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A specific antibody of major royal jelly protein MRJP1 and a preparation method thereof and Elisa quantitative detection thereof

A main protein and specific technology, applied in the field of royal jelly main protein MRJP1 specific antibody and its preparation and Elisa quantitative detection, can solve the problem of lack of MRJP1 detection index and detection method, and achieve the effect of strong specificity

Active Publication Date: 2013-04-24
ZHEJIANG UNIV
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  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although many attempts have been made, the current national and international standards for royal jelly still lack MRJP1 detection indicators and detection methods

Method used

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  • A specific antibody of major royal jelly protein MRJP1 and a preparation method thereof and Elisa quantitative detection thereof
  • A specific antibody of major royal jelly protein MRJP1 and a preparation method thereof and Elisa quantitative detection thereof
  • A specific antibody of major royal jelly protein MRJP1 and a preparation method thereof and Elisa quantitative detection thereof

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0033] The preparation method of royal jelly major protein MRJP1 specific antibody R2 comprises the following steps:

[0034] (1) Retrieve and download Apis mellifera from International GenBank ( Apis mellifera ) The amino acid sequences encoded by all 9 members (MRJP1-MRJP9) genes of the royal jelly major protein MRJPs family were analyzed by using the bioinformatics analysis software GENTYX, and a unique polypeptide of MRJP1 was selected according to the sequence information. These polypeptides are located at positions 51-57 (M1: QDAILSG), 73-80 (M2: HDKIFVTM), 340-346 (M3: NIRTVAQ), 360-371 (M4 :IKEALPHVPIFD). The common feature of the selected polypeptides is that the selected MRJP1 polypeptides share no more than 3 consecutive homologous amino acid residues with other 8 MRJPs members.

[0035] (2) New Zealand white rabbits were immunized with chemically synthesized MRJP1-specific polypeptide M4 to prepare polyclonal antibodies, including the following sub-steps:

[00...

Embodiment 1

[0070] Example 1: Screening of MRJP1-specific polypeptides

[0071] Log in to the international gene information database http: / / www.ncbi.nlm.nih.gov / , search and download the amino acid sequences encoded by the genes of the 9 members of the MRJPs family (MRJP1-MRJP9), the sequence number of MRJP1 is NM_001011579, and the sequence number of MRJP2 为NM_001011580、MRJP3的序列号为NM_001011601、MRJP4的序列号为NM_001011610、MRJP5的序列号为NM_001011599、MRJP6的序列号为NM_001011622、MRJP7的序列号为NM_001014429、MRJP8的序列号为NM_001011564、MRJP9的序列号为 NM_001024697. Input the amino acid sequence encoded by the MRJP1-MRJP9 gene of the above sequence into the GENTYX software for homology analysis. After the analysis is completed, the output is as follows figure 1 The homology analysis diagram shown ( figure 1 ).

[0072] according to figure 1 As a result of homology analysis, the specific polypeptides of MRJP1 were screened out, and these polypeptides were located at positions 51-57 (M1: QDAILSG), positions 73-80 (M2: HD...

Embodiment 2

[0073] Example 2: Preparation of MRJP1-specific polyclonal antibody

[0074] 1. Antigen Preparation

[0075] The M4 polypeptide sequence (IKEALPHVPIFD) in the example was selected for chemical synthesis. Conjugate 5 mg of synthetic peptide with KLH (the coupling agent is Sulfo-SMCC) as the immune antigen, and another 5 mg of the synthetic peptide is coupled with BSA (the coupling agent is glutaraldehyde) as the detection antigen; dilute with PBS respectively To a concentration of 1 mg / ml, aliquot and store at -20°C.

[0076] 2. Immunization of Rabbits

[0077] Two healthy New Zealand white rabbits were selected and immunized on days 1, 15, 29, and 43. The immunization procedure is as follows: Take 1 ml of immune antigen, add 1 ml of Freund’s complete adjuvant, emulsify, drop a drop of emulsified antigen solution into physiological saline, and inject it at multiple points (at least 8 points) subcutaneously on the back of the white rabbit’s neck with a syringe; On...

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Abstract

The invention discloses a specific antibody of major royal jelly protein MRJP1 and a preparation method thereof and Elisa quantitative detection thereof. First, homology analysis is performed to amino acid sequences of proteins of all members of the Apismellifera major royal jelly protein MRJPs family (MRJP1-MRJP9) to select a specific polypeptide amino acid sequence unlike other MRJPs family members in MRJP1. The related specific polypeptide is synthesized by using a chemical method, and is used as an antigen to immunize New Zealand white rabbits; taking serum, and performing Elisa assay obtaining polyclonal antibody R2 with a relatively high titer, then purifying the antibody by using an affinity column prepared from the synthesized MRJP1 polypeptide. The titer of antibody R2 is detected via Elisa assay by using MRJP1 as the antigen, and the titer of the antibody is greater than 1:20000. The present invention provides a very reliable new rapid detection method for the qualitative and quantitative detection of MRJP1 in royal jelly, and also provides a very reliable technical means for quality control, freshness detection, and identification of genuine products of royal jelly and honey products for bee product quality supervision departments and processing and trading enterprises.

Description

technical field [0001] The invention relates to a royal jelly major protein MRJP1 specific antibody, its preparation method and Elisa quantitative detection. Background technique [0002] The physical and chemical indicators stipulated in the current "National Standard of the People's Republic of China-Royal Jelly" (GB 9697-2008) are: moisture (67.5% for superior products, 69.0% for qualified products), 10-hydroxy-2-decenoic acid (10-hydroxy- 2-decenoic acid, referred to as 10-HDA; high-grade product content 1.8%, qualified product content 1.4%), protein (11-16%), total sugar (15%), ash (1.5%), acidity and starch. But among these indicators, except 10-HDA, other indicators can only reflect the content of a certain substance in bees, and cannot truly reflect the quality and authenticity of royal jelly. 10-HDA is a unique active ingredient of royal jelly, and its content has long been used as the most important indicator of quality and authenticity in the global royal jelly t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18C07K16/06G01N33/68G01N33/543
Inventor 沈立荣柳丹丹于张颖肖发李玫璐张瑮文
Owner ZHEJIANG UNIV
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