant plasmid labeled with the antigen is PET30a-GST-TPAG-TRX (containing HIS tag and GST peptide), the plasmid is a bicistronic expression plasmid, wherein TRX is a second cistron.
TPAG为TPN15-TPN17-TPN47嵌合表达抗原的表达序列，序列如SEQ ID No.1所示；TRX为嵌合表达多肽的表达序列，序列如SEQ ID No.2所示。TPAG is the expression sequence of the TPN15-TPN17-TPN47 chimeric expression antigen, and the sequence is shown in SEQ ID No. 1; TRX is the expression sequence of the chimeric expression polypeptide, and the sequence is shown in SEQ ID No. 2.
免疫源和杂交瘤细胞筛选抗原使用PGEX-2T、PGEX-6P-1、PGEX-5X-1、pET-41a质粒转化表达宿主菌ER2566。Immunogen and hybridoma cell selection antigens PGEX-2T, PGEX-6P-1, PGEX-5X-1, and pET-41a plasmids were used to transform the expression host ER2566.
取适量转化后宿主菌涂布于带抗性的LB固体培养平板上，37℃，培养过夜，第二天挑取7株在抗性培养平板上能生长的菌落，接种于3mL带抗性的LB液体培养基中，37℃培养5h，同时取转化有空载体的宿主菌做对照。加入终浓度为0.25mM的IPTG，30℃，180rpm诱导培养6h。离心收集菌体，用40μL 20mM PBS缓冲液重悬菌体，加入20μL 3×loadingBuffer，沸水煮10分钟后，SDS-PAGE电泳。考马斯亮兰染色，转化进重组质粒的菌株在预测的分子量位置有一条明显的蛋白表达带，而只转化进空质粒的菌株没有该条带。The appropriate amount of transformed host bacteria was coated on resistant LB solid culture plates, and cultured overnight at 37 ° C. On the next day, 7 colonies capable of growing on resistant culture plates were picked and inoculated into 3 mL resistant strains. The LB liquid medium was cultured at 37 ° C for 5 hours, and the host strain transformed with the empty vector was taken as a control. IPTG was added to a final concentration of 0.25 mM, and culture was induced for 6 h at 30 ° C and 180 rpm. The cells were collected by centrifugation, and the cells were resuspended in 40 μL of 20 mM PBS buffer, and 20 μL of 3×loading Buffer was added thereto, boiled in boiling water for 10 minutes, and then subjected to SDS-PAGE electrophoresis. Coomassie brilliant blue staining, the strain transformed into the recombinant plasmid had a distinct protein expression band at the predicted molecular weight position, whereas the strain transformed only into the empty plasmid did not have the band.
将1微升的转化宿主菌接种于500mL加有抗性的LB培养基中，37℃，200rpm培养过夜，第二天早上加入IPTG至终浓度为0.25mM，30℃，180rpm诱导4h。离心收集菌体，加入适量裂解缓冲液，超声破碎后离心取上清。分别进行硫酸铵梯度分析，经分析包被抗原用10％饱和硫酸铵进行沉杂蛋白，用20％硫酸铵沉目的蛋白；标记抗原用35％直接沉淀目的蛋白；免疫源和杂交瘤细胞筛选抗原用15％饱和硫酸铵进行沉杂蛋白，用30％硫酸铵沉目的蛋白。经硫酸铵处理后得到粗抗原。包被抗原经NI亲和介质纯化和SP离子交换层析获得目的蛋白并保证平衡液和洗脱缓冲液中含有0.15％吐温20或TritonX-100；标记抗原经NI亲和介质纯化和谷胱甘肽亲和介质纯化获得目的蛋白；两种抗原最终纯度均达到90％以上(见图5，包被抗原和标记抗原SDS-PAGE胶纯度电泳图：泳道1蛋白Marker；泳道2纯化后包被抗原；泳道3纯化后标记抗原)，-20℃保存备用。免疫源和杂交瘤细胞筛选抗原经谷胱甘肽亲和介质纯化获得目的蛋白，-20℃保存备用。 One microliter of the transformed host strain was inoculated into 500 mL of resistant LB medium, cultured overnight at 37 ° C, 200 rpm, and IPTG was added the next morning to a final concentration of 0.25 mM, 30 ° C, and induction at 180 rpm for 4 h. The cells were collected by centrifugation, and an appropriate amount of lysis buffer was added thereto. After ultrasonication, the supernatant was centrifuged. The ammonium sulfate gradient analysis was carried out separately, and the coated antigen was analyzed by using 10% saturated ammonium sulfate for the protein, and the protein was precipitated with 20% ammonium sulfate; the labeled antigen was directly precipitated with the target protein by 35%; the immunogen and the hybridoma cells were screened for the antigen. The protein was precipitated with 15% saturated ammonium sulfate and the protein was precipitated with 30% ammonium sulfate. The crude antigen was obtained after treatment with ammonium sulfate. The coated antigen was purified by NI affinity medium and SP ion exchange chromatography to obtain the target protein and ensured that 0.15% Tween 20 or TritonX-100 was contained in the balance solution and elution buffer; the labeled antigen was purified by NI affinity medium and glutathione The glycopeptide affinity medium was purified to obtain the target protein; the final purity of both antigens was over 90% (see Figure 5, coated antigen and labeled antigen SDS-PAGE gel purity electrophoresis map: lane 1 protein Marker; lane 2 purified and coated Antigen; labeled with antigen after washing in lane 3), stored at -20 ° C until use. The immunogen and hybridoma cell screening antigens were purified by glutathione affinity medium to obtain the target protein, and stored at -20 ° C until use.
实施例2Example 2
抗GST单克隆抗体的制备。Preparation of anti-GST monoclonal antibodies.
将以上获得的重组GST蛋白溶液用PBS透析之后，用PBS稀释到1.0mg/mL，与弗氏完全佐剂等体积混合，并充分乳化，得到油状乳液。将该乳液以0.2mL的剂量皮下施给6周龄雌性BALB/c小鼠背部位点。第一次免疫14天后腹腔增强免疫，即等量抗原与弗氏不完全佐剂等体积混合，增强免疫到四针后，采尾血，分离血清，用间接ELISA法测定效价，效价高于1∶10000即可用于融合。融合前3天，用相同剂量抗原与等体积0.9％氯化钠注射液混合腹腔注射追加免疫，免疫方法同上。The recombinant GST protein solution obtained above was dialyzed against PBS, diluted to 1.0 mg/mL with PBS, mixed with an equal volume of Freund's complete adjuvant, and fully emulsified to obtain an oily emulsion. The emulsion was administered subcutaneously to the back site of 6-week-old female BALB/c mice at a dose of 0.2 mL. After 14 days of the first immunization, the abdominal cavity was boosted, that is, the same amount of antigen was mixed with the Freund's incomplete adjuvant, and the immunization was increased to four needles. The tail blood was collected, the serum was separated, and the titer was determined by indirect ELISA. It can be used for fusion at 1:10000. Three days before the fusion, the same dose of antigen was mixed with an equal volume of 0.9% sodium chloride injection for intraperitoneal injection of booster immunization. The immunization method was the same as above.
以BALB/c鼠腹腔巨噬细胞作饲养细胞。在融合前1天，BALB/c鼠拉颈处死，75％酒精全身浸泡，超净台内，无菌操作下用剪刀剪开腹部皮肤，暴露腹膜，用注射器腹腔注入RPMI 1640基础培养液5mL，反复冲洗，回收冲洗液，1000rpm，离心5分钟，留沉淀，用RPMI 1640筛选培养液(含HAT的RPMI 1640完全培养液中)重悬，调整细胞浓度1×105个/mL，加入96孔板，150μL/孔，37℃，5％CO2培养过夜。BALB/c mouse peritoneal macrophages were used as feeder cells. One day before the fusion, the BALB/c mice were sacrificed by neck-stretching, 75% alcohol was immersed in the whole body, and the abdominal skin was cut with scissors under aseptic operation. The peritoneum was exposed, and 5 mL of RPMI 1640 basic culture solution was injected into the abdominal cavity with a syringe. Rinse repeatedly, recover the rinse solution, centrifuge at 1000 rpm for 5 minutes, leave a precipitate, resuspend with RPMI 1640 screening medium (in RPMI 1640 complete medium containing HAT), adjust the cell concentration to 1 × 10 5 /mL, and add 96 wells. Plates, 150 μL/well, cultured overnight at 37 ° C, 5% CO 2 .
小鼠末次免疫后三天，在无菌条件下取出脾脏，置于平皿中，RPMI 1640基础培养液冲洗一次，放于小烧杯的尼龙网上磨碎过滤，制成细胞悬液。离心，弃上清，RPMI 1640基础培养液重悬，如此重复三次，计数。Three days after the last immunization of the mice, the spleens were taken out under aseptic conditions, placed in a dish, rinsed once with RPMI 1640 base medium, and placed in a nylon beaker on a small beaker to be filtered to prepare a cell suspension. After centrifugation, the supernatant was discarded, and the RPMI 1640 base medium was resuspended, and this was repeated three times and counted.
小鼠骨髓瘤细胞Sp2/0经8-氮鸟嘌呤筛选后，培养至对数生长期，取两大瓶制成细胞悬液，离心，弃上清，用RPMI 1640基础培养液重悬，如些重复三次，计数。Mouse myeloma cells Sp2/0 were screened by 8-azaguanine and cultured to logarithmic growth phase. Two large bottles were prepared to make cell suspension, centrifuged, and the supernatant was discarded and resuspended in RPMI 1640 base medium. Repeat three times and count.
将骨髓瘤细胞与免疫脾细胞按1:10比例混合，在50mL塑胶离心管内用RPMI 1640基础培养液洗1次，1，200rpm，离心8分钟。弃上清，将细胞混匀，缓慢加入1mL 50％的PEG1500融合，融合1分钟后加入15mL的RPMI1640基础培养液终止细胞融合。1，000rpm，离心5分钟。弃上清，用50mL的RPMI 1640筛选培养液轻轻混悬，平分于10块96孔板(已铺饲养细胞)， 50μL/孔，37℃，5％CO2培养。培养至第六天，换HT培养液(含HT的RPMI1640完全培养液)两次。The myeloma cells and the immune spleen cells were mixed at a ratio of 1:10, and washed once with a RPMI 1640 base culture solution in a 50 mL plastic centrifuge tube, and centrifuged at 1,200 rpm for 8 minutes. The supernatant was discarded, the cells were mixed, 1 mL of 50% PEG1500 fusion was slowly added, and after 1 minute of fusion, 15 mL of RPMI1640 basal medium was added to terminate the cell fusion. Centrifuge for 5 minutes at 1,000 rpm. The supernatant was discarded and gently incubated with 50 mL of RPMI 1640 screening medium and aliquoted into 10 96-well plates (fed cells). Incubate at 50 μL/well, 37 ° C, 5% CO 2 . The culture was continued until the sixth day, and the HT medium (HTMI-containing RPMI1640 complete medium) was changed twice.
用0.06M pH9.6碳酸缓冲溶液稀释不同的GST重组蛋白(分别是：PGEX-2T、PGEX-6P-1、PGEX-5X-1、pET-41a、PET30a-GST-TPAG-TRX),同时pET-24a-TPAG作为阴性对照，使其终浓度为1μg/mL。每孔0.1mL加入96孔聚苯乙烯板，37℃2小时或4℃过夜。次日，用含10％小牛血清或1％脱脂奶粉的0.02M pH7.2PBS，0.15mL/孔，37℃封闭2小时，用于检测。重组融合后第七天，取细胞上清0.1mL于上述96孔检测板中，37℃30分钟，水洗六次后加入2000倍稀释的辣根过氧化酶标记的羊抗鼠IgG，37℃30分钟同上洗后，加TMB显色剂显色15分钟，加入稀硫酸溶液，每孔50μL，测450nm吸收值。PRMI 1640完全培养液作为阴性对照，以测定值与对照值得比≧2.0为阳性细胞孔。共检测有杂交瘤细胞的378孔，其中同时对上述五个含GST的蛋白有反应的阳性孔为77个。经过三次有限稀释克隆，最后获得12株稳定分泌抗GST蛋白的细胞株。Different GST recombinant proteins were diluted with 0.06 M pH 9.6 carbonate buffer solution (PGEX-2T, PGEX-6P-1, PGEX-5X-1, pET-41a, PET30a-GST-TPAG-TRX, respectively), while pET -24a-TPAG was used as a negative control to a final concentration of 1 μg/mL. 0.1 mL per well was added to a 96-well polystyrene plate at 37 ° C for 2 hours or 4 ° C overnight. The next day, it was blocked with 0.02 M pH 7.2 PBS containing 10% calf serum or 1% skim milk powder at 0.15 mL/well for 2 hours at 37 ° C for detection. On the seventh day after recombinant fusion, 0.1 mL of the cell supernatant was taken in the above 96-well assay plate, and washed at 37 ° C for 30 minutes, washed six times, and then added with 2000-fold diluted horseradish peroxidase-labeled goat anti-mouse IgG, 37 ° C After washing with the same amount of time, add TMB coloring agent for 15 minutes, add dilute sulfuric acid solution, 50 μL per well, and measure the absorption value at 450 nm. The PRMI 1640 complete medium was used as a negative control, and the measured value and the control value were compared with ≧2.0 as positive cell wells. A total of 378 wells of hybridoma cells were detected, of which 77 were positive for the above five GST-containing proteins. After three limiting dilution cloning, 12 cell lines stably secreting anti-GST protein were obtained.
选6-8周健壮的BALB/c小鼠，每只小鼠腹腔注射0.5mL的降植烷；10天后腹腔注射1×106个杂交瘤细胞。接种细胞7～10天后可产生腹水，密切观察动物的健康状况与腹水征象，待腹水尽可能多，而小鼠频于死亡之前，处死小鼠，用滴管将腹水吸入试管中，一般一只小鼠可获5～10mL腹水。收集腹水，离心取上清，放于-20℃冰箱保存。 Six to eight weeks of robust BALB/c mice were selected, and each mouse was intraperitoneally injected with 0.5 mL of pristane; after 10 days, 1 x 10 6 hybridoma cells were intraperitoneally injected. After inoculation of cells for 7 to 10 days, ascites can be produced, and the animal's health and ascites signs should be closely observed. As much as possible of ascites, while the mice are killed before death, the mice are sacrificed and the ascites is inhaled into the test tube with a dropper. The mice can obtain 5-10 mL of ascites. Ascites was collected, centrifuged, and stored in a refrigerator at -20 °C.
取腹水上清，用3倍体积的PBS稀释后滤纸过滤。将所得的滤液在1mL/min的流速下加到一个已用PBS平衡的蛋白G亲和层析柱。然后用PBS以1mL/min的流速洗涤未被蛋白G吸附的物质直至在OD280nm下的吸附值达到基线为止。再用0.2M的甘氨酸洗脱液(pH2.5)洗脱并回收该抗体。所回收的溶液用0.1M TRIS(pH8.8)中和，通过超滤将抗体浓度调节到一个合适的浓度，-20℃冻存。Clear the abdomen water, dilute with 3 volumes of PBS and filter the filter paper. The resulting filtrate was applied to a Protein G affinity chromatography column equilibrated with PBS at a flow rate of 1 mL/min. The material not adsorbed by protein G was then washed with PBS at a flow rate of 1 mL/min until the adsorption value at OD280 nm reached the baseline. The antibody was eluted and recovered with a 0.2 M glycine eluate (pH 2.5). The recovered solution was neutralized with 0.1 M TRIS (pH 8.8), and the antibody concentration was adjusted to a suitable concentration by ultrafiltration, and frozen at -20 °C.
实施例3 Example 3
GST单克隆抗体的胶体金标记工艺、TP间接标记检测***的摸索。The colloidal gold labeling process of GST monoclonal antibody and the exploration of TP indirect labeling detection system.
取1mL胶体金放小玻璃杯中，搅拌加入10μL、15μL、20μL的0.2M K2CO3调节pH至7.0，继续搅拌60秒；分别加入10μg、15μg不同株系GST单抗，继续搅拌60秒；加入20μL 10％BSA，继续搅拌60秒；5000g离心10分钟，弃掉上清，用多次尝试摸索的胶体金稀释液100u l(20mM PB，150mM NaCl，1％BSA，0.1％TritonX-100，2％Sucrose，0.01％Proclin300)复溶；最后在该100微升胶体金标记GST单抗复合物中加入0.15μg、0.25μg、0.3μg PET30a-GST-TPAG-TRX，充分混匀后4℃保存。该间接标记金标复合物命名为PET30a-GST-TPAG-TRX-GSTAb-AU。将上述金标复合物用胶体金稀释液10倍稀释后浸泡玻璃纤维，冻干，即制成金标垫。我们通过尝试发现1mL胶体金加15μL的0.2M K2CO3标记10μg、GSTAb258单抗时，产品灵敏度、特异性最高。Take a small glass discharge colloidal gold 1mL stirred added 10μL, 15μL, 20μL of 0.2M K 2 CO 3 pH was adjusted to 7.0 and stirring continued for 60 seconds; were added to 10μg, 15μg GST mAb different strains, stirring was continued for 60 seconds; Add 20 μL of 10% BSA, continue to stir for 60 seconds; centrifuge at 5000 g for 10 minutes, discard the supernatant, and use a colloidal gold dilution 100 μl (20 mM PB, 150 mM NaCl, 1% BSA, 0.1% Triton X-100, tried several times). 2% Sucrose, 0.01% Proclin 300) reconstituted; finally, 0.15 μg, 0.25 μg, 0.3 μg of PET30a-GST-TPAG-TRX was added to the 100 μl colloidal gold-labeled GST monoclonal antibody complex, fully mixed and stored at 4 ° C. . The indirect labeled gold standard complex was named PET30a-GST-TPAG-TRX-GSTAb-AU. The gold standard compound was diluted 10 times with a colloidal gold dilution solution, and the glass fiber was immersed and lyophilized to prepare a gold standard pad. When we tried to find 1mL colloidal gold plus 15μL of 0.2M K2CO3 labeled 10μg, GSTAb258 monoclonal antibody, the product has the highest sensitivity and specificity.
用检测线稀释液(10mM PBS+2％蔗糖+0.15％吐温20或Triton)稀释pET-24a-TPAG-TRX至0.8mg/mL制成检测线工作液，调整点膜仪，将其划到硝酸纤维素膜的相应位置上即为检测线(T线)，T线靠近金标垫端，距金标垫端约5mm；用同一稀释液稀释羊抗鼠单抗到0.5mg/mL制成对照线工作液，调整点膜仪，划线为C线，即为控制线，C线靠近吸收垫，距吸收垫约3mm。两线距离5～8mm，37℃烘干，封装备用。Dilute pET-24a-TPAG-TRX to 0.8 mg/mL with test line dilution (10 mM PBS + 2% sucrose + 0.15% Tween 20 or Triton) to prepare the test line working solution, adjust the spotting instrument, and mark it to The corresponding position of the nitrocellulose membrane is the detection line (T line), the T line is close to the gold standard pad end, about 5 mm from the gold standard pad end; diluted with the same dilution liquid to prepare the goat anti-mouse monoclonal antibody to 0.5 mg/mL Control the line working fluid, adjust the spotting instrument, and scribe the line C, which is the control line. The C line is close to the absorption pad and is about 3 mm away from the absorption pad. The distance between the two lines is 5~8mm, and it is dried at 37°C, and the package is ready for use.
将包被好的硝酸纤维素膜、金标垫、吸收垫、样品垫、聚酯板按图依次层叠起来，切成3mm宽的小条。每十小条一包，加入干燥剂，真空封装。4～30℃保存。The coated nitrocellulose membrane, the gold standard pad, the absorbent pad, the sample pad, and the polyester plate were sequentially laminated as shown in the figure, and cut into strips of 3 mm width. For every 10 strips, add a desiccant and vacuum package. Store at 4 to 30 °C.
加100μL待测样品到样品垫处，室温放置20分钟之后判定结果，当试纸条出现肉眼可见的***质控线，没有出现肉眼可见的***检测线，结果判为阴性；当试纸条同时出现肉眼可见的***质控线和***检测线，结果判为阳性；检测线颜色越深说明被检测样品的抗体水平越高；当试纸条没有出现肉眼可见的***质控线，不管有没有出现肉眼可见的***检测线，结果都判为检测试纸条失效，应废弃。 Add 100 μL of the sample to be tested to the sample pad, and leave it at room temperature for 20 minutes to judge the result. When the test strip has a purple-red color control line visible to the naked eye, there is no visible red-red detection line, and the result is negative; when the test paper At the same time, the purple-red quality control line and the purple-red detection line are visible to the naked eye, and the result is positive; the darker the detection line, the higher the antibody level of the sample to be tested; when the test strip does not have the purple-red quality control visible to the naked eye The line, whether or not there is a visible red-red detection line, is judged to be a test strip failure and should be discarded.
实施例4Example 4
TP间接标记的胶体金试剂盒的应用。Application of TP indirect labeled colloidal gold kit.
使用上述实施例3的间接标记双抗原夹心法金标检测***用于检测TP抗体，其灵敏度、特异性、重复性、稳定性、精密性等指标均优于现有商品化试剂盒。The indirect labeled double antigen sandwich method gold label detection system of the above Example 3 was used for detecting TP antibodies, and the sensitivity, specificity, repeatability, stability, precision and the like were superior to the existing commercial kits.
(1)灵敏度和特异性：250例血清标本经***核酸检测为阳性样本，2000例检出阴性样本，分别采用目前市售的梅毒检测试剂盒A与实施例4试剂盒B进行检测，20分钟内观察结果，结果见表1。(1) Sensitivity and specificity: 250 serum samples were positive samples detected by Treponema pallidum nucleic acid, and negative samples were detected in 2000 samples, respectively, using the currently commercially available syphilis detection kit A and the kit 4 of Example 4 for detection, 20 The results were observed in minutes and the results are shown in Table 1.
表1 快速诊断梅毒试剂盒的比较Table 1 Comparison of rapid diagnostic syphilis kits
Figure PCTCN2015098666-appb-000001
Figure PCTCN2015098666-appb-000001
由表1可以看出，试剂盒A的灵敏度为98.4％，试剂盒B灵敏度为100％，特异性试剂盒A为99.75％，试剂盒B为99.9％。说明本实施例的试剂盒在灵敏度和特异性两项指标上均优于现有的产品，完全可以用于常规的梅毒快速诊断。As can be seen from Table 1, the sensitivity of the kit A was 98.4%, the sensitivity of the kit B was 100%, the specific kit A was 99.75%, and the kit B was 99.9%. It is indicated that the kit of the present embodiment is superior to the existing products in both sensitivity and specificity, and can be completely used for rapid diagnosis of conventional syphilis.
(3)重复性：对阴、阳性待测样品各20份，采用不同时不同批次不同操作者进行10次检测，考查试剂盒的重复性，结果显示本实施例的金标检测试剂盒对阴、阳性判定结果的重复性为100％，表明试剂盒的再现性好，重复性高。(3) Repeatability: 20 samples of negative and positive samples to be tested, 10 tests were performed by different operators at different times and different batches, and the repeatability of the kit was examined. The results showed that the gold standard detection kit of this example The repeatability of the negative and positive determination results was 100%, indicating that the kit has good reproducibility and high reproducibility.
(4)稳定性：将本实施例间接标记制成的成品试剂盒分别37℃考核7天，取出后与同时4℃存放的成品试剂盒在同一条件下检测相同的20份阴性参考品和10份阳性参考品，计算其符合率，其结果见表2。 (4) Stability: The finished kits indirectly labeled in this example were examined at 37 ° C for 7 days, and after removal, the same 20 negative reference products and 10 were tested under the same conditions as the finished kit stored at 4 ° C simultaneously. For a positive reference, calculate the coincidence rate. The results are shown in Table 2.
表2 TP间接标记试剂盒稳定性实验结果Table 2 TP indirect labeling kit stability experiment results
Figure PCTCN2015098666-appb-000002
Figure PCTCN2015098666-appb-000002
由表2可以看出，本实施例的成品试剂盒稳定性好。As can be seen from Table 2, the finished kit of this example has good stability.
(5)精密性：在用本实施例的金标检测试剂盒检测同一份已知阳性标本，多次平行做10次重复检测，得到各检测条的结果均为阳性，且各检测条的显色程度也无显著差异，说明试剂盒精密性较好。(5) Precision: The same known positive specimen was detected by the gold label detection kit of the present embodiment, and 10 repeated tests were performed in parallel for multiple times, and the results of each test strip were all positive, and the test strips were displayed. There was no significant difference in the degree of color, indicating that the kit has good precision.
以上所述实施例仅表达了本发明的一种或几种实施方式，其描述较为具体和详细，但并不能因此而理解为对本发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。 The above-mentioned embodiments are merely illustrative of one or more embodiments of the present invention, and the description thereof is not to be construed as limiting the scope of the invention. It should be noted that a number of variations and modifications may be made by those skilled in the art without departing from the spirit and scope of the invention. Therefore, the scope of the invention should be determined by the appended claims.

Claims (15)

  1. 一种TP重组抗原，其特征在于，包括依次连接的嵌合表达多肽以及有助于可溶性表达的柔性链接肽，所述嵌合表达多肽为TPN15-TPN17-TPN47嵌合表达多肽。A TP recombinant antigen comprising a chimeric expression polypeptide ligated in sequence and a flexible linker peptide which facilitates soluble expression, the chimeric expression polypeptide being a TPN15-TPN17-TPN47 chimeric expression polypeptide.
  2. 根据权利要求1所述的TP重组抗原，其特征在于，所述TP重组抗原做为标记抗原时还包括GST标签，所述GST标签、所述嵌合表达多肽以及所述柔性链接肽依次连接。The TP recombinant antigen according to claim 1, wherein the TP recombinant antigen further comprises a GST tag as a labeled antigen, and the GST tag, the chimeric expression polypeptide, and the flexible linker peptide are sequentially linked.
  3. 根据权利要求1所述的TP重组抗原，其特征在于，所述TPN15-TPN17-TPN47嵌合表达抗原为(a)、由SEQ ID No.1所示的核苷酸序列组成的多核苷酸编码得到的多肽；(b)、与SEQ ID No.1所示的核苷酸序列组成的多核苷酸具有至少98％同源性的多核苷酸编码得到的多肽；或(c)、由SEQ ID No.1所示的核苷酸序列组成的多核苷酸，其中一个或多个碱基被缺失、替代或增加得到的多核苷酸编码得到的多肽。The TP recombinant antigen according to claim 1, wherein the TPN15-TPN17-TPN47 chimeric expression antigen is (a) a polynucleotide encoding a nucleotide sequence represented by SEQ ID No. 1. The obtained polypeptide; (b) a polypeptide encoded by a polynucleotide having at least 98% homology with a polynucleotide consisting of the nucleotide sequence shown in SEQ ID No. 1, or (c) by SEQ ID A polynucleotide consisting of the nucleotide sequence shown in No. 1, wherein one or more bases are deleted, replaced or increased by a polypeptide obtained by encoding the obtained polynucleotide.
  4. 根据权利要求1所述的TP重组抗原，其特征在于，所述柔性链接肽为(a)、由SEQ ID No.2所示的核苷酸序列组成的多核苷酸编码得到的多肽；(b)、与SEQ ID No.2所示的核苷酸序列组成的多核苷酸具有至少98％同源性的多核苷酸编码得到的多肽；或(c)、由SEQ ID No.2所示的核苷酸序列组成的多核苷酸，其中一个或多个碱基被缺失、替代或增加得到的多核苷酸编码得到的多肽。The TP recombinant antigen according to claim 1, wherein the flexible link peptide is (a) a polypeptide encoded by a polynucleotide consisting of the nucleotide sequence shown by SEQ ID No. 2; a polypeptide encoded by a polynucleotide having at least 98% homology with a polynucleotide consisting of the nucleotide sequence shown in SEQ ID No. 2; or (c), represented by SEQ ID No. 2. A polynucleotide consisting of a nucleotide sequence in which one or more bases are deleted, replaced or increased by a polypeptide encoded by the resulting polynucleotide.
  5. 一种TP重组抗原的制备方法，其特征在于，包括如下步骤：A method for preparing a TP recombinant antigen, comprising the steps of:
    步骤一、提供基因表达载体，所述基因表达载体用于表达TP重组抗原，所述TP重组抗原包括嵌合表达多肽以及有助于可溶性表达的柔性链接肽，所述嵌合表达多肽为TPN15-TPN17-TPN47嵌合表达多肽；Step 1. Providing a gene expression vector for expressing a TP recombinant antigen, the TP recombinant antigen comprising a chimeric expression polypeptide and a flexible link peptide capable of soluble expression, the chimeric expression polypeptide is TPN15- TPN17-TPN47 chimeric expression polypeptide;
    步骤二、将所述基因表达载体转化到宿主细胞中，所述宿主细胞为原核细胞；Step two, transforming the gene expression vector into a host cell, wherein the host cell is a prokaryotic cell;
    步骤三、对转化了所述基因表达载体的所述宿主细胞进行诱导表达，分离得到表达液； Step 3: inducing expression of the host cell transformed with the gene expression vector, and separating the expression liquid;
    步骤四、对所述表达液中进行饱和硫酸铵梯度分析，确定沉杂蛋白和沉目的蛋白的硫酸铵浓度，加入相应的沉杂的硫酸铵终浓度，充分静止后收集上清，接着在所述上清中加入饱和硫酸铵沉目的蛋白的终浓度，充分静止后收集沉淀，将所述沉淀重新溶解，得到粗产物；以及Step 4: performing a gradient analysis of saturated ammonium sulfate in the expression liquid, determining the ammonium sulfate concentration of the precipitated protein and the sinking protein, adding the corresponding final concentration of the ammonium sulfate, and collecting the supernatant after being completely still, followed by Adding the final concentration of the saturated ammonium sulfate sinking protein to the supernatant, collecting the precipitate after standing still, and re-dissolving the precipitate to obtain a crude product;
    步骤五、利用亲和柱纯化所述粗产物，再进行亲和柱及离子交换纯化，得到所述TP重组抗原。Step 5: purifying the crude product by using an affinity column, and then performing affinity column and ion exchange purification to obtain the TP recombinant antigen.
  6. 根据权利要求5所述的TP重组抗原的制备方法，其特征在于，所述TP重组抗原还包括GST标签，所述GST标签、所述嵌合表达多肽以及所述柔性链接肽依次连接。The method for producing a TP recombinant antigen according to claim 5, wherein the TP recombinant antigen further comprises a GST tag, and the GST tag, the chimeric expression polypeptide, and the flexible linker peptide are sequentially linked.
  7. 根据权利要求5所述的TP重组抗原的制备方法，其特征在于，所述TPN15-TPN17-TPN47嵌合表达抗原为(a)、由SEQ ID No.1所示的核苷酸序列组成的多核苷酸编码得到的多肽；(b)、与SEQ ID No.1所示的核苷酸序列组成的多核苷酸具有至少98％同源性的多核苷酸编码得到的多肽；或(c)、由SEQ ID No.1所示的核苷酸序列组成的多核苷酸，其中一个或多个碱基被缺失、替代或增加得到的多核苷酸编码得到的多肽。The method for producing a TP recombinant antigen according to claim 5, wherein the TPN15-TPN17-TPN47 chimeric expression antigen is (a) a multinuclear composed of the nucleotide sequence shown by SEQ ID No. 1. a polypeptide encoded by a nucleotide; (b) a polypeptide encoded by a polynucleotide having at least 98% homology with a polynucleotide consisting of the nucleotide sequence shown in SEQ ID No. 1; or (c), A polynucleotide consisting of the nucleotide sequence shown in SEQ ID No. 1, wherein one or more bases are deleted, replaced or increased by the resulting polynucleotide encoding the resulting polypeptide.
  8. 根据权利要求5所述的TP重组抗原的制备方法，其特征在于，所述柔性链接肽为(a)、由SEQ ID No.2所示的核苷酸序列组成的多核苷酸编码得到的多肽；(b)、与SEQ ID No.2所示的核苷酸序列组成的多核苷酸具有至少98％同源性的多核苷酸编码得到的多肽；或(c)、由SEQ ID No.2所示的核苷酸序列组成的多核苷酸，其中一个或多个碱基被缺失、替代或增加得到的多核苷酸编码得到的多肽。The method for producing a TP recombinant antigen according to claim 5, wherein the flexible link peptide is (a) a polypeptide encoded by a polynucleotide consisting of the nucleotide sequence shown by SEQ ID No. 2. (b) a polypeptide encoded by a polynucleotide having at least 98% homology with a polynucleotide consisting of the nucleotide sequence shown in SEQ ID No. 2; or (c) by SEQ ID No. 2 A polynucleotide comprising a nucleotide sequence as shown, wherein one or more bases are deleted, replaced or increased by a polypeptide encoded by the resulting polynucleotide.
  9. 一种梅毒检测试剂，其特征在于，所述梅毒检测试剂含有如权利要求1、3或4所述的TP重组抗原的溶液；A syphilis detecting reagent, characterized in that the syphilis detecting reagent contains a solution of the TP recombinant antigen according to claim 1, 3 or 4;
    所述TP重组抗原的溶液中含有质量百分浓度为0.1％～0.3％的吐温20或质量百分浓度为0.1％～0.3％的Triton X-100。The solution of the TP recombinant antigen contains Tween 20 in a mass percentage concentration of 0.1% to 0.3% or Triton X-100 in a mass percentage concentration of 0.1% to 0.3%.
  10. 根据权利要求9所述的梅毒检测试剂，其特征在于，所述TP重组抗原的溶液中含有质量百分浓度为0.15％的吐温20或质量百分浓度为0.15％的 Triton X-100。The syphilis detecting reagent according to claim 9, wherein the solution of the TP recombinant antigen contains 0.1% by mass of Tween 20 or 0.15% by mass. Triton X-100.
  11. 一种梅毒检测试纸，其特征在于，包括包被抗原、标记抗原、GST单克隆抗体及标记物；A syphilis test strip comprising a coating antigen, a labeled antigen, a GST monoclonal antibody and a label;
    所述标记抗原包括依次连接的GST标签、嵌合表达多肽以及有助于可溶性表达的柔性链接肽，所述嵌合表达多肽为TPN15-TPN17-TPN47嵌合表达多肽；The labeled antigen comprises a GST tag, a chimeric expression polypeptide, and a flexible link peptide which facilitates soluble expression, which is a TPN15-TPN17-TPN47 chimeric expression polypeptide;
    所述包被抗原包括依次连接的所述嵌合表达多肽以及所述柔性链接肽；The coating antigen includes the chimeric expression polypeptide and the flexible link peptide;
    所述标记物通过所述抗GST单克隆抗体与所述标记抗原间接结合。The label is indirectly bound to the labeled antigen by the anti-GST monoclonal antibody.
  12. 根据权利要求11所述的梅毒检测试纸，其特征在于，所述TPN15-TPN17-TPN47嵌合表达抗原为(a)、由SEQ ID No.1所示的核苷酸序列组成的多核苷酸编码得到的多肽；(b)、与SEQ ID No.1所示的核苷酸序列组成的多核苷酸具有至少98％同源性的多核苷酸编码得到的多肽；或(c)、由SEQ ID No.1所示的核苷酸序列组成的多核苷酸，其中一个或多个碱基被缺失、替代或增加得到的多核苷酸编码得到的多肽。The syphilis test strip according to claim 11, wherein the TPN15-TPN17-TPN47 chimeric expression antigen is (a) a polynucleotide encoding a nucleotide sequence represented by SEQ ID No. 1. The obtained polypeptide; (b) a polypeptide encoded by a polynucleotide having at least 98% homology with a polynucleotide consisting of the nucleotide sequence shown in SEQ ID No. 1, or (c) by SEQ ID A polynucleotide consisting of the nucleotide sequence shown in No. 1, wherein one or more bases are deleted, replaced or increased by a polypeptide obtained by encoding the obtained polynucleotide.
  13. 根据权利要求11所述的梅毒检测试纸，其特征在于，所述柔性链接肽为(a)、由SEQ ID No.2所示的核苷酸序列组成的多核苷酸编码得到的多肽；(b)、与SEQ ID No.2所示的核苷酸序列组成的多核苷酸具有至少98％同源性的多核苷酸编码得到的多肽；或(c)、由SEQ ID No.2所示的核苷酸序列组成的多核苷酸，其中一个或多个碱基被缺失、替代或增加得到的多核苷酸编码得到的多肽。The syphilis test strip according to claim 11, wherein the flexible link peptide is (a) a polypeptide encoded by a polynucleotide consisting of the nucleotide sequence shown by SEQ ID No. 2; a polypeptide encoded by a polynucleotide having at least 98% homology with a polynucleotide consisting of the nucleotide sequence shown in SEQ ID No. 2; or (c), represented by SEQ ID No. 2. A polynucleotide consisting of a nucleotide sequence in which one or more bases are deleted, replaced or increased by a polypeptide encoded by the resulting polynucleotide.
  14. 一种梅毒检测试剂盒，其特征在于，包括权利要求9或10所述的梅毒检测试剂。A syphilis detection kit comprising the syphilis detection reagent according to claim 9 or 10.
  15. 一种梅毒检测试剂盒，其特征在于，包括如权利要求11～13中任一项所述的梅毒检测试纸。 A syphilis test kit comprising the syphilis test strip according to any one of claims 11 to 13.
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CN115975023A (en) * 2022-12-16 2023-04-18 北京科跃中楷生物技术有限公司 Preparation method of recombinant TP antigen and antibody detection reagent prepared by preparation method

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CN110305927A (en) * 2019-08-07 2019-10-08 郑州伊美诺生物技术有限公司 Utilize the method for recombination bacillus coli fermentation preparation coating TP antigen
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