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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 and main protein technology, applied in the field of royal jelly main protein MRJP1 specific antibody and its preparation and Elisa quantitative detection, can solve the problems of lack of MRJP1 detection indicators and detection methods, and achieve strong specificity

Active Publication Date: 2014-07-30
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • 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 royal jelly major protein MRJP1 specific antibody and its preparation method and Elisa quantitative detection. Firstly, the amino acid sequences of all members of the royal jelly major protein MRJPs family (MRJP1-MRJP9) of the Italian honeybee (Apismellifera) were homologously analyzed, and the amino acid sequences of specific polypeptides of MRJP1 different from other MRJPs family members were selected. The related specific peptides were synthesized by chemical method, used as antigens, New Zealand white rabbits were immunized, the serum was collected, the polyclonal antibody R2 with high titer was detected by Elisa, and the antibody was purified by the affinity column prepared by the synthetic MRJP1 polypeptide. The titer of antibody R2 was detected by using MRJP1 as the antigen Elisa method, and the titer of the antibody was >1:20000. The present invention provides a very reliable and fast new detection method for the qualitative and quantitative detection of MRJP1 in royal jelly, and also provides quality control for bee product quality supervision departments, processing trade enterprises royal jelly and honey products, freshness detection, and identification of genuine and fake products. Very reliable technical means.

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 Patents(China)
IPC IPC(8): C07K16/18
Inventor 沈立荣柳丹丹于张颖肖发李玫璐张瑮文
Owner ZHEJIANG UNIV