KR20200082118A - Recombinant vector, transformed E.coli, vaccine composition comprising recombinant protein derived from heavy-chain C-terminal domain of Clostridium botulinum type D/C - Google Patents

Abstract

The present invention relates to a recombinant vector expressing a botulinum toxin D/C type heavy chain C-terminus, a transformed E. coli capable of mass-producing a botulinum toxin D/C type heavy chain C-terminal protein by inserting the recombinant vector, a vaccine for preventing livestock botulism D/C type toxicosis using a botulinum toxin D/C type heavy chain C-terminal recombinant protein isolated by culturing the transformed E. coli, and a kit for diagnosing livestock botulism D/C type toxicosis.

Description

Recombinant vector, transformed E. coli, vaccine composition comprising recombinant protein derived from heavy-chain C-terminal domain of Clostridium botulinum type D/C}

The present invention relates to a vaccine composition and a kit comprising a recombinant vector, a transformed E. coli, a recombinant protein derived from the botulinum D/C type heavy chain C terminal, and more specifically, a botulinum toxin D/C type heavy chain C terminal protein expressing Recombinant vector, the transformed E. coli capable of mass-producing the botulinum toxin D/C type heavy chain C terminal protein by inserting the recombinant vector, and culturing the transformed E. coli to mass produce the botulinum toxin D/C type heavy chain C terminal protein Method and a vaccine composition for preventing toxicosis caused by botulism D/C type of livestock containing the botulinum toxin D/C type heavy chain C terminal protein, and a kit for diagnosing botulism D/C type poisoning of livestock.

Botulism is a neurotoxicity caused by the neurotoxin produced by the anaerobic bacterium Clostridium botulinum . Clostridium botulinum is a Gram-positive bacterium that forms spores for long-term survival if exposed to oxygen or under poor conditions such as low humidity, high temperature, and malnutrition. Therefore, using these spores, Clostridium botulinum is widely distributed in the environment such as soil, and can survive for many years.

Botulinum toxin is the most deadly toxin on earth, with a lethal dose of 1 ng per kilogram of vertebrate, and is known to be more than 1 million times more potent than cyanide. Because of its high toxicity, the US Centers for Disease Control and Prevention (CDC) classified the biological agent category A as an anthrax, plague, and smallpox in consideration of risk and potential for mineralization (Non-Patent Document 1).

Cattle botulism mainly refers to diastolic paralysis. Initially, it begins with gait confusion and progresses for several hours to several days, showing a lateral stance and eventually dying of respiratory muscle paralysis. Botulism has occurred in hundreds of cattle in the region, which has become a major social problem. Since then, botulism has occurred sporadically in dozens of cattle every year, causing constant damage to farms.

Botulinum bacteria are widely distributed in the environment, and when the bacteria such as anaerobic conditions, proper temperature, and moisture are in good conditions to develop, they spread and spread toxins around them. Botulinum toxin mainly contaminates dead bodies or sealed bale silage. It is known to make it (non-patent document 2).

Botulinum neurotoxins are divided into 7 toxin types, from A to G. Humans are mainly addicted to A, B, and E, and cows and horses are mainly B, C, D, and birds are C, D. There are few reports of botulinum poisoning in older brothers and pigs, and fish and seafood are primarily affected by toxin E. Type G was discovered in the 1970s, but its mechanism of action and specificity were not clearly identified (Non-Patent Document 3).

Botulinum is divided into 4 groups according to the biochemical properties, and group III in which livestock is poisoned is C/D mosaic type and D/C mosaic type in which C and D type are fused (non-patent document 4). ).

Botulinum neurotoxin is a protein about 150 kDa in size and has three functional domains. According to its function, it can be divided into three domains: heavy chain C-terminal, heavy chain N-terminal (Hn), and light chain (LC).

The C-terminal of the heavy chain has a molecular weight of 50 kDa and specifically binds to a receptor present in a neuron to induce botulinum toxin to enter into the neuron. In addition, the N-terminal of the heavy chain has a molecular weight of 50 kDa, and serves to transport a light chain having actual toxicity in botulinum toxin that has been introduced into the cell.

On the other hand, the light chain by a zinc (Zinc, Zn ^{+ 2)} parameters present in the active region and serves to hydrolyze the synaptic vesicles (synaptic vesicle) protein. Botulinum toxin types B, D, F, and G degrade synaptobrevin present in synaptic vesicles, and toxin types A, C, and E degrade SNAP-25 attached to neuronal cell membranes, and toxin type C Decomposes syntaxin, and all toxin types interfere with the secretion of acetylcholine and cause muscle paralysis (non-patent document 5).

Since botulism is not a bacterial infection, but a toxin-induced poisoning, it is not possible to expect a great effect even if antibiotics are administered. Injecting an antitoxin against a confirmed toxin type can neutralize toxins in the blood to prevent the progression of the disease, but considering the price of the antitoxin, it is currently ineffective to use antitoxin in livestock.

In the case of botulism, it is difficult to diagnose early without group mortality, and there is no characteristic autopsy. The earliest botulinum vaccine is a toxinoid vaccine that inactivates toxins with formalin, and these are still in use.

However, toxoid vaccines that require large amounts of fungal culture currently require a biosafety level 3 facility, and because they have to deal with large amounts of toxins, there is a risk that they can cause serious side effects if not inactivated. Recently, studies on recombinant proteins using heavy and light chains of toxins have been attempted (Non-Patent Document 6).

Botulism in livestock is mainly caused by B, C, C/D, D, and D/C toxins, and a recombinant D/C type botul with safety and efficacy against D/C type botulism. Development of a vaccine for rhism is required.

Thus, the present inventors produced a recombinant vector using a heavy chain C-terminal sequence from the botulinum toxin D/C type isolated in Korea using recombinant technology, and vaccine for the recombinant protein produced in transformed E. coli using the recombinant vector And as a diagnostic composition, to prevent and diagnose toxicosis caused by botulinum toxin D/C type in livestock.

JAMA. 2001;285:1059-1070 J Vet Diagn Invest. 1999;12:453-455 Botulinum toxin. 2007, Chapter 16, 337-353 Appl Environ Microbiol. 2012;78:3120-3127 Ann Rev Biochem. 2010;79:591-617 Infect Immun. 1995;63:2738-2742

The present invention was derived by the necessity of the above, and an object of the present invention is to provide a recombinant vector in which the nucleotide sequence of the heavy chain C-terminal is inserted in the botulinum toxin D/C type.

Another object of the present invention is to provide a transformed E. coli capable of producing a recombinant protein derived from the botulinum toxin D/C type heavy chain C terminal.

Another object of the present invention is to provide a method for producing a large amount of recombinant protein derived from the botulinum toxin D/C type heavy chain C terminal using the transformed E. coli.

Another object of the present invention is to provide a vaccine composition comprising the botulinum toxin D/C type heavy chain C-derived recombinant protein as an active ingredient.

Another object of the present invention is to provide a kit containing a recombinant protein derived from the botulinum toxin D/C type heavy chain C terminal.

In order to achieve the above object, the present invention consists of a gene encoding maltose binding protein (MBP) and a nucleotide sequence of SEQ ID NO: 1, and a gene encoding a heavy chain C-terminal protein of botulinum D/C type is operably linked. Recombinant vectors are provided.

In addition, the present invention provides a transformed E. coli in which the recombinant vector is inserted.

In addition, the present invention provides a method for mass production of botulinum D/C type heavy chain C-derived recombinant protein comprising culturing the transformed E. coli.

In addition, the present invention provides a vaccine composition for the prevention of botulism D/C type poisoning of livestock containing recombinant protein derived from the C-terminal of the heavy chain of botulinum D/C type produced by the transformed E. coli.

In addition, the present invention provides a kit for diagnosing botulism type B in livestock containing recombinant protein derived from the C-terminal of the heavy chain of botulinum D/C type produced by the transformed E. coli.

The present invention is to produce a transformed E. coli that produces a botulinum toxin D/C type heavy chain C-terminal protein, and use the botulinum toxin D/C type toxin heavy chain C-terminal protein produced by the strain in livestock for vaccines and diagnostics. It can be safely used for prevention and diagnosis of botulism type D/C.

1 shows a cleavage map of a recombinant vector pMAL-c2X-HCR-D/C in the form of a maltose binding protein (MBP), a botulinum toxin D/C type heavy chain C terminal binding, according to one embodiment of the invention.
Figure 2 shows the results confirmed by SDS-PAGE after confirming and purifying the expression of the botulinum D/C type toxin heavy chain C-terminal recombinant protein induced by Example 2, lane M of A is a marker, S is water soluble Protein, P is an insoluble protein, and IPTG was used to confirm before and after expression of the protein, lane M of B is a marker, and lane 1 is a botulinum D/C type toxin heavy chain C-terminal protein.

The first embodiment of the present invention consists of a gene encoding a maltose binding protein (MBP) and a nucleotide sequence of SEQ ID NO: 1, and a recombinant vector operably linked to a gene encoding a heavy chain C-terminal protein of botulinum D/C type It is about.

In the recombinant vector of the present invention, the gene encoding the heavy chain C-terminal protein of the botulinum D/C type may be derived from a botulinum D/C type strain isolated in Korea.

In the recombinant vector of the present invention, the recombinant vector may be pMAL-c2X-HCR-D/C having the cleavage map of FIG. 1.

The recombinant vector of the present invention can be expressed in the form of a heavy chain C-terminal binding of maltose binding protein (MBP) and botulinum toxin D/C by amplifying the C-terminal portion of the heavy chain C of the botulinum toxin D/C isolated in Korea. It has features.

The second embodiment of the present invention relates to a transformed E. coli into which the above recombinant vector is inserted.

The transformed E. coli of the present invention is Escherichia coli coli ) pMAL-c2X-HCR-D/C (KCTC 13655BP).

The third embodiment of the present invention relates to a method for mass production of a recombinant protein derived from the botulinum type B heavy chain C-terminal comprising culturing the transformed E. coli.

In the mass production method of the present invention, the transformed E. coli is inoculated into a medium containing antibiotics, preferably ampicillin, preferably LB (Luria-Bertani) medium, and is 35 to 39°C, preferably 36 to 38°C In, it may include the step of shaking culture for 10 to 24 hours, preferably 10 to 18 hours, more preferably 12 hours.

The mass production method of the present invention further comprises the step of centrifuging after the culture is over, and recovering and purifying the pellets.

In the mass production method of the present invention, in order to purify the protein expressed by the culture of the transformed E. coli, the botulinum D/C type toxin heavy chain C-terminal recombinant protein is purified using a maltose binding affinity column. Can be removed.

The fourth embodiment of the present invention relates to a vaccine composition for the prevention of botulism D/C type poisoning of livestock, which contains a recombinant protein derived from the C-terminal of the heavy chain of botulinum D/C type produced by the transformed E. coli.

In the preventive vaccine composition of the present invention, the livestock may be selected from the group consisting of cow, horse, sheep, bird, dog, cat, mink and fox.

In the present invention, the protective efficacy against botulinum toxin D/C type was confirmed through a mouse experiment presented in the European Pharmacopoeia 5 ^th ed. Clostridium botulinum vaccine for veterinary use.

The fifth embodiment of the present invention relates to a kit for diagnosing botulism D/C type of livestock containing a recombinant protein derived from the C-terminal of the heavy chain of botulinum D/C type produced by the transformed E. coli.

In the kit of the present invention, the livestock may be selected from the group consisting of cow, horse, sheep, bird, dog, cat, mink and fox.

Hereinafter, the present invention will be described in detail through examples. However, these examples are only for describing the present invention in detail, and the scope of the present invention is not limited by these examples.

< Example 1> Construction of recombinant gene and recombinant vector

The base sequence for the C-terminal of the botulinum toxin D/C type heavy chain was analyzed by BLAST, and a heavy chain C-terminal portion of the botulinum toxin D/C type isolated in Korea was amplified using primers.

The amplified product was cloned into the pMAL-c2X vector, and the vector prepared above was finally named pMAL-c2X-HCR-D/C, and the cleavage map is shown in FIG. 1.

< Example 2> Recombinant gene protein expression

The recombinant vector pMAL-c2X-HCR-D/C prepared in Example 1 was inserted into E. coli, the host cell, to prepare a transformed E. coli to be used for expression of the recombinant protein.

At this time, E. coli BL21 (DE3) was used as the host cell for gene expression, and the insertion of the recombinant vector was thawed 100 μl aqueous E. coli BL21 on ice, and after mixing the 5 μl of the recombinant vector, Let it stand for 30 minutes on ice.

Then, heat shock was applied at 42°C for 45 seconds, and allowed to stand for 2 minutes on ice, and 1 ml of LB medium was added, followed by shaking culture at 37°C for 1 hour. The transformed E. coli was spread on agar plates containing ampicillin (50 μg/ml) and placed at 37° C. for 16 hours. Clones with ampicillin resistance were selected to prepare the transformed E. coli.

The transformed E. coli was deposited with the Korea Research Institute of Bioscience and Biotechnology's Biological Resource Center and was given a deposit number (KCTC 13655BP).

The transformed E. coli was inoculated into LB medium containing ampicillin and cultured with shaking at 37°C for 12 hours. The culture was inoculated at 1/50 in 500 ml of LB medium containing ampicillin and 2% glucose.

When the transformed E. coli grew to OD ₆₀₀ =0.6, the E. coli growth was stopped by incubating at 4°C for 1 hour. IPTG was added, and after shaking culture at 16° C. for 24 hours, centrifugation was performed at 6,000 rpm for 10 minutes, and pellets were recovered.

The pellets were suspended with a lysis buffer to disrupt the bacteria through ultrasonic crushing. The crushed solution was centrifuged at 17,000 rpm, 4° C. for 30 minutes, and divided into a soluble protein compartment and an insoluble protein compartment, and a protein expression pattern was confirmed on 12% SDS-PAGE (see FIG. 2 ).

In order to purify the expressed protein, a recombinant botulinum toxin D/C type heavy chain C-terminal protein was obtained using an MBP binding affinity column.

< Example 3> Protective effect of botulinum toxin D/C type recombinant protein on experimental animals

The mouse protective effect of the recombinant protein against botulinum toxin type D/C was confirmed. 18-20 g ICR mice were divided into 10 animals per experimental group, and the botulinum toxin D/C type recombinant protein was inoculated once in an amount of 1 μg/mouse, 5 μg/mouse, and 10 μg/mouse. Inoculation was performed at a dose.

After 3 weeks of inoculation of the recombinant protein, the botulinum toxin D/C type was challenged intraperitoneally with a dose of 25LD ₅₀ , respectively, and survival was confirmed for 1 week.

Table 1 below shows the results of observing the survival rate for one week after immunization with mice of various concentrations of recombinant protein once, followed by challenge with Botulinum toxin D/C type 25LD ₅₀ , respectively.

As shown in Table 1, when the botulinum toxin D/C type vaccination, both the negative control group inoculated with PBS and the recombinant protein 1 μg/mouse inoculated group died, and in the 5 μg/mouse inoculated group, 1 died 90 % Survival rate and 100% survival rate in the 10 μg/mouse inoculation group.

Table 2 below shows the results of observing the survival rate for one week after immunizing mice twice with various concentrations of recombinant proteins, and then vaccinating each with Botulinum toxin D/C type 25LD ₅₀ .

That is, in Table 2, 18-20 g ICR mice were divided into 10 groups per experimental group, and the botulinum toxin D/C type recombinant protein was first inoculated in an amount of 1 μg/mouse, 5 μg/mouse, and 10 μg/mouse. After 2 weeks of inoculation, 2 weeks after the first inoculation, Botulinum toxin type D/C was inoculated into the abdominal cavity at a dose of 25LD ₅₀ each, and the survival rate was confirmed for a week.

As shown in Table 2, all of the negative controls inoculated with PBS during the botulinum toxin D/C type vaccination were killed, but the group of 1 μg/mouse inoculated with the botulinum toxin D/C type recombinant protein of the present invention twice Two silver died and showed an 80% survival rate, and 5㎍/mouse and 10㎍/mouse inoculation group showed 100% survival rate.

Although described with reference to the preferred embodiment of the present invention as described above, those skilled in the art in the technical field of the present invention can be seen within the scope not departing from the spirit and scope of the invention as set forth in the claims below. It will be understood that the invention can be variously modified and changed.

The present invention uses a transformed E. coli that mass-produces a botulinum toxin D/C type heavy chain C-terminal recombinant protein, thereby using the botulinum toxin D/C type heavy chain C-terminal protein produced by the strain as an antigen for vaccines and diagnostics, domestic livestock It is expected to significantly reduce the mortality rate of livestock by effectively preventing and diagnosing D/C type botulism.

In addition, the development of new products by domestic vaccine manufacturers is expected to have a positive effect on the national economy, such as the reduction of foreign currency due to the substitution of imported vaccines and the export effects of domestic vaccines.

Korea Research Institute of Bioscience and Biotechnology KCTC13655BP 20181004

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Claims (10)

A gene encoding maltose binding protein (MBP), and
Recombinant vector consisting of the nucleotide sequence of SEQ ID NO: 1, the gene encoding the heavy chain C-terminal protein of botulinum D/C type is operably linked. The recombinant vector according to claim 1, wherein the gene encoding the botulinum D/C type heavy chain C-terminal protein is derived from a botulinum type B strain isolated in Korea. The recombinant vector according to claim 1, wherein the vector is pMAL-c2X-HCR-D/C having a cleavage map of FIG. 1. A transformed E. coli into which the recombinant vector of any one of claims 1 to 3 is inserted. The method of claim 4, Escherichia coli coli ) transformed E. coli, characterized in that it is pMAL-c2X-HCR-D/C (KCTC 13655BP). A method for mass production of botulinum D/C type heavy chain C-derived recombinant protein comprising culturing the transformed E. coli of claim 4. A vaccine composition for preventing botulism D/C type poisoning in livestock, comprising a recombinant protein derived from the C-terminal of the heavy chain of botulinum D/C type produced by the transformed E. coli of claim 4. 8. The vaccine composition according to claim 7, wherein the livestock is selected from the group consisting of cattle, horses, sheep, birds, dogs, cats, minks and foxes. A kit for diagnosing botulism type B in livestock, comprising a recombinant protein derived from the C-terminal of the heavy chain of botulinum D/C type produced by the transformed E. coli of claim 4. The kit according to claim 9, wherein the livestock is selected from the group consisting of cow, horse, sheep, bird, dog, cat, mink and fox.

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