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A kind of non-toxic Clostridium emphysematous gene engineering subunit vaccine strain and its application

A subunit vaccine and genetic engineering technology, applied in the field of veterinary biological products, can solve the problems of incompleteness, leakage and inactivation of bacterial strains, local inflammation and toxicity of animals, and achieve short time-consuming, good stability and good immunity Antigenic and immunoprotective effects

Active Publication Date: 2021-06-01
CHINA INST OF VETERINARY DRUG CONTROL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the vaccine has achieved certain effects in preventing animal emphysema, some defects are still exposed during the use of the vaccine. For example, vaccine immunization can easily cause local inflammation and toxic reactions in animals; The inactivation of Clostridium anthracis has biological safety hazards such as strain leakage or incomplete inactivation

Method used

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  • A kind of non-toxic Clostridium emphysematous gene engineering subunit vaccine strain and its application
  • A kind of non-toxic Clostridium emphysematous gene engineering subunit vaccine strain and its application
  • A kind of non-toxic Clostridium emphysematous gene engineering subunit vaccine strain and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Embodiment 1——Construction, expression and identification of Escherichia coli BL / MA strain

[0071] 1. Gene synthesis

[0072] According to the natural gene sequence of emphysematous gangrene FliA (C) (sequence 1) and CctA (sequence 3), after codon optimization, the coding gene containing FliA (C) and the middle region containing CctA (sequence 3) 95th to 261st amino acid) encoding genes were concatenated. At the same time, the amino acid tag coding sequence used for purification is added to the 3' end of the tandem gene, and the gene sequence GFliACctA is synthesized by chemical synthesis N (Sequence 5). See SEQ ID NO: 6 for the amino acid sequence.

[0073] sequence 1

[0074]

[0075] sequence 3

[0076]

[0077]

[0078] sequence 5

[0079]

[0080] sequence 6

[0081]

[0082]

[0083] 2. Construction of recombinant fusion protein expression vector

[0084] Synthetic GTTc-Tcnα nc As a template, the primer pair 1F / 1R (SEQ ID NO:7 / SEQ ID NO...

Embodiment 2

[0093] Example 2 - rFliACctA N expression and identification of

[0094] 1. rFliACctA N expression

[0095] Inoculate Escherichia coli (E.coli) BL / MA strain in 4 mL of LB liquid medium containing kanamycin, place at 37°C, when OD 600 When the temperature is 0.6-0.8, add IPTG solution with a final concentration of 0.5mM and place at 37°C and 15°C for induction and culture for 4h and 16h, respectively. After the bacterial culture is completed, collect the bacterial cells by centrifugation, add 10ml of lysate [0.02mol / LTris buffer (pH value 7.2), 0.3mol / LNaCl] to resuspend the bacterial cells according to the weight of each gram of bacterial body temperature, and sonicate in an ice-water bath The bacteria were crushed for 30 minutes, and the crushing conditions were: working for 9 seconds, resting for 9 seconds, and the ultrasonic power was 400W. The crushed bacterial liquid was centrifuged at 12000r / min for 10min at 4°C, and the supernatant was collected. Take 30 μL of the ...

Embodiment 3

[0098] Example 3 - rFliACctA N purification of

[0099] Inoculate the BL / MA strain of Escherichia coli in 1L LB liquid medium containing kanamycin for fermentation culture, shake culture at 37°C OD 600When the temperature is 0.6-0.8, lower the temperature to 15°C, and add IPTG solution with a final concentration of 0.5mM to induce culture for 16h. After the bacterial culture was completed, the bacterial cells were collected by centrifugation at 5000r / min for 5min, and the bacterial cells were resuspended at the ratio of adding 10ml of lysate (pH value 7.20.02mol / LTris buffer, 0.3mol / LNaCl) per gram of bacterial cell wet weight, Under the condition of 4°C, a low-temperature high-pressure homogenizer was used to disrupt the bacterial cells three times at a pressure of 800 bar. The lysate was centrifuged at 10,000 r / min at 4°C for 30 min, and the supernatant was collected. According to the instructions of the Ni-IDA affinity chromatography medium kit, the soluble expressed rFl...

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Abstract

The present invention relates to a non-toxic Clostridium aeruginosa genetically engineered subunit vaccine and its application. The strain used for the non-toxic Clostridium aeruginosa genetically engineered subunit vaccine prepared by the present invention is recombinantly expressed and codon-optimized. Escherichia coli, which is a recombinant fusion protein of Clostridium tumefaciens flagellin and cytotoxin A, and the genetically engineered subunit vaccine of Clostridium aeruginosa produced by this strain can ensure the safety of the vaccine to the greatest extent while maintaining its effectiveness. At the same time, the vaccine antigen expressed by this strain can be expressed in a soluble form. On the one hand, it avoids the impact of the complex process of inclusion body denaturation and renaturation on the immunogenicity of the antigen protein, and reduces the preparation time and production cost of the target protein; On the one hand, it can improve the antigenicity of the fusion protein, so it has the advantages of simple preparation process, low immune dose, good immune effect, etc., and is an ideal candidate vaccine for upgrading the current Clostridium emphysema vaccine in my country.

Description

technical field [0001] The invention relates to a strain for a non-toxic Clostridium aeruginosa gene engineering subunit vaccine and its application. It belongs to the field of veterinary biological products. Background technique [0002] Clostridium emphysema is also commonly known as black leg disease, mainly infecting ruminants, especially grazing newborn ruminants are most sensitive to Clostridium emphysema. The disease has a short course of disease, acute onset, and sudden death before treatment. For this reason, vaccination is the main means of preventing and controlling the disease, and the commercialized canker sore vaccines are mainly inactivated vaccines of two virulent strains, C54-1 and C54-2. Although the vaccine has achieved certain effects in preventing animal emphysema, some defects are still exposed during the use of the vaccine. For example, vaccine immunization is easy to cause local inflammation and toxic reactions in animals; The inactivation of Clost...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/70C12N15/62A61K39/08A61P31/04C12R1/19
CPCA61K39/08A61K2039/523A61K2039/552A61P31/04C07K14/33C07K2319/00C12N15/70
Inventor 杜吉革陈小云刘莹彭小兵薛麒朱真李启红印春生姚文生康凯
Owner CHINA INST OF VETERINARY DRUG CONTROL
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