Application of gram-positive bacterium expression system in expression of clostridium septium toxin, preparation method of clostridium septium alpha toxin and vaccine

A technology for Gram-positive bacteria and Clostridium putrefaction, applied in the direction of microorganism-based methods, biochemical equipment and methods, chemical instruments and methods, etc., can solve the problems of inclusion bodies without biological activity, complex endotoxin removal process, production High cost and other problems, to avoid complex production process, high protein expression and protein purity, and low production cost

Active Publication Date: 2020-11-17
天康制药股份有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In these reports, IPTG was used as the inducer to express in the E. coli system, and it was necessary to detect the growth of the recombinant E. coli and add the inducer; the α-toxin recombinant protein was expressed intracellularly, and complex procedures such as sonication and nickel column purification were required. process
Although the Escherichia coli expression system has the advantages of clear genetic background, easy operation, short production cycle, high expression level, low cost, and easy large-scale cultivation, it is the most commonly used and most economical exogenous protein expression system, but its expression of exogenous Proteins are usually expressed intracellularly as insoluble inclusion bodies or intracellular soluble proteins. Inclusion bodies do not have biological activity and need to be denatured and refolded. Intracellular soluble proteins are biologically active but contain more miscellaneous proteins. Complicated purification process is required; since Escherichia coli is a negative bacterium and contains a large amount of LPS (endotoxin), which causes vaccine side effects, endotoxin needs to be removed in vaccine production, but the endotoxin removal process is complicated and the production cost is high , which is currently a difficult point in the production of veterinary biological products

Method used

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  • Application of gram-positive bacterium expression system in expression of clostridium septium toxin, preparation method of clostridium septium alpha toxin and vaccine
  • Application of gram-positive bacterium expression system in expression of clostridium septium toxin, preparation method of clostridium septium alpha toxin and vaccine
  • Application of gram-positive bacterium expression system in expression of clostridium septium toxin, preparation method of clostridium septium alpha toxin and vaccine

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Experimental program
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Effect test

Embodiment 1

[0062] 1. Construction of pYL expression plasmid:

[0063] 1.1 Synthetic promoter, ori region, and Erm resistance gene: According to the requirements for expression vectors, Xyl / tet fragments, ori regions, and Erm resistance genes that can be screened with antibiotics are artificially chemically synthesized using tetracycline for expression regulation.

[0064] 1.2 Construction of PYL expression plasmid: Insert the gene fragment synthesized in the above 1.1 into the PRB373 plasmid by molecular biology method, the sequence is correct, and the expression plasmid PYL is obtained. The plasmid spectrum is as follows Figure 8 shown.

[0065] 2. Construction of Clostridium putrefactive alpha toxin mutants:

[0066]The amino acid sequence of the wild-type Clostridium putrefaciens alpha toxin gene as shown in SEQ ID NO.3 is optimized, the 86th cysteine ​​(C) is mutated into leucine (L), and the 296th asparagine (N ) is mutated into alanine (A), and the gene fragment is obtained thro...

Embodiment 2

[0079] Expression of Clostridium putrefaction alpha toxin mutants:

[0080] The recombinant staphylococcus epidermidis SE / PYL-ATX that embodiment 1 prepares C86L-N296A Inoculate in TSB liquid medium (containing 5 μg / ml Erm), the inoculation ratio is 1%, and induce the expression of the target protein. The induction conditions are: the final concentration of the inducer ATC is 300ng / ml, the induction temperature is 36-37°C, and the rotation speed of the incubator is 200r / m. min, culture time 18-24h. After induced expression culture, the supernatant was collected by high-speed centrifugation, and then the target protein in the supernatant was detected by SDS-PAGE and identified by Western Blot. The results of SDS-PAGE detection are as follows image 3 as shown, image 3 The middle lane M is the protein molecular weight marker; lanes a to d are the concentrations of 1000 μg / ml BSA, 500 μg / ml BSA, 250 μg / ml BSA and 125 μg / ml BSA in sequence; lane 1 is the α toxin mutant of Clos...

Embodiment 3

[0083] rATX protein mouse virulence experiment, the experimental method refers to "People's Republic of China Veterinary Pharmacopoeia" (2015 edition), including the following steps: select 18 ± 2g mice, divide them into 6 groups according to the way of random grouping, each group has 5 small mouse. The 6 groups include three experimental groups, negative control group, positive control group and blank control group. The three experimental groups were given 1μg, 10μg and 100μgrATX protein respectively; the negative control was given medium; the positive control was given 1MLD of natural Clostridium putrefaction alpha toxin. The administration method is tail vein injection, and the sample is diluted with gelatin buffer solution at the time of injection. The results of the experiment were: all the mice in the positive control group died, and the mice in the negative control group and the experimental group survived.

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Abstract

The invention provides application of a gram-positive bacterium expression system in expression of clostridium septium toxin, a preparation method of clostridium septium alpha toxin and a vaccine, andrelates to the technical field of biology. Extracellular secretion expression of the clostridium septium toxin is realized by expressing the clostridium septium toxin by adopting a gram-positive bacterium expression system, and supernate is collected centrifugally, so that a target protein with high expression quantity and relatively good purity can be obtained, and the production cost of a target protein purification and endotoxin removal process is reduced.

Description

technical field [0001] The invention relates to the technical field of biological products, in particular to an application of a Gram-positive bacteria expression system in expressing Clostridium putrefaction toxin, a preparation method of Clostridium putrefaction alpha toxin and a vaccine. Background technique [0002] Clostridium septium is a Gram-positive anaerobic bacillus that can cause malignant edema, muscle necrosis, gas gangrene, and necrotic enteritis in animals and humans. It is also the pathogen that causes sheep epidemics. [0003] Clostridium putrefaction can secrete various exotoxins such as α, β and γ, among which α toxin is the main pathogenic virulence factor and immune protective antigen. Clostridium putrefaciens α-toxin is one of the perforating toxins of the aerolysin family, and is the main lethal virulence factor of Clostridium putrefaciens, which has the effects of hemolysis, lethality and necrosis. The α-toxin secreted by Clostridium putrefaction ha...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/77C12N15/75C12N15/74C07K14/33C12N1/21G01N33/569G01N33/577A61K39/08A61P31/04C12R1/45C12R1/125C12R1/01
CPCC12N15/77C12N15/75C12N15/74C07K14/33G01N33/56911G01N33/577A61K39/08A61P31/04G01N2333/33G01N2469/20Y02A50/30
Inventor 贺笋王钢张飞何海丁占强周婷婷韩瑞凯杜晓杰李延涛
Owner 天康制药股份有限公司
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