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GnRH-defensin recombinant castrated vaccine and preparation method thereof

A castration vaccine, defensin technology, applied in the field of molecular vaccinology, can solve problems such as difficult preservation

Active Publication Date: 2018-11-30
广州源博医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the above defensins are used alone as adjuvants. The nature of their polypeptides makes them easy to degrade and not easy to store. As a result, the prepared vaccines need protective agents and special packaging methods to exert the adjuvant effect.

Method used

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  • GnRH-defensin recombinant castrated vaccine and preparation method thereof
  • GnRH-defensin recombinant castrated vaccine and preparation method thereof
  • GnRH-defensin recombinant castrated vaccine and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] The construction method of GnRH-defensin vaccine expression vector: the construction of plasmid pET28a GnRH-HDP: the DNA sequence with Nde ǀ and EcoR ǀ restriction sites synthesized from Suzhou Jinweizhi Biotechnology Co., Ltd. (the sequence is SEQ ID No. 5 and SEQ ID No.6) freeze-dried powder was dissolved in sterilized ultrapure water according to the instructions, and the dissolved DNA and pET28a plasmid were subjected to double enzyme digestion with Nde ǀ and EcoR ǀ at the same time. The double enzyme digestion system is shown in the following table:

[0025]

[0026] Digest for 2 hours at 37°C, separate the target fragments by nucleic acid electrophoresis, cut off the target bands with a scalpel, and purify them with a gel recovery kit.

[0027] Connect the GnRH hexademer / GnRH-HDP gene fragment and the pET28a vector fragment according to the following table

[0028]

[0029] Ligated overnight at 4°C, and the ligated mixture was transformed into Escherichia co...

Embodiment 2

[0031] Induced expression and purification of GnRH hexadecanoid protein and GnRH-defensin vaccine protein:

[0032] 1. Preparation of bacteria: transform the above-mentioned plasmid pET28a GnRH hexadecameres and plasmid pET28a GnRH-HDP plasmid pET28aGnRH-HDP into Escherichia coli BL21 by conventional methods, + (50ug / ml) LB solid medium and cultivated overnight for one hour, picked a transformant, and named them BL21 / pET28a GnRH hexadexadecimal and BL21 / pET28aGnRH-defensin respectively. Incorporate BL21 / pET28a GnRH hexademer and BL21 / pET28a GnRH-defensin into kan + (50ug / ml) LB liquid medium for overnight culture; take 1ml of overnight culture solution, add 100mL fresh containing kan + (50ug / ml) LB liquid medium, shake culture at 200rpm at 37°C until the OD600 is 0.6, add IPTG with a final concentration of 0.4mM, continue to shake at 160rpm at 37°C for 2-4h, with 5000g, Centrifuge at 4°C for 10 min to collect the bacterial pellet.

[0033] 2. Bacteria breaking: add 5ml of ...

Embodiment 3

[0037] Vaccine application

[0038] Preparation of vaccine formulation: The vaccine was prepared with water-in-oil adjuvant.

[0039] GnRH-defensin vaccine protein expression is high, 0.1g GnRH-defensin vaccine protein can be obtained from 100ml bacterial liquid (dissolved according to 1g / 2ml) is 2ml, this vaccine has strong immunogenicity, 25ug / dose, can produce 400 tubes, according to each dose The dose is 100ul, so the total volume is 400ml, and the GnRH-defensin vaccine protein is finally diluted 200 times. Due to the high dilution factor, the inclusion body solution can be directly dropped into the water-in-oil adjuvant for dilution and refolding and simultaneously prepared into a vaccine.

[0040] Immunization application of GnRH-defensin vaccine: The ability of the GnRH-defensin vaccine to induce an immune response against GnRH will be tested in males and females (cats). Forty animals were divided into four groups, each group consisting of 10 animals (5 males and 5 fe...

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Abstract

The invention belongs to the technical field of the molecular biology and describes a fusion vaccine containing an encoded immunocontraceptive protein gonadotropin-releasing hormone (GnRH) and a heterologous defensin polypeptide (HDP). The invention discloses a method for construction, transformation expression and purification of the recombinant GnRH-HDP fusion protein vaccine. Through inoculation of animals, a contraceptive peptide-specific antibody is produced through immunization and controls the animal fertility. The GnRH-HDP defensin immunogenic composition can effectively control the fertility of the animals.

Description

technical field [0001] The invention relates to the field of molecular vaccinology, in particular to a GnRH-defensin recombinant castration vaccine and a preparation method thereof. Background technique [0002] The castration technique used for animals has always been surgical castration, which is very stressful to animals. It usually takes 7-10 days to recover, and sometimes pets die due to wound infection. In addition, the surgical castration of female pets not only has a higher risk of infection, but also requires high technical and physical strength of the operator. In addition, animal welfare activists have been pressuring to stop surgical castration in many countries. An alternative to surgical castration is to use GnRH castration vaccination to eliminate the influence of gonadotropin-releasing factor (GnRH, also known as LHRH) in the body, so that the animal cannot be sexually mature. Compared with surgical castration , Safe and convenient, reduce the chance of inf...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/00A61P15/16A61P15/18C07K19/00C12N15/70
CPCA61K39/0006A61K2039/552A61P15/16A61P15/18C07K7/23C07K14/4723C07K2319/00C12N15/70
Inventor 刘昕王弋皮川真汪业军林少妹
Owner 广州源博医药科技有限公司
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