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Shewanella-smarisflavi-resistant egg yolk antibody and preparation method thereof

A technology of Shewanella and egg yolk antibodies, which is applied in the fields of antibodies, food preparation, antibacterial drugs, etc., to achieve the effect of low cost, convenient and low cost, and simple and easy preparation methods

Active Publication Date: 2015-12-30
DALIAN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the yolk antibody has been widely concerned and applied in animal husbandry, but the research and development in the aquaculture industry is still in the basic stage. At present, the patents applied for publication and authorization in aquaculture include anti-prawn virus, anti-Vibrio anguillarum, anti- parahaemolyticus and anti-Aeromonas hydrophila yolk antibody, but there is no report on the anti-Shewanella yellowsea yolk antibody and its preparation method

Method used

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  • Shewanella-smarisflavi-resistant egg yolk antibody and preparation method thereof
  • Shewanella-smarisflavi-resistant egg yolk antibody and preparation method thereof
  • Shewanella-smarisflavi-resistant egg yolk antibody and preparation method thereof

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Effect test

Embodiment 1

[0027] A preparation method for anti-Shewanella yellowsea yolk antibody, comprising the following steps:

[0028] (1) Inoculate Shewanella marisflavi with the deposit number CGMCC No. 7303 in 20 ml of 2216E liquid medium, culture at 28° C. for 12 hours, and revive the bacteria. After recovery of the bacteria, inoculate 100 μL of the bacterial liquid into the TSA solid agar medium, incubate it upside down at 28°C for 24 hours, pick a single colony and inoculate it in the 2216E liquid medium, and culture it on a shaking table at 28°C and 140 rpm for 10 hours , adjust the amount of Shewanella yellowsea bacteria in 2216E liquid medium to be 3×10 9CFU / mL. Afterwards, 8000r / min, centrifuge at 4°C for 5min to collect the bacterial cells. The collected bacteria were resuspended with PBS, centrifuged at 8000r / min for 5min, the supernatant was discarded, the pellet was resuspended with PBS, and this process was repeated three times. Then add formaldehyde with a volume fraction of 0.5...

Embodiment 2

[0036] In vitro agglutination test of anti-Shewanella yellowsea egg yolk antibody

[0037] Dilute the pure culture of Shewanella flavumii to 10 6 CFU / mL, take 20ml and divide into five 50mL centrifuge tubes, then add 20ml of 0mg / ml, 0.5mg / ml, 1mg / ml, 5mg / mL Huang Haixi after filtration and sterilization with 0.22μm microporous membrane Varnella specific egg yolk antibody (dissolved in PBS) and 5 mg / ml non-specific egg yolk antibody (dissolved in PBS), cultivated on a shaker at 26°C, took 100 μL of coated TSA agar plate every two hours, and set 1 replicate for each , after 24 hours of inverted culture at 26°C, the cells were photographed and counted.

[0038] The results of agglutination experiments showed that ( image 3 ): Adding a group of specific yolk antibodies can effectively agglutinate Shewanella flavus, which supports the mechanism of action of yolk antibodies reported in the literature, and the agglutination is dose-dependent.

Embodiment 3

[0040] Specificity of anti-Shewanella yellowsea egg yolk antibody detected by transmission electron microscope

[0041] Dissolve the lyophilized powder of anti-Shewanella yellowsea yolk antibody in sterile PBS (pH7.2) at a concentration of 1mg / mL, pass through a 0.22μm microporous membrane to sterilize and add Shewanella yellowsea to make the bacterial concentration for 10 8 cfu / ml. The same concentration of non-specific IgY was used as control. The above mixture was incubated at 37°C for 2h, centrifuged at 5000rpm for 5min, and the pellet was washed twice with PBS. The pellet was resuspended in 400 μL colloidal gold-labeled rabbit anti-chicken IgG (1:30 dilution) in PBS dilution, incubated at 37°C for 2 h, and the sample was centrifuged and washed. 10 μL of the obtained resuspension solution was fixed on the copper grid used for the observation of the transmission electron microscope sample, and after natural drying, the sample was observed under the transmission electron ...

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Abstract

The invention discloses a shewanella-smarisflavi-resistant egg yolk antibody and a preparation method thereof. The shewanella-smarisflavi-resistant egg yolk antibody is obtained by immunizing a laying hen by using inactivated shewanella smarisflavi as an antigen, collecting eggs of the immunized laying hen, separating yolk to obtain a water-soluble component, and separating and purifying to obtain the shewanella-smarisflavi-resistant egg yolk antibody. The shewanella-smarisflavi-resistant egg yolk antibody has features of good specificity and high titer, is used for preventing diseases caused by shewanella smarisflavi and immunological detection, and has good effects. In a preparation process of the egg yolk antibody, blood sampling is not required, immunized animals are not injured, and modern animal rights protection rules are met; and each egg contains more than 100mg of the vibrio-splendidus-resistant egg yolk antibody approximately, and 3g of the vibrio-splendidus-resistant egg yolk antibody can be obtained in a month, and is 10-20 times greater than an antibody extracted from blood of an immunized rabbit. The shewanella-smarisflavi-resistant egg yolk antibody has the advantages of safety, environmental protection, high efficiency, low cost and the like, and is easy to industrialize.

Description

technical field [0001] The invention relates to the field of preparation technology and application of biological products, in particular to a preparation method of an anti-Shewanella yellow sea yolk antibody. Background technique [0002] Shewanella marflavi was named by Jung-Hoon Yoon in 2004 and belongs to the genus Shewanella. Shewanella is one of the major marine pathogens. It is reported that Shewanella yellowsea can infect marine organisms such as oysters and sea cucumbers, and is a highly harmful strain. [0003] At present, the most important means of prevention and treatment of diseases caused by Shewanella yellow sea are the following: antibiotics, Chinese herbal medicines, monoclonal antibodies, inactivated vaccines, etc. Antibiotics and chemical drugs are easy to cause stress reactions, have different degrees of toxicity to organisms, and are prone to drug resistance after repeated use, and there are drug residues. Today, when advocating green agriculture, the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/12C07K16/02A61K39/40A61P31/04A23L1/305A23K1/16G01N33/569
Inventor 徐永平徐乐李晓宇王丽丽李淑英
Owner DALIAN UNIV OF TECH
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