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Preparation method of apis cerana antimicrobial peptide AccRoyalisin polyclonal antibody and use thereof

A polyclonal antibody, the technology of Apis cerana, applied in the fields of genetic engineering and immunology, achieves the effect of simple operation, high sensitivity and low cost

Inactive Publication Date: 2013-04-17
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, Royalisin, in addition to reports on the Royalisin gene sequence and amino acid sequence of Apis mellifera, and the antibacterial test of Royalisin isolated from natural royal jelly, there is no use of Royalisin antibodies to detect Royalisin in bee species and bee colony hemolymph at home and abroad. content, and Royalisin content in royal jelly, as the basis for evaluating the disease resistance of bee species and bee colonies, and the antiseptic performance of royal jelly

Method used

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  • Preparation method of apis cerana antimicrobial peptide AccRoyalisin polyclonal antibody and use thereof
  • Preparation method of apis cerana antimicrobial peptide AccRoyalisin polyclonal antibody and use thereof
  • Preparation method of apis cerana antimicrobial peptide AccRoyalisin polyclonal antibody and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Example 1 Recombinant expression of AccRoyalisin gene and separation and purification of fusion expression product

[0067] 1. Recombinant expression and identification of AccRoyalisin gene

[0068] Using the bee head cDNA library plasmid containing the AccRoyalisin gene (GenBank acceptance number: EF660337) as a template, its precursor fragment was amplified by PCR and cloned into the expression vector pGEX-4T-2. The recombinant pGEX-4T-2 / Accroyalisin obtained by screening was transformed into Escherichia coli BL21 and fused with glutathione (GST) for expression. Both the antimicrobial peptide AccRoyalisin and AccRoyalisin precursor of royal jelly and the precursor of Apis mellifera antimicrobial peptide AmRoyalisin are composed of 95 amino acid residues, with a homology of 90%-92%; the mature peptides of both are composed of 51 amino acid residues , only 3-4 amino acid residues are different, and the homology is above 90%.

[0069] The recombinant bacteria were...

Embodiment 2

[0075] Example 2 Preparation of GST-AccRoyalisin polyclonal antibody

[0076] Take the GST-AccRoyalisin purified and eluted by AKTA column, put it into a dialysis bag, dialyze overnight, transfer the dialysis desalted solution into a 1.5 mL eppendorf tube, freeze-dry, dissolve the purified protein with 0.9% normal saline, and use as Antigen back up.

[0077] Two healthy male rabbits aged 14-16 weeks were selected, and each rabbit was injected with about 0.5 mL of the purified fusion protein antigen by subcutaneous injection. Before immunization, blood samples were drawn from rabbit ears as negative controls. The fusion protein antigen was dissolved in 700 μL of normal saline, emulsified with 700 μL of Freund’s complete adjuvant and appropriate amount of penicillin and streptomycin, and injected subcutaneously at multiple points for immunization. Three weeks later, a booster immunization was performed with the same antigen, adjuvant and penicillin emulsion. After 5 weeks, ...

Embodiment 3

[0078] Example 3 Detection of GST-AccRoyalisin polyclonal antibody titer by Elisa method

[0079] Using the recombinant GST-AccRoyalisin isolated in Example 1 as the antigen, and the GST-AccRoyalisin antibody prepared in Example 2 as the primary antibody, follow the steps below:

[0080] (1) Coating: use the recombinant GST-AccRoyalisin described in Example 1 as the antigen, and use carbonate buffer (CBS, Na 2 CO 3 1.59g, NaHCO 3 2.93g,ddH 2 O 950 ml, adjust pH to 9.6, add ddH 2 O dilute to 1000 ml, store at 4°C), dilute to 1 μg / ml, add 100 μl / well into a 96-well plate, and store overnight at 4°C;

[0081](2) Blocking: Discard the coating solution in the small wells of the enzyme-linked plate the next day, use PBST (PBS+0.05% Tween-20) as the washing solution, wash 5-10 times in each step, and pat dry. Then add 100 μL of PBST solution containing 5% BSA or skimmed milk powder to block at 37 °C for 1.5 h.

[0082] (3) Discard the blocking solution in the small wells of...

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Abstract

The invention discloses a preparation method of an apis cerana antimicrobial peptide AccRoyalisin polyclonal antibody and the use thereof. Apis cerana royal jelly antimicrobial peptide AccRoyalisin recombined and expressed by escherichia coli is taken as an antigen, and a big rabbit is immunized to prepare a polyclonal antibody, the valence of the antibody is larger than 40,000 times, the polyclonal antibody is taken as a primary antibody, recombined and expressed GST-AccRoyalisin is taken as a standard protein antibody to perform doubling dilution, the absorbance of GST-AccRoyalisin protein samples with different concentrations is detected through an Elisa method by a microplate reader, and the standard curve between the absorbance and the MRJPI protein concentration is established. Different bee species or different bee swarms of hemolymph and lyophilized royal jelly powder serve as antibodies after being diluted, and the absorbance of the obtained sample is detected through adopting the Elisa method, and is introduced into the established standard curve to calculate the content of Royalisin of the obtained sample. The invention provides the new use for the Royalisin antibody, and provides a novel quick, qualitative and quantitative detection method for evaluating premunition of the bee species and bee swarms in the bee-keeping field, breeding the antiviral bee swarms and evaluating the antiseptic and antibiosis performance of royal jelly.

Description

technical field [0001] The present invention relates to the field of genetic engineering and immunology, particularly, the present invention relates to a kind of Apis cerana cerana ) A polyclonal antibody prepared from the fusion expression product of the royal jelly antimicrobial peptide AccRoyalisin gene, GST-AccRoyalisin. More specifically, the present invention relates to the preparation method of AccRoyalisin antibody, and using the antibody Elisa method to detect the Royalisin content in the hemolymph of Apis mellifera and Apis mellifera, as a basis for judging the disease resistance of bee species or bee colonies; and using the antibody Elisa The content of Royalisin in royal jelly was detected by this method, which was used as the basis for evaluating the antiseptic performance of royal jelly. Background technique [0002] Royal jelly is a biologically active substance secreted by the royal jelly glands on the head of bees. It has many functions such as promoting hu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18C07K16/06C07K19/00C12N15/12C12N15/70G01N33/68G01N33/53
Inventor 沈立荣李玫璐王一然谭量量金凤丁美会陈正贤
Owner ZHEJIANG UNIV
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