Search

CN-122011178-A - Specific monoclonal antibody of AKK bacterium efficacy protein Amuc-1100, kit, detection method and application thereof

CN122011178ACN 122011178 ACN122011178 ACN 122011178ACN-122011178-A

Abstract

The invention belongs to the field of biological detection, and particularly relates to a specific monoclonal antibody of AKK bacterium efficacy protein Amuc-1100, a kit, a detection method and application thereof. The antibody pair is 3E10 and 2F7 monoclonal antibodies, the amino acid sequences of the variable regions of the heavy chain and the light chain of the 3E10 are SEQ ID NO.2, SEQ ID NO.4 and 2F7 are SEQ ID NO.6 and SEQ ID NO.8. The antibody has strong pairing specificity and high affinity, and the rapid screening and accurate quantification of Amuc _1100 protein are respectively realized based on the colloidal gold rapid detection and double-antibody sandwich ELISA quantitative detection kit constructed by the antibody, so that the antibody has good detection linearity, high precision and excellent accuracy, can be used for detecting the protein in the mucus Ackermansis and controlling and evaluating the activity of relevant probiotic preparations, and provides key technical support for relevant basic research and industrialization application.

Inventors

  • MA XIN
  • WEN HAIXIA
  • YU YANG
  • YU XUEPING

Assignees

  • 善恩康生物科技（苏州）有限公司

Dates

Publication Date
20260512
Application Date
20260414

Claims (10)

  1. A specific monoclonal antibody pair of AKK bacterial active protein Amuc-1100 is characterized by comprising a 3E10 monoclonal antibody and a 2F7 monoclonal antibody, wherein the amino acid sequence of a heavy chain variable region of the 3E10 monoclonal antibody is shown as SEQ ID NO.2, the amino acid sequence of a light chain variable region of the 2F7 monoclonal antibody is shown as SEQ ID NO.4, and the amino acid sequence of a heavy chain variable region of the 2F7 monoclonal antibody is shown as SEQ ID NO.6, and the amino acid sequence of a light chain variable region of the 2F7 monoclonal antibody is shown as SEQ ID NO. 8.
  2. 2. A nucleic acid molecule encoding a specific monoclonal antibody pair according to claim 1, wherein the nucleotide sequence encoding the heavy chain variable region of the 3E10 monoclonal antibody is shown in SEQ ID No.1 and the nucleotide sequence encoding the light chain variable region is shown in SEQ ID No. 3; the nucleotide sequence of the heavy chain variable region of the 2F7 monoclonal antibody is shown as SEQ ID NO.5, and the nucleotide sequence of the light chain variable region is shown as SEQ ID NO. 7.
  3. 3. An expression vector comprising the nucleic acid molecule of claim 2.
  4. 4. A host cell comprising the nucleic acid molecule of claim 2 or the expression vector of claim 3.
  5. 5. A kit for detecting Amuc _1100 protein comprising the specific monoclonal antibody pair of claim 1.
  6. 6. The kit of claim 5, wherein the kit is a colloidal gold detection kit, and the kit further comprises one or more of a sample extraction solution, a sample pad treatment solution, and a multiplex solution.
  7. 7. The kit of claim 5, wherein the kit is a double-antibody sandwich ELISA detection kit, and the kit further comprises one or more of an enzyme-labeled secondary antibody, a chromogenic solution, a stop solution, a washing solution, a blocking solution and a coating buffer solution.
  8. 8. A method for detecting Amuc _1100 protein, wherein the detection is performed using the kit of any one of claims 5 to 7.
  9. 9. Use of a specific monoclonal antibody pair according to claim 1 or a nucleic acid molecule according to claim 2 or an expression vector according to claim 3 or a host cell according to claim 4 or a kit according to any one of claims 5-7 for detecting Amuc _1100 protein in akkermansia mucilaginosa.
  10. 10. Use of a specific monoclonal antibody pair according to claim 1 or a nucleic acid molecule according to claim 2 or an expression vector according to claim 3 or a host cell according to claim 4 or a kit according to any one of claims 5-7 for quality control and activity assessment of a probiotic preparation of akkermansia mucilaginosa.

Description

Specific monoclonal antibody of AKK bacterium efficacy protein Amuc-1100, kit, detection method and application thereof Technical Field The invention belongs to the field of biological detection, and particularly relates to a specific monoclonal antibody of AKK bacterium efficacy protein Amuc-1100, a kit, a detection method and application thereof. Background Amuc _1100 protein is used as a core functional protein of mucus Ackermansia (AKKERMANSIA MUCINIPHILA, AKK bacteria for short), plays a key role in physiological processes such as intestinal microenvironment steady state regulation, host metabolism balance maintenance and the like, and related research has become an important direction in the fields of microbiology, clinical medicine and bioengineering. Along with the continuous deep research on the mechanism of AKK bacteria probiotics, the functional value of Amuc _1100 protein is continuously mined, so that a key target point is provided for analyzing the interaction between intestinal flora and a host, and a foundation is laid for the development of related functional products, and therefore, the technical requirements on the efficient preparation and accurate detection of the protein are increasingly urgent. In the aspect of protein preparation technology, the acquisition of the existing microorganism functional protein mainly depends on the extraction of natural strains or the preparation of a recombinant expression system. For Amuc-1100 protein, the culture condition of natural AKK bacteria is harsh, the growth period is long, the natural expression quantity of the protein in the strain is extremely low, the process is complex, time and labor are consumed, the extraction cost is high, the purity is difficult to control and the like when the protein is directly extracted from the natural strain, and the requirements of subsequent experiments and application on high-purity and large-scale protein cannot be met. While the conventional prokaryotic expression system is widely applied to the preparation of various recombinant proteins, specific problems are often faced to the expression of Amuc _1100 protein, such as low protein solubility expression proportion, easy formation of inclusion bodies of the expression products, easy loss of activity in the purification process and the like, so that the obtained recombinant protein is difficult to meet the requirements of subsequent experiments such as antibody preparation, functional verification and the like in terms of concentration, purity and activity, and the promotion of related researches is restricted. In the technical field of detection, the existing protein detection methods such as Western Blot, common ELISA and the like have obvious limitations when applied to Amuc _1100 protein detection. The Western Blot technology has the advantages of complicated operation, long detection period and poor quantitative accuracy, is only suitable for qualitative analysis of a small amount of samples, cannot meet the rapid quantitative detection requirement of a large amount of samples, and is difficult to realize accurate quantification and specific recognition of Amuc _1100 protein in samples due to insufficient detection sensitivity and poor specificity caused by the fact that a common ELISA method lacks a high-specificity and high-affinity exclusive antibody aiming at Amuc _1100 protein and is easy to cross-react with other intestinal flora proteins or hybrid proteins in biological samples. In addition, the application of the main stream high-efficiency immune detection technologies such as colloidal gold immunochromatography, double-antibody sandwich ELISA and the like in Amuc-1100 protein detection does not form a standardized scheme, the core antibody pairing screening lacks systematic optimization, and key parameters (such as antibody coating concentration, reaction temperature and time, buffer solution formula, sample processing conditions and the like) of a detection system are not explicitly and reasonably set, so that the reliability, stability and repeatability of the related detection technology are difficult to guarantee. The technical bottlenecks not only seriously prevent the deep development of basic researches such as physiological functions, action mechanisms and the like of Amuc _1100 protein, but also enable the activity evaluation and quality control of functional products (such as AKK bacteria probiotic preparations and related biological medicine products) based on the protein to lack effective technical means in the research and development process, and limit the industrialized transformation and market application of the functional products. Therefore, a complete technical system which covers Amuc-1100 protein efficient recombinant expression, high-purity purification, specific monoclonal antibody preparation and standardized detection methods is developed, the blank of the current protein special research tool can be filled, key tech