WO2000047613A1

WO2000047613A1 - Modified treponema pallidum outer membrane protein, its immunoassay use and immunoassay kit

Info

Publication number: WO2000047613A1
Application number: PCT/CN1999/000110
Authority: WO
Inventors: Jian Wang; Feng Mu; Zhijie Li; Cunji Gao
Original assignee: Jian Wang; Jin Hongjun; Cunji Gao; Zhijie Li; Zhang Yuanming; Feng Mu
Priority date: 1999-02-08
Filing date: 1999-08-06
Publication date: 2000-08-17
Also published as: AU5147199A

Abstract

The present invention provides proteins of a general formula: (Xaa)nTpp(Xaa)m, wherein Tpp is selected from the outer membrane proteins of Treponema pallidum Tpp15, Tpp17 and Tpp47, and any immunoactive fragments and derivatives thereof that can bind to human immunoglobulins; Xaa represents a natural amino acid residue, wherein at least one of Xaas which are nearest to the two ends of Tpp and at least one terminal Xaa of the formula are selected from the group consisting of Lys, Arg and Trp; n and m represent independently an integer from 1 to 10, providing n+m≥2. A novel method that assays anti-Treponema pallidum antibodies using the proteins, particularly ba the double-antigen-sandwich immunoassay is provided. An immunoassay kit for detecting human anti-Treponema pallidum antibodies by the method of present is also provided.

Description

Modified syphilis spiral outer membrane protein, its immunoassay should be

FIELD OF THE INVENTION

The present invention relates to some modified outer membrane proteins of Treponema pallidum, an immunological method for detecting antibodies to Treponema pallidum using these proteins, and a kit containing them. Background of the invention

Syphilis is a disease caused by Treponema pallidum (hereinafter referred to as Tp). With the development of protein purification technology, DNA recombination technology and monoclonal antibody technology, favorable conditions have been provided for the development of highly specific and sensitive detection technology for Treponema pallidum. US4514498 and 4740467 have prepared ELISA kits for serological detection of syphilis, but their sensitivity and specificity have not been satisfactory. Immunogenic syphilis spiral outer membrane proteins Tppl5, Tρρ17, Tρρ47 are known. BK Purcell et al. Molecular Microbiology (1990) 4 (8), 1371-1379; RA Darrin et al. Infection and Immunity 1993 (4), 1202-1210 and LM Weigel et al. 1992 (4), 1568-1576, basically clarified the structure and composition of the outer membrane proteins Tppl5, Τρρ17, Τρρ47 of the syphilis spiral outer membrane, and given their amino acid sequences and nucleotide sequences encoding them. Japanese Patent Application Publication Hei-2-500403 describes a method for preparing a 47 kDa Treponema pallidum antigen and a method for immunodetection of anti- Treponema pallidum antibodies using this antigen. US5578456 prepared a fusion protein of the syphilis spiral outer membrane proteins Tppl5, Τρρ17, Τρρ47 and GST S transferase based on the DNA recombination technology, and proposed to use a syphilis spiral outer membrane protein Tppl5-and / or Tppl7-GST S transferase The fusion protein is used as an antigen to detect human Treponema pallidum antibody, and it is said that the sensitivity of serological detection of syphilis is improved compared with the method using the 47 kDa antigen. However, most of the fusion proteins other than Treponema pallidum antigens account for the majority, which will definitely increase the non-specific immune binding activity of the antigen and reduce the Low detection specificity for Treponema pallidum antibodies. Therefore, there is still a need for an immunological method for detecting Treponema pallidum antibodies with higher sensitivity and specificity.

The present invention is based on the surprising discovery that the syphilis spiral outer membrane protein antigen

Tppl5, Τρρ17, Τρρ47 are modified without affecting their immune binding specificity, which significantly improves the sensitivity of immunodetection of human Treponema pallidum antibodies; in addition, it can retain the binding specificity of natural antigens. Get higher detection specificity. Summary of invention

One aspect of the present invention relates to a protein having the general formula (Xaa) _n Tpp (Xaa) _m , wherein Tpp is selected from the syphilis outer membrane protein Tppl5, Τρρ17, Τρρ47 or a fragment thereof having an immunological activity that binds human immunoglobulin or Derivatives; Xaa represents a natural amino acid residue, wherein at least one Xaa closest to each side of Tpp and at least one terminal protein of the general formula Xaa is an amino acid residue selected from Lys, Arg and Trp; n and m are 0 10 Integer, provided that n + m≥2.

Another aspect of the present invention relates to a method for immunodetection of human Treponema pallidum antibodies using the protein of the general formula, and in particular to a method for quantitative detection of human Treponema pallidum antibodies by the double-antigen sandwich method. ELISA method for quantitative detection of human Treponema pallidum antibodies.

Another aspect of the present invention relates to a kit for detecting Treponema pallidum antibody by the above-mentioned immunological method. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 Amino acid sequence and nucleotide sequence of the modified outer membrane protein KTppl5 of syphilis spiralis.

Figure 2 Amino sequence and nucleotide sequence of the modified syphilis spiral outer membrane protein shield KTPpρΠ. FIG. 3 Amino sequence and nucleotide sequence of the outer membrane protein KTpp47 of the modified syphilis spiralis.

Fig. 4 is a schematic diagram showing the construction of a recombinant plasmid containing a nucleotide sequence encoding a modified syphilis spiral outer membrane protein ktρρ15.

Fig. 5 is a schematic diagram showing the construction of a recombinant plasmid containing a nucleotide sequence encoding a modified syphilis spiral outer membrane protein ktpρ17.

Fig. 6 is a schematic diagram showing the construction of a recombinant plasmid containing a nucleotide sequence encoding a modified syphilis spiral outer membrane protein ktρρ47.

Figure 7 SDS-PAGE results of purified syphilis outer membrane protein KTPpρ after purification, Coomassie blue staining. Wherein: Lane 1 is the low molecular protein standard; Lane 2 is the bacterial lysate containing KKTρρ15 before purification; Lane 3 is the lysate of bacterial strain containing KTPpρ15 before purification; ΚΤρρ17; lane 8 is the lysate of bacterial cells containing KTPp47 before purification; lane 9 is the purified KTPp47.

FIG. 8 uses Western blotting to detect the activity of the purified syphilis spiral outer membrane protein KTpp of the present invention after binding to Treponema pallidum antibody. Among them: the right side is the molecular weight standard of amino black staining; 1 is κρρρ15 that reacts with human Treponema pallidum positive serum; 2 is κΤρρΠ that reacts with human Treponema pallidum positive serum; 3 is κΤρρ47 that reacts with human Treponema pylori positive serum.

Figure 9 Comparison of HRP-labeled recombinant Treponema pallidum antigen and unmodified Treponema pallidum antigen labeling efficiency. The upper picture shows the spectrum of HRP labeled Τρρ17 after passing through the Superdex75 gel chromatography column, and the lower picture shows the spectrum of HRP labeled KTppl7 after passing through the Superdex75 gel chromatography column.

FIG. 10 uses the double antigen sandwich method to respectively take the modified recombinant syphilis spiral outer membrane protein KTpp and the unmodified recombinant syphilis spiral outer membrane protein Tpp of the present invention as coating antigens, and compare the enzyme-labeled modified recombinant syphilis spiral outer membrane protein HRP-KTpp and Targeted unmodified recombinant protein HRP-Tpp as enzyme-labeled antigen for detection of human Treponema pallidum antibody fruit. The comparison of the enzyme-labeled recombinant syphilis outer membrane protein HRP-KTppl5 with the enzyme-labeled unmodified recombinant protein HRP-Tppl5; the enzyme-labeled recombinant syphilis outer membrane protein> HRP-KTppl7 and the enzyme-labeled unmodified Comparison of the recombinant protein HRP-Tppl7; The following is a comparison of the enzyme-labeled recombinant syphilis outer membrane protein HRP-KTpp47 and the enzyme-labeled unmodified recombinant protein HRP-Tpp47; Detailed description of the invention

A protein having the general formula (Xaa) nTpp (Xaa) _m , wherein Tpp is selected from the syphilis outer membrane protein; Tppl5, Tppl7, Tρρ47 or a fragment or derivative thereof having an immunological activity that binds human immunoglobulin; Xaa stands for Natural amino acid residues, wherein at least one Xaa closest to each side of Tpp and the terminal Xaa of at least one formula protein are amino acid residues selected from Lys, Arg, and Trp; n and m are integers from 0 to 10, The condition is that n + m≥2. The modified recombinant syphilis outer membrane protein of the present invention, hereinafter sometimes referred to as KTpp, refers to the N of the amino acid sequence on the known outer membrane proteins of syphilis helix Tρρ15, Τρρ17, Τρρ47 or its immunologically binding active fragment. The protein modified at the terminal and C-terminus is wide, thereby improving the immunological binding activity of the protein or fragment thereof to human Treponema pallidum antibody. The syphilis spiral outer membrane proteins Tρρ15, Tρρ17, and Tρρ47 according to the present invention include intact proteins that respectively contain their own secretion signal peptides, as well as proteins that do not contain mature forms of the secretion signal peptide sequence. In addition, the syphilis spiral outer membrane proteins Tρρ15, Tρρ17, and Tρρ47 according to the present invention may also each carry heterologous signal peptides for protein secretion and expression commonly used by those skilled in the art from various other sources.

In a preferred embodiment of the present invention, KTpp is MetLysLys-Tpp-LysLysLeuGluLys, and Tpp is the syphilis outer membrane protein Tppl5 or a fragment or derivative thereof having an immunological activity that binds human immunoglobulin.

In another preferred embodiment of the present invention, KTpp is MetLysLys-Tpp- LysLysLeuGluLys, and Tpp is the syphilis outer membrane protein Tppl7 or has Fragments or derivatives that bind to the immunological activity of human immunoglobulins.

In another preferred embodiment of the present invention, KTpp is MetLysLys-Tpp- LysLysLeuGluLys, and Tpp is the outer membrane protein of syphilis spiralis ρρρ47 or a fragment or derivative thereof having an immunological activity that binds human immunoglobulin. As used herein, the term "immune binding activity" refers to the ability of an antigenic protein to recognize and specifically bind to Treponema pallidum antibodies.

In order to obtain the modified recombinant syphilis spiral outer membrane protein ktpρ, PCR amplification technology and recombinant DNA technology known to those skilled in the art can be used. The corresponding PCR primers were designed, and at the same time, the corresponding PCR primers were added with bases encoding the corresponding number of amino acid residues, and the DNA fragments of the three proteins were amplified from the clinical strains of Treponema pallidum (Nichols). The obtained PCR product is introduced into a suitable expression vector by a method known to a person skilled in the art to construct a recombinant DNA construct. An appropriate host cell is transformed with the obtained DNA construct, the host cell thus transformed is cultured under conditions suitable for expression of the target protein, and the target protein is recovered and purified by a technique known in the art.

These expression vectors include chromosomal, non-chromosomal and synthetic DNA sequences, such as derivatives of SV40, bacterial plasmids, phage DNA, yeast plasmids, vectors derived from the combination of plasmid and phage DNA, viral DNA, baculovirus, vaccinia Virus, adenovirus, fowlpox virus and pseudorabies virus. However, as long as they can replicate and survive in the host, other vectors can be used.

Suitable DNA sequences can be inserted into the vector by a number of methods. Generally, the DNA sequence is inserted into a suitable restriction enzyme site using methods known in the art. These methods are believed to be within the knowledge of those skilled in the art.

The DNA sequence in the expression vector is effectively linked to a suitable expression control sequence (promoter) to guide mRNA synthesis. The following are representative of such promoters: the LTR or SV40 promoter, the Lac or trp promoter of E. coli, the PL promoter of phage, and those known to control gene expression in prokaryotic or eukaryotic cells or their viruses Other promoters. The expression vector also contains a ribosome binding site and a transcription terminator required for translation initiation. The vector may also have suitable sequences that enhance expression.

In addition, the expression vector preferably contains one or more selectable marker genes that can provide phenotypic characteristics to facilitate selection of transformed host cells. For example, eukaryotic cells can be cultured with dihydrofolate reductase or neomycin resistance. You can use tetracycline or ampicillin resistance.

Vectors containing the appropriate DNA sequences, suitable promoters or control sequences described above can be used to transform a suitable host and allow the host to express the protein.

Listed below are representatives of suitable hosts: bacterial cells, such as E. coli, Streptomyces, Salmonella typhimurium; fungal cells, such as yeast; insect cells, if fly S2 and Spodoptera frugiperda Sf; animal cells, such as CHO, COS (monkey Renal fibroblast cell line) or Bowes melanoma; adenovirus; plant cells, etc. From these teachings, the selection of a suitable host should be within the knowledge of those skilled in the art.

Methods for introducing constructs into host cells are: PEG-mediated DNA transformation, calcium phosphate transfection, DEAE-dextran-mediated transfection, electroporation, or gene gun methods.

Generally, a recombinant expression vector contains an origin of replication and a selection marker for transforming a host cell, such as an ampicillin resistance gene of E. coli and a TRP1 gene of Saccharomyces cerevisiae, and a promoter derived from a highly expressed gene, thereby mediating the downstream structure. Transcription of the sequence. These promoters can be derived from operons encoding glycolytic enzymes, such as glyceryl triphosphate glycerate kinase (PGK :), factor α, acid sulfonate, or heat shock proteins. The heterologous structural sequence is assembled in the appropriate orientation with translation initiation and termination sequences and other preferred sequences. Alternatively, the DNA sequence may encode a fusion protein that contains an N-terminal confirmation peptide and exhibits desired characteristics, such as stabilization and purification simplification of the expressed recombinant product.

Effective expression vectors for bacteria are constructed by inserting a structural DNA sequence encoding the protein of interest into an operable reading frame with a functional promoter, along with appropriate translation initiation and termination signals. The vector should contain one or more phenotypic selection markers and duplicates The origin of replication and the origin of replication ensure that the vector can be retained in the host, and more ideally, the vector can be amplified. Prokaryotic hosts suitable for transformation include E. coli, Bacillus subtilis, Salmonella typhimurium, and various bacteria of the genus Pseudomonas, Streptomyces, and Staphylococcus. Other hosts can also be selected.

After transforming the appropriate host strain and the host strain to the appropriate cell density, the selected promoter is induced by an appropriate method (such as temperature shift or chemical induction), and the cells are cultured for a period of time.

The cells are usually harvested by centrifugation, the cells are disrupted by physical or chemical methods, and the crude extract is retained for further purification. The microbial cells used to express the protein can be disrupted by any convenient method, including freeze-thaw cycles, ultrasound, mechanical disruption, or the use of cytolytic agents, all of which are well known to those skilled in the art.

Various mammalian cell culture systems can also be used to express recombinant proteins. Examples of mammalian expression systems are the COS-7 cell line of monkey kidney fibroblasts described by Gluzman (Cell 23: 175, 1981), and other cell lines capable of expressing compatible vectors, such as C127, 3T3, CHO, HeLa And BHK cell lines. Mammal expression vectors include origins of replication, appropriate promoters, enhancers and any necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, transcription termination sequences and 5, flanking non-transcription sequence. DNA sequences and polyadenylation sites derived from SV40 splicing sequences can be used to provide the required non-transcribed genetic elements.

The expressed can be recovered and purified from the recombinant cell culture. The methods of recovery and purification include ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphate cellulose chromatography, hydrophobic interaction chromatography, affinity Chromatography, hydroxyapatite chromatography and lectin chromatography. High performance liquid chromatography (HPLC) was used for the final purification step.

In a preferred embodiment of the present invention, the expression vector GB-syl plasmid is used to construct a recombinant DNA construct, which is transformed into E. coli ER2566, and the target protein expression is induced by IPTG.

In a preferred embodiment of the present invention, the cultured The transformed E. coli host was separated and purified using a metal nickel-Sepharose affinity chromatography column.

The present invention also includes proteins or peptides obtained by modifying the outer membrane protein fragment or derivative of Treponema pallidum with immunological activity that binds human immunoglobulin.

The modified recombinant syphilis outer membrane protein KTpp according to the present invention can also be obtained by chemically modifying τρρ15, Τρρ17, Τρρ47, or a fragment thereof of the outer membrane protein of syphilis spiralis.

It has been found that the modified protein of the present invention not only has higher labelability than an unmodified protein, but also has unexpectedly higher immunodetection activity.

"High ^{labelability π} means that using the same conventional chemical cross-linking method with modified proteins significantly improves the yield of the labeled complex. This can be confirmed in Example 3.

"High immunodetection activity" means that the use of modified proteins significantly improves the sensitivity and / or specificity of the immunodetection of Treponema pallidum antibodies compared with unmodified proteins. This was confirmed by the indirect ELISA method of Example 10 and the double-antigen sandwich method of Example 11.

Therefore, the present invention also relates to a method for immunodetection of human Treponema pallidum antibody using the modified recombinant syphilis spiral outer membrane protein shield KTpp of the present invention.

It is known that the epitopes of Treponema pallidum are mainly located on the outer membrane proteins Tpp47, Tρρ17 and Tρρ15. Using at least one of the modified recombinant syphilis spiral outer membrane proteins KTpp of the present invention as an antigen, an immune-binding complex is formed by an antigen-antibody immune binding reaction, thereby detecting human Treponema pallidum antibodies in a biological sample of an individual.

In one embodiment of the invention, this method includes the following steps:

1) coating a solid phase support with the purified at least one modified protein of the present invention;

2) adding the biological sample to be tested onto the coated solid support, and incubating for a period of time under conditions suitable for forming an antigen-antibody immune binding complex; 3) Moderate cleaning to remove any unbound Treponema pallidum antibodies;

4) adding a labeled secondary antibody against human antibodies, and incubating for a period of time under conditions suitable for forming an antigen-antibody immune complex;

5) Moderate washing to remove any unbound secondary antibodies;

6) Quantitatively detect the presence of antigen-antibody immune binding complex in the incubation mixture.

In the above scheme, the secondary antibody against human antibody may be labeled with a radioisotope (ie, RIA), or may be performed with horseradish peroxidase, alkaline phosphatase, P-galactosidase or acetylcholinesterase. Labeled (ie ELISA :).

The second antibody may be an anti-human-IgG or / and-M antibody. These secondary antibodies are commercially available, such as sheep anti-human-IgG or / and-IgM, goat anti-human-IgG or / and-IgM, mouse anti-human-IgG or / and-IgM, rat anti-human -IgG or / and-IgM, rabbit anti-human-IgG or / and-IgM, etc.

The use of different secondary antibodies can detect the presence of specific antibodies in the sample and the relative amounts of different antibodies. Because the relative amounts of G and IgM in the human body are different at different stages of syphilis infection, multiple tests with different secondary antibodies can help determine the course of the patient.

The biological samples described in this specification refer to blood, serum, plasma, urine samples, body fluids, saliva and other secretions or excretions, and tissue or cell extracts from subjects (including patients and negative controls).

In a preferred embodiment of the present invention, the so-called double antigen sandwich immunoassay (DAGS EIA) is used to detect human Treponema pallidum antibodies. Typically, the method includes the following steps:

1) Coating a solid support with a purified modified recombinant syphilis spiral outer membrane protein KTpp;

2) Pre-labeled modified recombinant syphilis spiral outer membrane protein KTpp corresponding to the type of antigen KTpp used for coating the solid support;

3) Add the biological sample of the individual to be tested and the protein labeled by step 2) to the coating Incubate for a period of time on a solid phase support under conditions suitable for the formation of antigen-antibody immune binding complexes;

4) Moderate cleaning to remove any unbound Treponema pallidum antibodies;

5) Quantitatively detect the presence of antigen-antibody immune complexes in the incubation mixture.

The "purified antigen" means that the purity of the outer membrane protein of the modified syphilis spiralis as an antigen is higher than 95%, and more preferably higher than 98%.

The "solid support" includes organic and inorganic polymers well known in the art, such as including but not limited to: dextran, natural or modified cellulose, polyethylene, polystyrene, polyacrylamide , Agarose, latex, inner walls of containers such as test tubes, titration plates, glass cups, etc.

The solid support can be coated using a variety of methods known in the art. For example: activation using bifunctional reagents, see US Patent No. 5,399,501.

Particularly in the embodiment employing the double-antigen sandwich method, the modified syphilis outer membrane protein coated with the solid phase support preferably includes more than one modified recombinant outer membrane protein ^. That is, two or three types selected from the group consisting of KTPp15, KTPp17, and T47p are used for solid-phase coating.

Correspondingly, according to the type of the modified outer membrane protein of the syphilis spiral coat coated with the solid support in step 1), the corresponding kind of modified outer membrane protein of the syphilis spiral coat is labeled by a technique commonly used in the art. It is preferred to use an enzyme that can be measured by colorimetry, spectrophotometry, or fluorescence, including but not limited to oxidoreductases, such as catalytic enzymes, peroxidase, horseradish peroxidase, glucose gadolinium, β -Galactosidase, alkaline phosphatase, acetylcholinesterase, etc. are labeled. A preferred embodiment of the present invention is to label the corresponding species of antigen with horseradish peroxidase. It is also possible to use a radioisotope-labeled modified syphilis outer membrane protein as an antigen.

The method of labeling the above hydrazone and the modified syphilis spiral outer membrane protein of the present invention is well known in the art, for example, by using a known method of peptide chemistry to label by forming a sulfonamide bond. Labeling with radioisotopes can also be performed using methods known in the art. The "conditions suitable for forming an antigen-antibody immune complex" are well known in the art and refer to incubating a mixture of an antigen and an antibody-containing sample under appropriate temperature and time conditions, for example, at about 0-50 degrees Celsius, preferably about 4 At -30 degrees Celsius, the labeling is about 30 minutes to about 48 hours, and preferably about 30 minutes to about 4 hours.

After incubation to form the antigen-antibody immune complex, the detection plate is washed several times with a suitable washing solution commonly used in the art to remove unbound labeled antigen. The cleaning solution used is generally a phosphate buffer solution having a pH of about 5-8, preferably about 7.4.

The formation of the antigen-antibody complex can be quantified using a variety of methods known in the art. Select the appropriate quantitative detection protocol based on the method used to label the modified syphilis spiral outer membrane protein. In a preferred embodiment of the present invention, horseradish peroxidase is used when labeling an antigen, so a substrate with a peroxide solution as liquid A and a benzidine substance as liquid B and a labeled horseradish The antigen-antibody immune complex of oxide tritium acts as an immunological complex. After color development, the absorbance is read at a wavelength of 450 nm (reference wavelength 630 nm) using a light source. Compare the readings with those of the negative control to determine the results of the test sample.

The method for detecting the human Treponema pallidum antibody in a biological sample of an individual by an immunological method using the modified recombinant syphilis outer membrane protein KTpp of the present invention as an antigen also includes other methods routinely used in the art to detect antibodies using specific antigens.

The present invention also relates to a detection kit for detecting human Treponema pallidum antibodies using these methods. A detection kit for detecting human Treponema pallidum antibodies according to the present invention includes: using a purified recombinant T. pallidum outer membrane protein KTpp of the present invention as an antigen-coated solid support; and instructions for using the kit.

In one embodiment, the kit of the invention further comprises a second antibody labeled as described above. The second antibody may be an anti-IgG antibody and / or an anti-M antibody.

When used in the double-antigen sandwich method, the kit further includes a labeled recombinant syphilis spiral outer membrane protein KTpp corresponding to the kind of protein KTpp coated with the solid support in step 1). In the above detection kit, the labeled syphilis outer membrane protein may be packaged in a suitable container in a liquid form, or may be placed in a suitable package in the form of a lyophilisate that is convenient for reconstitution. In the latter case, the detection kit also preferably contains a suitable buffer for reconstitution of the labeled outer membrane protein of the syphilis spiralis.

The above-mentioned detection kit may further include a corresponding range for providing a judgment detection result.

In the following examples, KTpp represents SEQ ID No. 1, SEQ ID No. 2, and SEQ ID

The protein of No. 3 is taken as an example to illustrate the present invention, but this is not meant to limit the scope of the present invention. Examples

Example 1 Amplification of the gene of the outer membrane protein KTpp of the helical toxin by PCR method Experimental materials and methods

The Treponema pallidum Nichols wipes and E. coli ER2566 strains were provided by the Chinese Academy of Sciences Microbiology. The GB-Syl plasmid is a gift from the Institute of Genetics of the Chinese Academy of Sciences. This plasmid contains a NdeL Xhol single-cleavage site, and a recombinant characteristic histidine tag is attached to one end of the Xhol 瑭 site. Aspartic acid sequence tag. Restriction enzymes Ndel, Xhol, and T ₄ DNA ligase were provided by the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences. TaqDNA polymerase, digestive protease, and enterokinase were provided by the Molecular Biology Laboratory of the Institute of Genetics, Chinese Academy of Sciences. IPTG is a product of PROMEGA. Preparation and purification of Treponema pallidum DNA

For the culture of Treponema pallidum Nichols strain and the preparation and purification of chromosomal DNA, refer to the methods mentioned in Zhampionce et al. Infection and Immunity 58, 1697-1704, 1990. Preparation and purification of plasmid DNA

E. coli plasmid DNA was prepared and purified according to the method provided by Sambrook in Molecular Cloning, Laboratory Manual, 2nd Edition, Cold Spring Harbour Laboratory Press, 1989.

PCR primer design PCR primers were designed based on nucleoside sequences encoding Tppl5, Τρρ17 and Τρρ47 proteins reported in known literature (Β Purcell et al., Molecular Microbiology (1990) 4 (8), 1371-1379; RA Darrin et al., Infections And Immunization 1993 (4), 1202-1210 and LM Weigel et al., Infection and Immunity 1992 (4), 1568-1576), in which the restriction sites Ndel and Xhol for introduction of expression vectors and lysine residues are added Codon.

The primer sequences used for PCR amplification are as follows:

KTppl5: SEQ ID NO: 4 and SEQ ID NO: 5

Upstream primer: 5 'GGG TCGT CATATG AAG AAG ATG GTG AAA AGA GGT 3,

Ndel Lys Lys

Downstream primer: 5 TTCG CTCGAG TTT TTT CCT GCT AAT AAT GGC 3 '

Xhol Lys Lys

Tppl5: SEQ ID NO: 6 and SEQ ID NO: 7

Upstream primer: 5, GGG TCGT CATATG ATG GTG AAA AGA GGT 3 '

Ndel

Downstream primer: 5, TTCG CTCGAG CCT GCT AAT AAT GGC 3,

Xhol

KTppl7: SEQ ID NO: 8 and SEQ ID NO: 9

Upstream primer: 5, GATTGAG CAI41G AAG AAG ATG AAA GGA TCT GTC3 '

Ndel Lys Lys

Downstream primers: 5, GTCAG CTCGAG TTT TTT TTT CTT TGT TTT TTT3,

Xhol Lys Lys

Tppl7: SEQ ID NO: 10 and SEQ ID NO: 11

Upstream primer: 5, GATTGAG CATATG ATG AAA GGA TCT GTC3 '

Ndel

Downstream primer: 5, GTCAG CTCGAG TTT CTT TGT TTT TTT 3 '

Xhol

KTpp47: SEQ ID NO: 12 and SEQ ID NO: 13

Upstream primers: 5, GTACTAG CATATG AAG AAG ATG TTC GAT GCA GTT 3,

Ndel Lys Lys

Downstream primers: 5, CTGT CTCGAG TTT TTT CTG GGC GAC TAC CTT3,

Xhol Lys Lys

Tpp47: SEQ ID NO 14 and SEQ ID NO: 15

Upstream primers: 5, GTACTAG CATATG ATG TTC GAT GCA GTT 3,

Ndel Downstream primers: 5, CTGT CTCGAG— CTG GGC GAC TAC CTT3,

PCR amplification of Xhol gene encoding antigen protein KTpp

Using the total DNA of Treponema pallidum Nichels strain obtained as above as a template, 50 pmmol of primers, 2.5 mmol / L dNTP and 1U Taq DNA polymerase were added, and water was added to the total reaction volume 100 μl, and the surface was covered with paraffin oil. The PCR-90AD PCR instrument produced by the Institute of Genetics of the Chinese Academy of Sciences was used to amplify according to the following conditions:

Denaturation at 95 ° C for 60 seconds

55 ° C annealing for 60 seconds (KTppl5), 58 X annealing for 60 seconds (ΚΤρρ17), 60 X annealing for 60 seconds (ΚΤρρ47)

Extend at 72 ° C for 90 seconds for a total of 30 cycles.

After the last cycle was completed, it was extended for another 10 minutes at 72 ° F. 41: Store the reaction mixture for later use. The reaction mixture was extracted with an equal volume of phenol / chloroform to remove the paraffin oil. Take 5μ1 of the reaction solution containing the PCR product and check the amplification by 1% agarose gel electrophoresis. Add 0.1 volume of 3M potassium acetate solution to the rest of the product. Precipitate absolute ethanol in-and collect the product by centrifugation.

The obtained PCR amplification product was checked by 1% agarose gel electrophoresis. The reaction products corresponding to the nucleotide sequences encoding KTpp, K15Tppl7, and Τρρ47 correspond to DNA bands of about 0.4 Kb, about 0.5 Kb, and about 1.1 Kb, respectively This indicates that the primer design is correct and the corresponding target gene fragment is obtained. Example 2 Construction and identification of recombinant plasmids

Cloning of PCR amplification products

The GB-Syl plasmid DNA of lg was completely digested with Ndel and Xhol enzymes at 37 *, extracted with chloroform, and precipitated with -20t of absolute ethanol overnight. After centrifugation, the pellet was dissolved in 13μ1 Η ₂ 0. The PCR product fully digested with Ndel and Xhol alcohol was ligated with the aforementioned pre-digested plasmid GB-Syl, transformed into E. coli ER2566, and coated with LB selection plate of 200 μιηη1 ampicillin. 37Ό overnight culture. See Figure 4, Figure 5 and Figure 6 for the construction process. For the methods and conditions of the ligation and transformation, see "Molecular CloningiA Laboratory Manual" P672-849 (Science Press, 1998) by J. Sambrecht et al.

Selection and identification of recombinant plasmids

Resistant colonies were picked from the transformation plates, plasmids were extracted and checked by electrophoresis, and recombinants were screened. Restriction endonucleases Ndel and Xhol digestion analysis were used to screen recombinants containing the target gene inserted correctly. Sequencing the obtained recombinant plasmid containing the inserted fragment confirmed that no mutation or error occurred in the target gene fragment during the PCR process.

Expression and purification of target protein

The selected recombinant strains were inoculated in an LB medium containing 20 _μg / ml ampicillin, cultured on a constant temperature shaker, and cultured at 37X until the OD value of the bacterial solution was about 0.6, and then induced by ImM IPTG, 30 ° C. The culture was continued for 2 hours. Then the fermentation culture was centrifuged to collect the bacterial cell pellets. The obtained bacterial cells were broken with ultrasonic waves and centrifuged again to collect the supernatant. The protein was subjected to affinity chromatography using a metal nickel-Sepharose (Pharmacia) affinity column. Isolation and purification (eluting conditions 0.02M pH 7.4 phosphate buffer solution (PBS), washing J degree lml / min). After fully washing to remove non-specifically bound proteins, the expression of metal nickel-Sepharose binding was separated and collected The protein was subjected to SDS-PAGE electrophoresis, purity identification and ELISA serological test. Purification method refer to the literature Arnild, FH (1991) Bio / Tedmology 9, 151-156 and Dekker, N. (1992) Nature 362, 852-855.

The target protein containing the histidine tag was collected, and 1U enterokinase was added to cleave the histidine. The target protein was further purified by DEAE-Sepharose (Pharmacia) and Superdex 75 (Pharmacia) columns. The specific operation process is as follows: Collect the target protein solution with 6 histidine tags cut on the M-Sepharose column, add 1U enterokinase in the proportion of 1L to the cleavage enzyme, 4X overnight, and then place the cleavage protein solution on Samples were buffered with Tris-HCl, pH 7.2, 0.01M The pre-equipped DEAE-Sepharose column was collected to collect the individual components. Identification of the target protein's immune activity by ELISA method: Dilute the collected components with 0.05M pH9.6 carbonate buffer (CB) 1:10 into a coating working solution. C overnight. Then add 1% bovine serum albumin (BSA) and 1% skimmed milk powder in 0.02M pH7.4 phosphate buffered saline (PBS), 4: blocked for 2 hours and discard. Add Treponema pallidum 10 microliters / well of positive patient serum, using 100 microliters of 1% BSA in PBS as the sample dilution, 37 "C, after 30 minutes, discard. Wash twice with 0.05M PBS washing solution (PBS-T) containing 0.5% TW-20, and add dilute standard antibody (20% goat serum (produced by Zhejiang Sanli Blood Products Co., Ltd.), 1% casein (Sigma ), 0.02M PBS pH 7.4)) Sheep anti-human IgG (manufactured by DAKO) diluted 1: 5000 100 μl / well, 37 ° C, 30 minutes, and then containing 0.5% Tween 20 (TW-20 ) Was washed twice with 0.02M pH7.4 PBS washing solution (PBS-T), and finally 50 μl of 0.06% urea peroxide (Sigma) 0.01M acetate buffer pH 4.5 was added to each well. Substrate A solution and 50 μ1 of enzyme-acting substrate 37 consisting of 0.06% 3,3,5,5,5, -tetramethylbenzidine (TMB) (Sigma), 0.01M citrate buffer pH 4.5 , Develop color for 10 minutes, and terminate by adding 1M H ₂ S0 _{4 at} last, and read the absorbance value (reference wavelength 630nm) at 450nm. If the absorbance value is high, it means that the collected fraction contains the target recombinant Treponema pallidum protein. After concentrating these high absorbance fractions at low temperature, they were purified through a Superdex75 column (using a 0.02M pH7.4 PBS equilibrium column), and a single component was collected, and further immunoassay was performed by ELISA method, the method is the same as above.

Identification of recombinant antigens

The genes encoding the modified syphilis spiral outer membrane protein KTpp of the present invention were expressed in E. coli, separated and purified by affinity chromatography, and then identified by SDS-PAGE electrophoresis. The modified treponema spiral outer membrane proteins κΤρρ15, ΚΤρρΠ and The molecular weights of KTpp ⁴⁷ are about 15KD, 17KD, and 47KD, respectively (see Figure 7); by Western blotting, it was confirmed that the resulting protein has the activity of binding to human Treponema pallidum antibodies (see Figure 8). Example 3 Enzymatic labeling of recombinant antigen protein KTpp

The KTPp protein purified and prepared by the above method was confirmed to have good immune activity by the indirect ELISA method known in the art. Horseradish peroxidase (HRP), NaI04, NaBH ₃ CN, ethanolamine, and methyl-α-D-mannoside were purchased from Sigma Company for enzyme labeling. Recombinant antigen protein KTpp was enzymatically labeled using a modified periodate method. Refer to Tijssien (Analytical Biochem 136,451-457,1984). The specific steps are as follows:

1) Activate HRP: Add lml 5mg / ml with ΙΟΟμΙ 0.1M NaI0 ₄ of the HRP, thoroughly mixed at room temperature, the reaction was placed on a shaker full, dark shock 150 rev / min for 20 minutes, to the ImM pH4.4 Fully dialyze the acetate buffer for 24 hours;

2) Cross-linking with Treponema pallidum antigen: add 1/4 volume of 0.2M pH 9.6 carbonate buffer (CB) to 1 ml of the activated HRP to adjust the pH value to 9.6, and add Treponema pallidum antigen (KTppl5, KTppl7 Or KTpp47 3mg / ml) Add 1/4 volume of 0.2M pH9.6CB to 1ml to adjust the pH value to 9.6, then mix the two liquids thoroughly, put them on a shaker at 200 rpm, and fully react in the dark at room temperature 2 hours;

3) Reduction of double bond Shiff base: 25 μ1 5M NaBH ₃ CN is added to the above cross-linked product, and after being thoroughly mixed, 4 * stand in the dark for 2 hours;

4) Blocking unreacted groups: Add 125 μ1 1M ethanolamine to block excess aldehyde groups on the HRP that is not cross-linked with Treponema pallidum antigen, and mix thoroughly. 4 Let stand in the dark for 1 hour;

5) removing small molecular substances from the cross-linked product: the above-mentioned cross-linked product is fully dialyzed against 0.02 MρΗ7.4 PBS for 24 hours;

6) Remove the uncrosslinked free enzyme: Take the liquid from the dialysis bag and add an equal volume of 50% saturated (NH4) ₂ S0 _4. After mixing thoroughly, leave it for 4 hours, centrifuge at 2000 rpm for 20 minutes, and collect the precipitate. Dilute the pellet with 0.02M pH7.4 PBS, and dialyze 0.02M pH7.4 PBS for 24 hours;

7) Remove uncrosslinked free antigen: Prepare a 2ml ConA-Sepharose pure A chemical column (produced by Pharmaca) was equilibrated with a 0.02M pH7.4 PBS column, and the dialysis-enzyme-labeled antigen was added. The PBS was eluted with 0.02M pH7.4, and then methyl-α-D was used. -Mannoside in 0.02M pH 7.4 PBS as the eluent, collect the eluted peak of the enzyme-labeled antigen containing glycosylated protein;

8) Preservation of purified target antigen: Add the collected enzyme-labeled antigen to an equal volume of glycerin, and store at -20 ° C to prepare the Treponema pallidum antigen stock solution.

In order to compare the labeling efficiency of the modified recombinant Treponema pallidum antigen and the unmodified Treponema pallidum antigen using the above method, take the same concentration of KTppl7 and Tρρ17 antigen (3mg / ml) as an example, and follow exactly the amount used in the above labeling method. In parallel experiments, the purified labeled product (before adding glycerol) was analyzed on a Superdex75 gel column (loading amount 100 l) (produced by Pharmaca) using an AKTO purifier protein purification analyzer (produced by Armersham pharmaca), The change in the amount of antigen protein in the conjugate was detected with a UV detector at ²⁸⁰ nm, and the change in the amount of horseradish peroxidase in the conjugate was detected with a UV detector at 403 nm (Ref. Tijssien (Analytical Biochem 136, 451-457, 1984). The ratio of the main peak area of 403nm to 280nm is used to detect the extent to which the recombinant Treponema pallidum antigen molecule can be labeled with horseradish peroxidase. The peak results of the product are shown in Figure 9. The integration results are listed below:

Table 1 Comparison of HRP-labeled recombinant Treponema pallidum antigen Γρρ17 and unmodified Treponema pallidum antigen Τρρ17

From the experimental results, the labeling efficiency of the modified Treponema pallidum antigen KTppl7 was significantly higher than that of the unmodified Treponema pallidum antigen Tρρ17. Example 4 Determination of the optimal concentration of the solid-phase-supported antigen and the enzyme-labeled antigen and the optimal compatibility of the types of antigens used

96-well slats were made of polystyrene plates (produced by Shenzhen Liji Co., Ltd.) (the difference between wells, plates and batches CV <10%), and 0.05M pH9.6 carbonate buffer (CB ) For the coating solution, add 100 microliters of coating solution prepared with the antigen according to the experimental design concentration, and coat at room temperature overnight. Then the antigen solution was discarded, the coated plate was washed twice with 0.02M PBS washing solution (PBS-T) containing 0.5% TW-20, patted dry, and added with 1% bovine serum albumin (BSA) and 1% skim milk powder. 0.02M pH7.4 Linate buffer (PBS) was blocked at room temperature for 2 hours. The blocking solution was discarded, the coated plate was air-dried, and the aluminum foil bag was vacuum-packed 41 for storage. By selecting KTppl5 antigen coating concentration of 5, 12.5, 25, 50, 75, 100 μg / ml for coating, select 0, 10, 20, 30, 40, 60, 80, 100 / ml Enzyme labeling

Mind experiment. The results showed that when KTppl5 was coated at a concentration of 50 μg / ml and the alcohol-labeled antigen was 40 μg / ml, the positive serum was evaluated (the positive serum was confirmed by Western Blot as Tppl ⁵ , Tppl7, and ⁴⁷ Tpp syphilis). (Positive serum) reached the maximum OD value, and the mean of negative response serum was less than 0.05. Based on this, we chose 50 μg / ml as the coating concentration of KTPp15 and 40 μg / ml as the enzyme-labeled antigen concentration. Using the same method, the coating concentration of KTppl7 was determined to be 20 yg / ml, and the concentration of the enzyme-labeled antigen was 40 μg / ml. The coating concentration of KTpp47 was determined to be 40 yg / ml, and the concentration of the enzyme-labeled antigen was 40 μg / ml.

Selection of the compatibility of the type of antigen used

See the ratio of coated antigen to enzyme-labeled antigen as shown in the following table. Select the appropriate compatible concentration between each antigen by square matrix test. Table 2. Dual antigen sandwich results (S / CO)

& ¾Anti

\ Γρρ15: Γρρ17: ΚΓρρ47 original Γρρ17 ΚΓρρ47

§ ¾¾¾ί5-, \ Γρρ15 (μ ^ πιΐ)

\, (Μ ^ ιηΐ)

Quality

Comment

Blood 50:50:50 50:20:40 20:20:20 50 20 40

HRP- ΚΓρρ15 Positive 5.0 7.0 45 6.8 6.7 62

40

HRP- sex

40

Γρρ17

40

HRP- Yin 025 0.19 0.12 0.15 0.1 ΚΓρρ47

Yang

Γρρ15 20 4.6 8 · 5 6.8 7.6 7.8 7.4

Sex

Γρρ17 40

Overcast

Γρρ47 20 023 0.1 02 0.1 0.09 0.12

Sex

Yang

6.9 65 5.0 4.4

Sex

40

HRP- Yin

0.1 0.1 0.1 0.1

Γρρ15 sex

Yang

6.4 7.0 6.8 63

Sex

HRP- 40

Overcast

Κ ρρ17 0.1 0.1 0.1 0.1

Sex

Yang

5.6 5.7 6 · 5

Sex

HRP- 40

Overcast

ΚΤρρ47 0.1 0.1 0.1 0.1

Sex From the above data, we select the compatible concentrations of the antigens as follows: The coating concentrations of KTppl5, Τρρ17, ΚΤρρ47 are 50, 20, and 40 μ g / ml respectively; the target antigen concentrations are 20, 40, and 20 μ g / mL, respectively. This compatibility selection highlights the role of ΓρρΠ, and also takes into account the possible complementary effects of ktρρ15 and ktρρ47.

The optimized coating antigen composition is called syphilis S-3 series antigens, and the optimized target antigens are collectively referred to as enzyme-labeled S-3 antigens. Example 5 Determination of appropriate antigen-antibody reaction conditions and time

At the concentrations of the coating antigen and the enzyme-labeled antigen determined in the above examples, the conditions suitable for the antigen-antibody reaction to form an immune-binding complex were studied using the enzyme-labeled plate and the reaction buffer as described above. Three weak, medium, and strong positive sera and negative controls were selected, and the double-antigen sandwich method experiment was performed according to the operation method described in detail in Example 7. By reacting at 37, 20, 40, 60, 90, and 120 minutes, and developing color for 15 minutes, the measured OD value reached the highest value at 60 minutes. Therefore, the reaction time is chosen to be 60 minutes. Example 6 Determination of Positive Threshold Values of Double Antigen Sandwich Immunoassay Samples According to the immunoassay method described in the previous example, 300 serums of normal volunteer blood donors determined to be negative by indirect ELISA were detected, and the normal distribution value was taken. conduct statistical analysis. The average value was 0.021 and SD was 0.004. The positive threshold was set to 0.18 + the mean of negative controls. Example 7 Detection of Treponema pallidum antibodies by the double-antigen sandwich method To a 96-well microtiter reaction plate was added κρρρ15, ΚΤρρ17, ΚΤρρ47 to the optimal concentration (Syphilis S -3 series of antigen) coating solution, let stand for 24 hours, add 0.02M pH7.4 PBS washing solution (PBS-T) containing 0.1% TW-20 to fill each well, leave it for a few seconds, discard it, and repeat washing twice. Add 200 μl of 1% bovine serum albumin (BSA) and 1% skimmed milk powder to 0.02M ρ7.4 phosphate buffered saline (PBS) per well, and block for 2 hours. Discard the blocking solution, dry at room temperature, seal the plate with a thin aluminum bag, and store it in 4X until use. Add pre-used enzymes to the wells of the microwell reaction plate Standard antigen dilution solution (20% goat serum, 1% casein, 0.02M PBS, pH7.4) Dilute the corresponding alcohol-labeled antigen of syphilis S-3 series antigen with optimized concentration, add 50μ1 per well, and add 15 Sera from different subjects to be tested were incubated at 37 ° C for 60 minutes. Then each well was filled with 0.02M PBS-T washing solution containing 0.1% TW-20, left for a few seconds, discarded, and repeated washing 5 times. Add 50 μ1 of enzyme substrate A solution consisting of 0.06% urea peroxide (Sigma) O.OlM pH 4.5 pH solution to each well and 50 μ1 of lemon with 0.06% TMB (Sigma) 0.01M pH 4.5 Enzyme substrate B solution consisting of acid buffer, incubated at 37 ° C for 15 minutes. The reaction was stopped by adding 50 μΐ of 2M H ₂ S0 _{4 to} each well. Read the absorbance at 450nm (using 630 as the reference wavelength) on a target reader (Dyntech MR 4100). The positive control and the negative control are human serum that has been reviewed by TPHA, RPR, and FTA-ABS methods. The results of the samples are determined according to the threshold determined in Example 6. Samples higher than 0.18 + 0.024 are positive for Treponema pallidum. The serum samples were obtained from 49 syphilis patients with clinically confirmed sera from patients with dermatology in the First Affiliated Hospital of Beijing Medical University, including 11 syphilis patients (I 1--I 11) and 38 syphilis patients (Π 12― — Π 49) (of which 4 were post-treatment sera, indicated by Π *). The RPR kit used in the parallel experiment was an Organon product (batch number: 0349), and the TPPA kit was a product of Fuji Biological Co., Ltd. (batch number: VN81204). The results are as follows:

Table 3.Dual antigen sandwich method for detecting sera of patients with syphilis

ELISA (¾¾ sandwich, method) RPR

Discrimination titer Discrimination TPPA Positive time photo 2231 + 1:32 + + shade tree photo 0.024---

I 1 1.892 + 1:16 + +

I 2 0.54 +-+

I 3 1.766 +-+

I 4 1.869 + 1: 1 + +

I 5 1.935 + 1: 2 + +

I 6 1.436 + 1: 1 + +

I 7 0 · 557 +-+

I 8 0 · 505 +-+

I 9 1238 + 1: 1 + +

I 10 0.658 +-+

I 11 1.154 + 1: 4 + +

Π12 1.133 + 1: 8 + +

Π 13 * 2.159 + 1: 4 + +

Π14 76 + 1: 8 + +

Π15 0530 + 1:16 + +

Π16 0.869 + 1:16 + +

Π17 0.901 + 1: 32 + +

Π18 0.898 + 1: 8 + +

Π19 0.760 + 1:16 + +

Π20 1.018 + 1:32 + +

II 21 0564 + 1:16 + +

Π22 72 + 1: 8 + +

Π23 1.932 + 1:16 + +

Π24 0.821 + 1: 8 + +

Π25 0.741 + 1:32 + +

Π26 * 0.785 + 1: 4 + +

Π27 0.682 + 1:16 + +

Π28 0.676 + 1:32 + + Table 3 (continued)

Example 8 Composition of the double antigen sandwich method for Treponema pallidum immunoassay kit The preferred double antigen sandwich method for Treponema pallidum immunoassay kit consists of the following parts:

Coated antigens: syphilis S-3 series antigens; that is, KTppl5, ΚΤρρ17, ΚΤρρ47 at concentrations of 50, 20, and 40 μg / ml, respectively.

Antigen coating plate: a domestic 96-well detachable plate, coated with syphilis S-3 series antigen as described in Example 4, dried under vacuum and sealed;

Enzyme-labeled antigen: Horseradish peroxidase-labeled syphilis S-3 series antigen. Ie: concentration Enzyme-labeled antigens HRP-KTppl5, HRP-KTppl7, HRP-KTpp47, 20, 40, and 20 μg / ml, respectively

Negative control and positive control serum: 0.2 ml each, containing 0.05% thimerosal, 0.01% sodium azide, 50 μ1 per well when used;

Enzyme-labeled S-3 antigen 6ml working solution: Enzyme-labeled S- 3 Dilute the antigen to the appropriate titer.

Substrate solution A: 6ml 0.06% urea peroxide (Sigma) 0.01M pH 4.5 citric acid (Sigma) buffer;

Substrate solution B: 6ml 0.06% TMB (Sigma) 0.01M pH 4.5 citric acid (Sigma) buffer solution;

Stop solution: 6ml lM sulfuric acid

Washing solution: 20ml 50 X PBS, containing 0.1% Tween-20;

Anti- Treponema pallidum positive serum OlM should be above the mean of 0.18+ negative control.

The anti- Treponema pallidum negative serum OD value should be below 0.18.

One copy of the double antigen sandwich immunoassay kit for Treponema pallidum antibody kit. Example 9 A double-layered sandwich method and an indirect ELESA method for detecting females in the drug industry. A 96-well plate coated with the modified recombinant Treponema pallidum antigen protein KTpp according to the conditions of the foregoing embodiment, and the antigen protein KTpp enzymatically labeled in Example 3 was used. According to the method of Example 7, the clinical sera obtained from the Institute of Dermatology, Chinese Academy of Medical Sciences, Beijing STD Prevention and Treatment Center, Beijing Red Cross Blood Center, Department of Laboratory Medicine, China-Japan Friendship Hospital, Department of Dermatology, First Affiliated Hospital of Beijing Medical University, The double antigen sandwich method was used for immunodetection of Treponema pallidum antibodies, and the results were compared with the conventional indirect ELISA method. The evaluation results of 30 positive sera showed that the two antigen sandwich method and the indirect ELISA method were used to detect weak positive samples, and the ratio (S / CO) of the sample OD value to the cut-off value was compared. * Indicates) and early syphilis (indicated by ** in the table; higher than the indirect ELISA method. The results are shown in the table below.. Comparison of double antigen sandwich method and indirect method (S / CO)

The above experimental results show that the dual antigen sandwich immunoassay for Treponema pallidum kit can detect I _g G, IgM and IgA simultaneously, and an indirect ELISA for detecting IgG antibodies. Compared with the method, it can not only diagnose the incubation period of syphilis early, but also reduce the probability of missed detection.

In addition, through the determination of 400 clinical negative serum samples, the dual antigen sandwich method Treponema pallidum kit implemented by the present invention is significantly better than the indirect ELISA method. The 00 value measured by the dual antigen sandwich method is <0.02, and the indirect ELISA method is Right and left, the false positive rate of the indirect ELISA method is 5% o, and the double antigen sandwich method is 0. Example 10 Comparison of the effects of recombinant KTpp antigen and unmodified Treponema pallidum antigen Tpp indirect ELISA on the detection of Treponema pallidum antibodies

In order to compare the antigenicity of the modified Treponema pallidum antigens KTppl5, KTPp17, and KTPp47 prepared by the present invention with the Treponema pallidum antigen with no lysine residues at the N-terminus and C-terminus, the indirect ELISA method was used for comparison. That is, the two recombinant protein antigens Tppl5 and KTppl5, Τρρ17 and Γρρ17, Τρρ47 and Κρρρ47 were respectively coated with solid-phase enzyme plates at various concentrations, and the same syphilis positive serum samples were detected by indirect ELISA. For antigen detection of Treponema pallidum antibodies, specific experimental operations and results are shown in the table below.

Recombinant KTpp antigen and Tpp antigen at a concentration of 0, 2.5, 5, 10, 15, 20, 25, 30, 40, 50 (μg / ml) were used in 0.05M pH9.6 carbonate buffer (CB ) Prepare a coating working solution and coat the 96-well microtiter plate 4 * C overnight. Then 1% bovine serum albumin (BSA) and 1% skimmed milk powder were added in the same 0.02M pH7.4 phosphate buffered saline (PBS), and blocked at 4X for 2 hours and discarded. ^ Add the same volume of Treponema pallidum positive serum (Tppl5, Tppl7, and Tρρ47 band positive serum confirmed by Western Blot) 10 μl / well, using 100 μl of PBS solution containing 1% BSA as the sample dilution Discard after 37, 30 minutes. Wash twice with 0.05M PBS washing solution (PBS-T) containing 0.5% TW-20, and add the same diluted solution with drunk standard antibody (20% goat serum (produced by Zhejiang Sanli Blood Products Co., Ltd.), 1% casein (Sigma), 0.02M PBS pH7.4)) 100 μl / well of sheep anti-human IgG (manufactured by DAKO) diluted 1: 5000 for 30 minutes, and then use 0.5% Tween 20 (TW-20) 0.02M Wash twice with pH 7.4 PBS washing solution (PBS-T), and finally add 50 μ1 of tritiated substrate A solution composed of 0.06% urea peroxide (Sigma) 0.01M acetate buffer pH 4.5 to each well. And 50 μ1 of the substrate B, which is 0.06% 3,3,5,5, -tetramethylbenzidine (TMB) (Sigma), 0.01M citrate buffer pH 4.5, 37 €, significantly Color for 10 minutes, stop by adding 1M H ₂ S0 _{4 at} last, read the absorbance at 450nm (reference wavelength 630nm), and list the experimental results: Table 5: Indirect ELISA method Tppl5 antigen and KTPpρ15 antigen detection Comparison (450nmOD value, reference wavelength 630nm)

Table 6: Comparison of indirect ELISA Tppl7 antigen and KTPpρΠ antigen for detection of Treponema pallidum positive serum ( ⁴⁵⁰ nmOD value, reference wavelength 630nm)

Coating concentration (μ g / ml) ΤρρΠ ΚΤρρ17

0 0 0

2.5 0.3 0.3

5.0 0.615 0.689

15 1.04 1.28

20 1.28 1.58

25 1.44 1.74

30 1.52 1.86

40 1.61 1.973

50 1.64 2 Table 7: Comparison of indirect ELISA Tpp47 antigen and KTPpρ47 antigen for detection of Treponema pallidum positive serum (450nmOD value, reference wavelength 630nm)

The experimental results show that the antigenic sensitivity of KTPp15, KTPp17, and KTPp47 is higher than that of Tρρ15, Tρρ17 and Tρρ47. Example 11 Comparison of Enzyme-labeled Recombinant TKρρ Antigen and Enzyme-labeled Unmodified Treponema Pallidum Antigen Tρρ Dual Antigen Sandwich Method for the Determination of T. pallidum Antibodies

In order to compare the effects of the modified Treponema pallidum antigens KTPpρ15, KTPpρΠ, and KTPpρ47 prepared by the present invention with the N-terminal and C-terminal T. pylori antigen-enzyme-labeled complexes without lysine residues, a double antigen sandwich ELISA method was used. Compare. That is, the optimized protein antigens Tppl5 and KTppl5, Τρρ17 and ΚΤρρ17, Τρρ47, and ΚΤρρ47 were respectively coated with the solid-phase enzyme plate, and then a series of concentrations of the recombinant protein antigens τρρ15 and κΤρρ15, Τρρ17 and κρρ17, Τρρ47, and κρρ47 were used. Enzyme-labeled complex to detect the same Treponema pallidum positive serum samples, and compare the effect of two enzyme-labeled antigens on detection of Treponema pallidum antibodies.

Recombinant κρρρ antigen (concentrated optimized κρρ15 50 μ g / ml, KTppl7 20 / ml, KTpp47 40 μ g / ml) and Tp antigen (concentrated optimized Τρρ15 50 μ g / ml, Tppl7 30 μ g / mI, KTpp47 (40 μg / ml) were prepared with 0.05M pH9.6 carbonate buffer (CB) to coat the working solution, 4 "C coated on 96-well target plate Was overnight. Then, 1% bovine serum albumin (BSA) and 1% skimmed milk powder were added in the same 0.02M pH 7.4 phosphate buffered saline (PBS), and blocked at 4 ° C for 2 hours and discarded. Then add diluted with enzyme-labeled antigen (20% goat serum (produced by Zhejiang Sanli Blood Products Co., Ltd.), 1% casein (Sigma), 0.02M PBS pH 7.4)) and dilute to 0, 2.5, 5, 10, 15, 20, 25, 30, 40, 50 (μg / ml) series of enzyme-labeled antigen complexes HRP-KTppl5, HRP-KTppl7, HRP-KTpp47 and HRP-Tppl5, HRP-Tppl7, HRP-Tpp47, 50 Microliter / well, 37 ^, 60 minutes, wash twice with 0.02M pH7.4 PBS washing solution (PBS-T) containing 0.5% Tween 20 (TW-20), and finally add 50μ1 to each well by 0.06% Tritium substrate A solution consisting of urea peroxide (Sigma) 0.01M acetate buffer pH 4.5 and 50 μ1 of 0.06% 3,3,, 5,5, -tetramethylbenzidine (TMB) ( Sigma), 0.01M substrate B consisting of 0.01M citrate buffer, pH 4.5, 37 €, color development for 10 minutes, stop by adding 1M H ₂ S0 ₄ and read the absorbance at 450nm (reference wavelength 630nm), the experimental results are shown in Figure 10.

The experimental results show that whether the modified recombinant KΓρρ15, ktρρ17, ktρρ47 is coated with the antigen or the unmodified recombinant τρρ15, Τρρ17, Τρρ47 is coated with the enzyme-labeled recombinant antigens HRP-KTppl5, HRP-KTppl7, HRP -KTpp47's dual-antigen sandwich method for detecting syphilis-positive shielded serum is more sensitive than the use of enzyme-labeled unmodified recombinant antigens HRP-Tppl5, HRP-Tppl7, and HRP-Tpp47.

Claims

Rights request

1. A protein having the general formula (Xaa) _n Tpp (Xaa) _m , wherein Tpp is selected from the outer membrane proteins of syphilis spiralis Tρρ15, Tρρ17, Tρρ47 or a fragment or derivative thereof having an immunological activity that binds human immunoglobulin; Xaa represents a natural amino acid residue, wherein at least one Xaa closest to each side of Tpp and the terminal Xaa of at least one formula protein are amino acid residues selected from Lys, Arg, and Trp; π and m are integers of 0 to 10, The condition is that n + m≥2.

2. The protein according to claim 1, wherein the protein is MetLysLys-Tpp-LysLysLeuGluLys, and Tpp is a syphilis outer membrane protein Tppl5 or a fragment or derivative thereof having an immunological activity that binds human immunoglobulin.

3. The protein according to claim 1, wherein the protein is MetLysLys-Tpp-LysLysLeuGluLys, and Tpp is a syphilis outer membrane protein Tppl7 or a fragment or derivative thereof having an immunological activity that binds human immunoglobulin.

4. The protein according to claim 1, wherein the protein is MetLysLys-Tpp-LysLysLeuGIuLys, and Tpp is a syphilis outer membrane protein Tpp47 or a fragment or derivative thereof having an immunological activity that binds human immunoglobulin.

5. The protein of claim 2 having the amino group of SEQ ID No. 1.

6. The protein of claim 3, which has the amino acid sequence of SEQ ID No. 2.

7. The protein of claim 4, which has the amino acid sequence of SEQ ID No. 3.

8. A method for immunodetection of Treponema pallidum antibodies, comprising using an antigen-antibody reaction between at least one protein according to any one of claims 1 to 7 and Treponema pallidum antibodies in a biological sample.

9. The method of claim 8, comprising the steps of:

1) coating a solid support with at least one purified protein according to any one of claims 1 to 7;

2) Add the biological sample to be tested to the coated solid support, Incubate for a period of time under conditions where antigen-antibody immune binding complexes are formed;

3) Moderate cleaning to remove any unbound Treponema pallidum antibodies;

4) adding a labeled secondary antibody against human antibodies, and incubating for a period of time under conditions suitable for forming an antigen-antibody immune binding complex;

5) Moderate washing to remove any unbound secondary antibodies;

6) Detect the presence of antigen-antibody immune binding complex in the incubation mixture.

10. The method of claim 9, wherein the second antibody against the human Treponema pallidum antibody is labeled with an enzyme selected from the group consisting of horseradish peroxidase, alkaline phosphatase, β-galactosidase, and acetylcholinesterase.

11. The method of claim 8, comprising the steps of:

2) Preparing labeled proteins corresponding to those used in 1) in advance;

3) adding the biological sample of the individual to be tested and the protein labeled in step 2) to the coated solid support, and incubating for a period of time under conditions suitable for forming an antigen-antibody immune binding complex;

4) Moderate cleaning to remove any unbound Treponema pallidum antibodies;

5) Detect the presence of antigen-antibody immune complexes in the incubation mixture.

12. The method of claim 11, wherein the protein in step 2) is labeled with a hydrazone selected from the group consisting of horseradish peroxidase, alkaline phosphatase, β-galactosidase, and acetylcholinesterase.

13. A detection kit for immunodetection of human Treponema pallidum antibodies, comprising a detection plate coated with a purified at least one protein according to any one of claims 1-7.

14. The detection kit according to claim 13, further comprising a labeled protein according to any one of claims 1 to 7 corresponding to a kind of the coated detection plate.

15. The detection kit according to claim 13, wherein the protein is labeled with an enzyme selected from the group consisting of horseradish peroxidase, alkaline oxidase, galactosidase, and acetylcholinesterase.

16. The detection kit of claim 13, further comprising a labeled secondary antibody against a human antibody.

17. The detection kit according to claim 13, wherein the second antibody against the human antibody is labeled with an enzyme selected from the group consisting of horseradish peroxidase, alkaline phosphatase, P-galactosidase, and acetylcholinesterase.