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Connection method of paralytic shellfish poison and carrier protein

A carrier protein and paralytic technology, which is applied in the field of connection between paralytic shellfish poison and carrier protein, and can solve the problems of no shellfish poison connection and insufficient immunogenicity.

Inactive Publication Date: 2013-04-03
SHANGHAI BIOCHIP +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it is necessary to obtain specific antibodies against shellfish poison, and shellfish poison is a small molecular compound, which is not immunogenic enough to directly stimulate the body to produce specific antibodies. It needs to be linked with a carrier protein to immunize animals. Currently, there is no shellfish poison C1&C2 and carrier Successful case of protein ligation

Method used

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  • Connection method of paralytic shellfish poison and carrier protein
  • Connection method of paralytic shellfish poison and carrier protein

Examples

Experimental program
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Effect test

Embodiment approach

[0031] As a preferred embodiment of the present invention, the method includes: dissolving 1 mg of paralytic shellfish poison C1 & C2 in 500 ul of coupling buffer with a pH value of 5.0-6.0, adding 1-10 mg of carrier protein, mixing, and then adding 37% formaldehyde Solution 20ul, mixed reaction for 72 hours; and paralytic shellfish poison C1 & C2 and carrier protein coupling product into a dialysis bag, dialyzed with pH7.2 PBS solution at 4 ° C for 72 hours, during which the dialysate was changed 3 times. Finally, a step of identifying the dialysis product by electrophoresis may also be included.

[0032] The inventors found in experiments that an appropriate pH value is critical for the successful coupling of paralytic shellfish poisons C1 & C2 to carrier proteins. When the pH is lower than 5, the coupling efficiency is very low, and it is difficult to obtain stable coupling products.

[0033] In the present invention, the C1&C2-carrier protein coupling product prepared by ...

Embodiment 1

[0039] Example 1, the extraction and purification of paralytic shellfish toxin

[0040] 1. Reagents and materials

[0041] Eluent: 2mM tetrabutylammonium phosphate solution, adjust the pH to 6.0.

[0042] Oxidant: 50mM dipotassium hydrogen phosphate buffer containing 7mM periodate, pH 9.0;

[0043] Acidifying agent: 0.5M acetic acid solution.

[0044] Other conventional chemical reagents: glacial acetic acid, acetone, absolute ethanol.

[0045] 2. Culture of algae

[0046] Alexandrium.Tamarense is used for the production of toxin C (paralytic shellfish poison C1 & C2). Take 25ml of algae liquid, centrifuge at 4000g, 20°C for 10min, discard the supernatant, add 500ul of 50mM acetic acid to the lower layer of algae cells and store at -20°C or extract the toxin immediately, and determine the toxin content by HPLC analysis. When the toxin content reached the highest level, the algae cells were collected with a 10-micron sieve and stored at -20°C for the next step of extractin...

Embodiment 2

[0066] Embodiment 2, the coupling method of paralytic shellfish poison C1&C2 and carrier protein

[0067] (1) Dissolve 1mg of paralytic shellfish poison C1&C2 in 500ul of coupling buffer (weigh 16.4g of sodium acetate, dissolve in 1000ml of deionized water, adjust the pH value to 5.0 with glacial acetic acid), add 5mg of carrier protein BSA, mix Uniformly, then add 20ul of 37% formaldehyde solution, mix and react for 72 hours.

[0068] (2) The paralytic shellfish poison C1&C2-carrier protein coupling product was put into a dialysis bag, and dialyzed with PBS solution of pH 7.2 at 4°C for 72 hours, during which the dialysate was changed 3 times.

[0069] (3) The paralytic shellfish poison C1&C2-carrier protein coupling product after dialysis is identified by electrophoresis, and it is determined that the successfully coupled protein has been obtained efficiently, such as figure 1 . From figure 1 It can be seen from the figure that in the sample after the reaction, there are ...

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Abstract

The invention relates to a connection method of paralytic shellfish poison and carrier protein. By adopting the connection method, a coupling product of the paralytic shellfish poison C1 and C2 and the carrier protein under stable coupling can be obtained, thus an effective way is provided for preparing a paralytic shellfish poison antibody and immunologically detecting and analyzing paralytic shellfish poison C1 and C2.

Description

technical field [0001] The invention belongs to the field of biotechnology; more specifically, the invention relates to a connection method between paralytic shellfish poison and carrier protein. Background technique [0002] Since the 1950s, marine red tides have occurred frequently, and the hazards caused by red tide toxins to humans have also increased. At present, there are nearly 20 species of dinoflagellates and a small number of diatoms in red tides that can produce phycoxanthin. Shellfish feed on toxic microalgae (mainly phycoxanthin), and bioaccumulate and magnify into organic toxins, namely shellfish poisons. The hazards of shellfish poisoning are sudden and widespread. Because of its high toxicity, quick response and lack of suitable antidotes, many difficulties have been brought to the prevention and control. Therefore, it is of great significance to carry out research on shellfish poisoning to ensure the safety of aquatic products and human life and property. ...

Claims

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Application Information

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IPC IPC(8): C07K14/795C07K14/765C07K14/77C07K1/107C07K16/44G01N33/53
Inventor 邵钧吴杨蔡兴锋
Owner SHANGHAI BIOCHIP
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