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CN-121991180-A - Streptococcus mutans phage gp15 protein and application thereof in rapid diagnosis of streptococcus mutans

CN121991180ACN 121991180 ACN121991180 ACN 121991180ACN-121991180-A

Abstract

The invention discloses a streptococcus mutans phage gp15 protein and application thereof in rapid diagnosis of streptococcus mutans, and firstly discovers and proves that the gp15 protein in the streptococcus mutans phage is a novel RBP, and the gp15-mNG fusion protein system is constructed to prove that the protein can efficiently and specifically identify and bind to a surface receptor of the streptococcus mutans, the binding rate is up to more than 99%, and an ideal molecular tool is provided for developing rapid diagnosis technology of the streptococcus mutans.

Inventors

  • LI YONGLIANG
  • ZHANG MINGRUI
  • CHEN XIAOLIN
  • CHEN YUXING
  • WANG XIAOYAN
  • MA ZHE
  • XU MINGMING

Assignees

  • 北京大学口腔医学院

Dates

Publication Date
20260508
Application Date
20260309

Claims (10)

  1. 1. A streptococcus mutans phage receptor binding protein gp15 is characterized in that the amino acid sequence is shown in SEQ ID NO. 1.
  2. 2. A nucleic acid molecule encoding the receptor binding protein gp15 of claim 1, wherein the nucleotide sequence is as shown in SEQ ID No. 2 or has more than 90% identity to SEQ ID No. 2.
  3. 3. A fusion protein gp15-mNG, wherein the fusion protein gp15-mNG consists of the receptor-binding protein gp15 of claim 1 and a fluorescent protein mNeonGreen; preferably, the amino acid sequence of the fusion protein gp15-mNG is shown in SEQ ID NO. 3.
  4. 4. A recombinant expression vector for encoding fusion protein gp15-mNG is characterized in that the recombinant expression vector is pET-28a, gp15-mNG, which consists of gp15 gene fragment, fluorescent protein mNeonGreen and linearized pET-28a vector; Preferably, the nucleotide sequence of the recombinant expression vector is shown as SEQ ID NO. 4 or has more than 90% of identity with SEQ ID NO. 4.
  5. 5. A recombinant host cell comprising the fusion protein gp15-mNG of claim 3 and/or the recombinant expression vector of claim 4.
  6. 6. A colloidal gold-gp 15 conjugate, wherein the colloidal gold-gp 15 conjugate comprises the receptor-binding protein gp15 of claim 1 and colloidal gold; Preferably, the colloidal gold-gp 15 conjugate is prepared by coupling the receptor binding protein gp15 with colloidal gold.
  7. 7. Use of any one of the receptor binding protein gp15 of claim 1, the fusion protein gp15-mNG of claim 3 and/or the colloidal gold-gp 15 conjugate of claim 6: 1) The application in preparing products for specifically detecting streptococcus mutans; 2) The application in preparing caries risk assessment or oral cavity cariogenic bacteria screening products.
  8. 8. The use according to claim 7, wherein the product comprises a diagnostic reagent, a kit, a test strip, a microfluidic chip, a biosensor, a system, a device, a readable medium; preferably, the diagnostic reagent is labeled with a detectable label; preferably, the detectable label is selected from the group consisting of colloidal gold, fluorescent protein, enzyme, quantum dot, magnetic bead, or radioisotope; Preferably, the kit further comprises at least one of a diluent, a quality control product, a buffer solution and instructions; Preferably, the test strip takes the colloidal gold-gp 15 conjugate as a conjugate, and comprises a detection line coated with a receptor binding protein gp15 specific antibody, a quality control line coated with an anti-receptor binding protein gp15 polyclonal antibody, and a chromatographic carrier consisting of a sample pad, a binding pad, a nitrocellulose membrane and a water absorption pad; Preferably, the test strip is a colloidal gold immunochromatographic test strip.
  9. 9. A method for preparing the fusion protein gp15-mNG of claim 3, comprising the steps of amplifying the receptor binding protein gp15 of claim 1, recombining with fluorescent protein mNeonGreen and linearized pET-28a vector through a Gibson ligation reaction, and transforming into competent cells; preferably, the competent cells are competent escherichia coli; preferably, the competent cell is competent E.coli BL21 (DE 3).
  10. 10. A product, characterized in that it comprises any one of the following: 1) A colloidal gold immunochromatographic kit for specific detection of streptococcus mutans, comprising the colloidal gold-gp 15 conjugate of claim 6, a detection line coated with a gp 15-specific antibody of a receptor binding protein, a quality control line coated with a gp 15-polyclonal antibody of an anti-receptor binding protein, and a chromatographic carrier consisting of a sample pad, a binding pad, a nitrocellulose membrane and a water absorbing pad; 2) A fluorescence detection kit for specific detection of streptococcus mutans, the fluorescence detection kit comprising the fusion protein gp15-mNG of claim 3, and at least one of a diluent, a quality control, a buffer, and instructions; Preferably, the fluorescence detection kit further comprises a microplate, a glass slide or a microfluidic chip for carrying samples; Preferably, the sample is a sample comprising oral flora of a subject.

Description

Streptococcus mutans phage gp15 protein and application thereof in rapid diagnosis of streptococcus mutans Technical Field The invention belongs to the technical fields of microbiology, immunology and clinical diagnosis, and particularly relates to a streptococcus mutans bacteriophage gp15 protein and application thereof in rapid diagnosis of streptococcus mutans. Background Streptococcus mutans is a common pathogenic bacterium in the oral cavity of humans and is closely related to occurrence and development of dental caries. Currently, detection methods for streptococcus mutans mainly comprise a traditional culture method, PCR molecular detection, immunological detection and the like. The traditional culture method has long time consumption and low sensitivity, the PCR detection method depends on professional equipment and technicians, is easy to influence the accuracy due to nucleic acid pollution, and the sensitivity and the specificity of immunological methods such as ELISA and the like are limited by the antibody preparation quality. Therefore, the development of a rapid, sensitive and easy-to-operate streptococcus mutans detection method has important significance. Phages are viruses that specifically infect microorganisms such as bacteria, and the infection process begins with specific adsorption to the surface of the host bacteria. The adsorption process relies on specific recognition and binding of the receptor binding protein (Receptor Binding Protein, RBP) at the tail of the phage to the host surface receptor. As a core functional molecule of a phage recognition host, RBP naturally has the remarkable advantages of strong binding specificity, high affinity, small molecular weight, good stability and easiness in genetic engineering modification, and has become a novel recognition element with great potential in the field of rapid diagnosis of microorganisms. However, current research on the RBP of mutans phage remains blank, and functional RBP molecules of mutans phage have not been clearly identified in the prior art. The technical gap causes that the streptococcus mutans lacks a single molecular recognition tool with high specificity and high affinity, and the defects of long time consumption, low sensitivity and the like exist in detection, so that the breakthrough and clinical transformation of the rapid diagnosis technology of the streptococcus mutans are seriously limited, and a novel functional RBP needs to be discovered and a related application system is established. Disclosure of Invention In view of the above, the invention aims to provide a streptococcus mutans bacteriophage gp15-mNG fusion protein, which is used for defining the biological function of gp15 protein as Receptor Binding Protein (RBP), realizing visual characterization of protein binding activity by means of fluorescent characteristics of mNG, and simultaneously establishing a rapid diagnosis method of streptococcus mutans based on the protein, and is suitable for rapid screening and diagnosis of streptococcus mutans in the fields of clinical samples, oral health monitoring, food safety and the like. The invention adopts the following technical scheme to realize the purposes: in a first aspect, the invention provides a Streptococcus mutans phage receptor binding protein gp15, which has the amino acid sequence shown in SEQ ID NO. 1. The streptococcus mutans bacteriophage is one kind of virus dependent on streptococcus mutans (Streptococcus mutans) to grow and belongs to the field of long tail bacteriophage. The head part is an equiaxed icosahedral capsid, the tail part is a non-shrinkage long tail, the diameter of the capsid is estimated to be (67+/-0.4) nm, the length of the non-shrinkage tail is estimated to be (283+/-7) nm, and the width is estimated to be (8.3+/-0.1) nm. Phages are a class of viruses that specifically infect bacteria, whose host recognition relies on high affinity, high specificity interactions of tail receptor binding proteins (Receptor Binding Protein, RBPs) with bacterial surface receptors. Functional RBP molecules of Streptococcus mutans phages have not been identified explicitly in the prior art. In the present invention, the Streptococcus mutans phage comprises a phage that has been reported as M102, UA140, UA159,APCM01、SM1, etc., also contains unreported phages such as 1-7 in the examples of the present invention. In a second aspect, the invention provides a nucleic acid molecule encoding the receptor binding protein gp15 of the first aspect, which has a nucleotide sequence as shown in SEQ ID NO. 2 or has more than 90% identity with SEQ ID NO. 2. The term "identity" also known as "homology" refers to an amino acid sequence or nucleotide sequence that is at least 80% identical in sequence to the sequences provided herein. To determine sequence identity, sequence alignments can be performed in various ways known to those skilled in the art, for example, using BLAST, BLAST-2, ALIGN, NEEDLE, megalign