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CN-121991188-A - Mutant antigen for improving activity of TP antigen

CN121991188ACN 121991188 ACN121991188 ACN 121991188ACN-121991188-A

Abstract

The invention provides a mutant antigen for improving TP antigen activity. The invention aims to provide a TP recombinant antigen mutant which can detect TP antibodies in a sample with high sensitivity. Specifically, the invention provides a TP recombinant antigen mutant, which comprises a TP47 antigen fragment, a TP17 antigen fragment and a TP15 antigen fragment, wherein at least any one amino acid of R60, K110 and K391 positions of the TP47 antigen fragment, K114 and K143 positions of the TP17 antigen fragment and/or R29 and K88 positions of the TP15 antigen fragment is mutated to G or A.

Inventors

  • LIU WEI
  • XI JIANHONG
  • WAN HUAN
  • WANG WENFENG
  • Hou Sulin
  • YUAN HUIMEI
  • HE BIN
  • ZHONG XINGHUA

Assignees

  • 杭州检一生物技术有限公司

Dates

Publication Date
20260508
Application Date
20251229

Claims (13)

  1. 1. A TP recombinant antigen mutant comprising a TP47 antigen fragment, a TP17 antigen fragment and a TP15 antigen fragment, characterized in that at least any one of amino acids at positions R60, K110, K391 of the TP47 antigen fragment, positions K114, K143 of the TP17 antigen fragment, and/or positions R29, K88 of the TP15 antigen fragment is mutated to G or a.
  2. 2. The TP recombinant antigen mutant of claim 1, wherein the TP47 antigen fragment, the TP17 antigen fragment, and the TP15 antigen fragment are connected by a flexible linker.
  3. 3. The TP recombinant antigen mutant according to claim 1 or 2, wherein the wild-type sequence of the TP47 antigen fragment is amino acid residues 26-223 and amino acid residues 352-434 of SEQ ID No.1, the wild-type sequence of the TP17 antigen fragment is amino acid residues 22-156 of SEQ ID No.2, and the wild-type sequence of the TP15 antigen fragment is amino acid residues 18-141 of SEQ ID No. 3.
  4. 4. The TP recombinant antigen mutant according to claim 1 or 2, comprising a TP47 antigen fragment, a TP17 antigen fragment and a TP15 antigen fragment in order from the N-terminus to the C-terminus.
  5. 5. The TP recombinant antigen mutant according to claim 1 or 2, having an amino acid sequence as shown in any one of SEQ ID NOs 4 to 16.
  6. TP antigen mutant comprising at least one of a TP47 antigen fragment, a TP17 antigen fragment and a TP15 antigen fragment, characterized in that at least any one of the amino acids at the R60, K110, K391 positions, the K114, K143 positions of the TP17 antigen fragment, and/or the R29, K88 positions of the TP15 antigen fragment is mutated to G or a.
  7. 7. A polynucleotide encoding the TP recombinant antigen mutant of any one of claims 1-5, or the TP antigen mutant of claim 6.
  8. 8. An expression vector comprising the polynucleotide of claim 7.
  9. 9. A composition comprising the TP recombinant antigen mutant of any one of claims 1-5, or the TP antigen mutant of claim 6, optionally further comprising a pharmaceutically acceptable carrier, or a solid support or detectable label conjugated to the TP recombinant antigen mutant or TP antigen mutant.
  10. 10. A kit comprising the TP recombinant antigen mutant of any one of claims 1-5, or the TP antigen mutant of claim 6, or the composition of claim 9.
  11. 11. Use of a TP recombinant antigen mutant according to any one of claims 1-5, or a TP antigen mutant according to claim 6, or a composition according to claim 9 for the preparation of a kit for in vitro detection of TP antibodies in a sample or for detection/diagnosis of syphilis.
  12. 12. Use according to claim 11, wherein the sample comprises a body fluid, tissue or cell, such as a blood sample, urine sample.
  13. 13. A method of detecting TP antibodies in a sample in vitro, the method being for non-diagnostic purposes, comprising detecting using the TP recombinant antigen mutant of any one of claims 1-5 or the TP antigen mutant of claim 6, optionally the method being a double antigen sandwich method, optionally the TP recombinant antigen mutant or the TP antigen mutant as a labeling antigen or a coating antigen.

Description

Mutant antigen for improving activity of TP antigen Technical Field The present invention relates to the field of immunodetection. In particular, the invention relates to a mutant antigen for improving TP antigen activity, more particularly, the invention relates to a TP recombinant antigen and a mutant thereof, and also relates to nucleic acid for encoding the TP recombinant antigen and the mutant thereof, and related vectors, host cells, immunoassay methods and detection kits. Background Syphilis is a systemic sexually transmitted disease caused by treponema pallidum (Treponema pallidum, TP), which severely jeopardizes human health. Early diagnosis and effective treatment are of great significance in blocking disease transmission and preventing late serious complications. Among the various treponema pallidum polypeptide antigens, tpN15, tpN17, tmpA and TpN47 have proven diagnostic. TpN15 and TpN17 are diagnostic antigens commonly used in early stages, and recent researches show that the TpN47 antigen shows better immunoreactivity and is expected to become an important marker for early diagnosis of syphilis. The TP diagnostic reagent mainly uses gene fragments such as TpN15/TpN17/TpN47 on different detection platforms, and in the diagnostic reagent, the TP diagnostic raw materials are mostly in modes such as TpN17+TpN47, tpN15+TpN17+TpN47 chimeric and the like. Serological detection is the main means of current syphilis diagnosis, wherein chemiluminescence immunoassay (CLIA) has become the mainstream detection platform by virtue of high sensitivity, wide linear range, convenient operation, easy automation and the like. In this method, the labeling efficiency of the antigen and its immunological activity directly determine the core indicators of the detection performance, such as sensitivity, specificity and signal-to-noise ratio. The labeling method commonly used at present relies on carbodiimide (such as EDC) mediated crosslinking reaction of carboxyl groups and amino groups, so that the carboxyl groups on the surface of the magnetic beads are covalently bonded with free amino groups (mainly from epsilon-amino groups of lysine (K) and guanidine groups of arginine (R)) on antigen molecules. However, TP antigens (including TpN15, tpN17, tpN47, etc.) have reduced detection sensitivity, poor specificity bias and poor signal-to-noise ratio, severely limiting the development and use of high performance diagnostic reagents for syphilis. In order to alleviate the technical bottlenecks, the current stage is generally improved by optimizing labeling conditions or performing strategies such as pretreatment on magnetic beads. However, these methods are generally based on the adjustment of technological parameters, and do not fundamentally overcome the inherent limitations of the composition of the self amino acids of the TP antigen. Thus, there is an urgent need for a mutant antigen that enhances the activity of TP antigen labeling. Disclosure of Invention The invention aims to provide a TP recombinant antigen mutant which can improve the detection activity of TP recombinant antigens, so that the sensitivity and the specificity of TP antibody detection in a subject sample are improved, and the false negative rate is reduced. Specifically, the invention relates to the following technical scheme: 1. A TP recombinant antigen mutant comprising a TP47 antigen fragment, a TP17 antigen fragment and a TP15 antigen fragment, characterized in that at least any one of amino acids at positions R60, K110, K391 of the TP47 antigen fragment, positions K114, K143 of the TP17 antigen fragment, and/or positions R29, K88 of the TP15 antigen fragment is mutated to G or a. 2. The recombinant antigen mutant of TP according to item 1, wherein the TP47 antigen fragment, the TP17 antigen fragment and the TP15 antigen fragment are connected by a flexible linker. 3. The TP recombinant antigen mutant according to item 1 or 2, wherein the wild-type sequence of the TP47 antigen fragment is amino acid residues 26 to 223 and amino acid residues 352 to 434 of SEQ ID No. 1, the wild-type sequence of the TP17 antigen fragment is amino acid residues 22 to 156 of SEQ ID No. 2, and the wild-type sequence of the TP15 antigen fragment is amino acid residues 18 to 141 of SEQ ID No. 3. 4. The TP recombinant antigen mutant according to item 1 or 2, which comprises a TP47 antigen fragment, a TP17 antigen fragment and a TP15 antigen fragment in order from the N-terminus to the C-terminus. 5. The TP recombinant antigen mutant according to item 1 or 2, which has an amino acid sequence as shown in any one of SEQ ID NOs 4 to 16. 6. TP antigen mutant comprising at least one of a TP47 antigen fragment, a TP17 antigen fragment and a TP15 antigen fragment, characterized in that at least any one of the amino acids at the R60, K110, K391 positions, the K114, K143 positions of the TP17 antigen fragment, and/or the R29, K88 positions of the TP15 antigen fragment is mutated