KR20030096980A - Food grade vector comprising PstⅠ-SpeⅠ fragment of pMMH1 plasmid derived from wild-type Bacillis - Google Patents

Abstract

PURPOSE: An edible vector comprising Pst I-Spe I restriction sites of plasmid pMMH1 isolated from Bacillus mesentericus KCTC 0750BP is provided, thereby easily supplying bio-stimulating material such as vitamins and proteins to food. CONSTITUTION: An edible vector comprises a 2.3 kb fragment obtained by digesting a plasmid pMMH1 isolated from Bacillus mesentericus KCTC 0750BP with restriction enzymes Pst I and Spe I. The edible vector pPS is constructed by inserting the 2.3 kb fragment obtained by digesting the plasmid pMMH1 isolated from Bacillus mesentericus KCTC 0750BP with restriction enzymes Pst I and Spe I into a plasmid pGEM5zf(+).

Description

Food grade vector comprising Pst I-Spe I fragment of pMMH1 plasmid derived from Bacillus mesentericus (JPC 0750 PF)

The present invention relates to an edible vector, and more particularly, to an edible vector containing a cleavage fragment of plasmid pMMH1 isolated from the wild species Bacillus mesentericus .

Efforts and research for the development of edible vectors have not been actively carried out to date and have often been reported in Europe, where only a large number of dairy farmers are involved. This is because it is not easy to select a vector harmless to the human body, it is difficult to select a suitable selection marker (selection marker), the selection of a vector of a suitable size stable, has been tried to be a very low. These past R & D achievements show that the development of food vectors is not easy. Currently, the development of food vectors is underway using plasmids isolated from Lactobacillus , Lactococcus lactis , and the like. However, it is still more of an animal food vector than a human.

pMMH1 is a plasmid isolated from Bacillus ( Bacillus subtilis and its similar strains), which has been edible for a long time as a major fermentation strain of soybean-derived fermented foods and is internationally recognized for its safety. Based on these advantages, the present invention constructs several vectors using pMMH1 to develop an edible vector technique, finds the stable part while including the smallest part of pMMH1, and produces a physiologically useful substance therefrom. We tried to establish a secretion system that can

Therefore, an object of the present invention is to provide an edible vector that can be ingested by humans using a plasmid isolated from wild Bacillus inhabiting traditionally ingested food products.

The object of the present invention is to isolate plasmid pMMH1 from Bacillus mesentericus (KCTC 0750BP), remove each part of pMMH1 one by one to confirm that the isolated plasmid is suitable for use as an edible vector, and to E. coli plasmid pGEM5z ( Connect +) to construct a vector that can be used by both Bacillus and Escherichia coli, transform it into Bacillus, a host of the constructed vector, and continuously culture it for up to 100 generations in the absence of antibiotics to confirm stability. This was achieved by confirming that the fragment ( Pst I- Spe I) is best suited for development as an edible vector.

Hereinafter, the configuration and operation of the present invention.

1 is a schematic diagram showing the result of constructing a vector by removing each part of pMMH1 to construct an edible vector.

Figure 2 is a table listing the bacteria and plasmids used in the present invention.

3 is a schematic diagram showing a vector pPS constructed including only 2.3 kb DNA fragment ( Pst I- Spe I) of pMMH1.

4 is a schematic diagram of pJSN vector production.

Figure 5 is a schematic diagram showing the nucleic acid and amino acid sequence of the fusion (fusion) position.

6 is a graph showing the growth of cells cultured in CPGY medium.

7 is a graph showing the activity of beta-glucosidase measured every 3 hours.

FIG. 8 is a photograph showing SDS-PAGE analysis of oat beta-glucosidase produced from Bacillus subtilis DB104 containing pJSN vector.

The present invention constructs a vector by constructing a vector by removing one of the five portions identified as a result of pMMH1 DNA sequencing for the purpose of developing an edible vector technique using pMMH1 plasmid isolated from Bacillus mesentericus (KCTC 0750BP). It consists of a partial search step that is stable to use as an edible vector for the constructed vector.

Vector construction was performed by the following procedure.

Portions of the pMMH1 plasmid were cut to determine which regions are needed for stable vector maintenance with high copy numbers. For convenient DNA manipulation, the pMMH1 was inserted into the Nco I position of the pGEM5zf (+) vector (Promega, USA) to prepare pMMG.

Meanwhile, 1.4 kb Sal I fragment of cat (chloramphenicol acetyltransferase) gene, which is used as a selection marker in Bacillus , was cloned at Sal I position of pGEM5zf (+) to construct pGEMC.

The EcoR V- Nco I fragment of pGEMC was inserted into the Pvu II- Nco I fragment of pMMH1 to construct pEN. PEN was cleaved with Nde I and a 4.0 kb fragment containing the resulting DNA binding protein coding region, replication origin, and secretion protein coding region was prepared using pGEM5zf ( PMN was constructed by binding to +). PME was prepared by cleaving DNA binding protein coding region of pEN by EcoR I and then self-bonding.

Meanwhile, 2.4 kb Pst I- Pst I fragments and 2.3 kb Pst I- Spe I fragments containing the replication region of pMMH1 were isolated from pGEMC and inserted into pGEM5zf (+), respectively, to construct pPP and pPS. It became.

Since the RCR plasmid pMMH1 isolated from B. mesentericus is very stable under nonselective conditions, efforts have been made to develop it as a stable vector in Bacillus. Sequence analysis showed that pMMH1 had a replication origin, two open reading frames (ORFs) of γ-GTP, and a type I signal peptidase (sipP). To identify the regions necessary for the stability of the pMMH1 vector, each region was removed from pMMH1. As a result, five vectors (pPS, pPP, pEN, pMN, pME) were prepared with B. subtilis- E. Coli shuttle vectors. The pEN from which the γ-GTP coding sequence was removed showed more than 98% stability over 100 generations, suggesting that the γ-GTP coding sequence is unnecessary for plasmid stability. In addition to ORF2 (pME) or γ-GTP coding sequences, type I signal peptidase (pMN) also did not critically affect the stability of the plasmid. For both pPP and pPS, the γ-GTP coding sequence, ORF1, ORF2, type I signal peptidase was completely eliminated, but ORF1 was more removed from pPS than pPP. Thus pPS was the smallest vector with 2.3 kb of pMMH1 plasmid.

All the constructed plasmids showed over 90% stability even after 100 generations. Removal of the γ-GTP coding sequence, ORF1, ORF2, type I signal peptidase did not significantly affect the stability of the plasmid, and the single strand origin (SSO) between the origin of replication and ORF2 did not affect the plasmid. It seemed most important in terms of stability.

Type I signal peptidase of the pMMH1 plasmid is important for stability in Bacillus . B. subtilis has a well-developed extracellular secretion system and type I signal peptidase (type I SPases) is a secretion via translocase or numerous internal molecules. Plays an important role in processing Type I signal peptidases are present on chromosomes (SipS, SipT, SipU, SipV, SipW) and plasmids (SipP). Of these, SipS and SipT are very important for preprotein processing and activity. SipP is also very important because it can functionally replace damaged SipS and SipT.

However, in the present invention, deletion of the type I signal peptidase did not affect the stability of the plasmid. This indicates that this is not a necessary factor for stability.

Therefore, as described above, as the size of the vector increases in size, it is unstable in the cell. Therefore, the unnecessary portion for the stable replication in the cell is deleted and the necessary portion for the stable replication, 2.3 kb DNA fragment of pMMH1 ( Pst I- Spe I PPS containing) is more suitable for use as an edible vector than the above-mentioned vectors.

The expression vector pJSN was prepared to confirm whether the foreign protein is expressed through the pPS. The expression vector pJSN is the smallest and most stable pPS among the plasmids, and an alpha-amylase promoter-signal sequence is used. Was constructed (FIG. 3). Β-glucosidase to locate the beta-glucosidase gene (1.5 kb) under the control of the alpha-amylase promoter-signal sequence. The gene was amplified by PCR from pET24-Glu1 and cloned into the BamH I and Sal I positions of p8A1 to construct p8A1-α25. Forward primer (forward primer; 5'- GGATCC GAAT TCGCGCTTGAAAGT C-3 ') is for the easy cloning was designed to contain a BamH I position (shown in underline), a nucleic acid C (underlined), the alpha-amylase signal It was designed for in-frame fusion between the α-amylase signal sequence and the N-terminus of the beta-glucosidase mature gene. Reverse Bang ?? The reverse primer (5'-CTGCAG CTCGAGTCATCA CGCGGT-3 ') is designed to include the Xho I position (underlined) and any TGA stop codons (underlined). p8A1-α25 was digested with EcoR I- Hind III and the 2.5 kb-fragment containing alpha-amylase promoter-signal sequence-β-glucosidase was pKS-α25 Subclones were constructed with pBluescript II KS (+) at the same location. Finally, pKS-α25 was cleaved with Sac II- Apa I and then inserted into the same position of the pPS vector to construct a pJSN expression vector.

When the β-glucosidase gene of oats was inserted, β-glucosidase was secreted successfully. The activity of β-glucosidase in the supernatant increased steadily after 15 hours and peaked at 27 hours. In SDS-PAGE analysis of the proteins in the supernatant, beta-glucosidase with an expected size of about 60 kD was found only from transformations containing expression / secretion vectors. This confirms that beta-glucosidase is secreted.

Hereinafter, the configuration of the present invention will be described in more detail with reference to Examples, but the scope of the present invention is not limited only to the following Examples.

Example 1 Bacillus mesentericus Bacillus mesentericus Vector construction using pMMH1 plasmid isolated from

Bacillus mentericus (KCTC 0750BP), obtained from the Biotechnology Research Institute, was incubated overnight in LB medium to isolate the pMMH1 plasmid, cleaved this plasmid with restriction enzyme Nco I, and then the E. coli plasmid pGEM5zf (+). Enzyme cleavage was linked. This is called pMMG and based on this, the five parts contained in pMMH1 are: replication origin, open reading frame 1 (orf1), type I signal peptidase, and gamma-tipi. GTP), secretion protein coding region (5) (pEN, pMN, pME, pPS, pPP) vector was constructed by reducing the size of the plasmid by removing one by one except for the replication origin. , 2).

More detailed vector construction was performed by the following procedure.

Portions of the pMMH1 plasmid were cut to determine which regions are needed for stable vector maintenance with high copy numbers. For convenient DNA manipulation, pMMH1 was inserted into the Nco I position of the pGEM5zf (+) vector (Promega, USA) to prepare pMMG. A 1.4 kb Sal I fragment of the cat (chloramphenicol acetyltransferase) gene used as a selection marker in Bacillus was cloned into Sal I of pGEM5zf (+) to construct pGEMC. The EcoR V- Nco I fragment of pGEMC was inserted into the Pvu II- Nco I fragment of pMMH1 to construct pEN.

PEN was cleaved with Nde I and a 4.0 kb fragment containing the resulting DNA binding protein coding region, replication origin and secretion protein coding region was prepared using pGEM5zf (+ ) To construct pMN. PME was prepared by cleaving the DNA binding protein coding region of pEN by EcoR I and then self-bonding.

Meanwhile, 2.4 kb containing the replication region of pMMH1PstI-PstI intercept and 2.3 kbPstI-SpeI segment was isolated from pGEMC and each Inserted into pGEM5zf (+) to construct pPP and pPS

Example 2: searching for the most stable part of the constructed vector

In order to investigate the stability of the vector constructed in Example 1 and find a part to be used as an edible plasmid, a plasmid consisting of each part of pMMH1 was transformed into Bacillus, and the transformant was inoculated in 3 ml LB liquid medium. Incubated overnight at 37 ° C. incubator and then inoculated 2% (1 ml) in a 250 ml Erlenmeyer flask containing 50 ml LB liquid medium. Thereafter, up to 20 generations were transferred to fresh medium and cultured. Subsequently, they were batch-cultured in a medium free of antibiotics for up to 100 generations under the same conditions, passaged in a previously warmed medium. Cultures were transferred to fresh medium every 20 generations. After 100 generations, only 200 μl of 50 ml culture was taken and plated with antibiotic-free medium and incubated overnight. Then, the cells grown here (colony) were transferred to a medium containing antibiotics (chloramphenicol), and about 200 cells were collected and cultured again overnight. Finally, the stability of the plasmid was confirmed by looking at how many cells survived in the medium containing antibiotics. The constructed vectors all exhibited high stability of at least 90% (Table 1).

Results showing stability of plasmids constructed based on pMMH1 Built vector Stability Test Results pPS 98% pPP 94% pME 97% pMN 98% pEN 95%

Dual pMMH1 the 2.3kb DNA fragment (Pst I- Spe I) are eopeumyeonseo a problem in stability because it contains the smallest part of the pMMH1 it is determined to be suitable for use as a food vector. The reason why small ones are useful as stable vectors is that they become unstable intracellularly after their size. In addition, when the target gene is inserted into the vector, the vector becomes larger than necessary, which exceeds the size that the vector can accommodate and eventually loses stability.

Therefore, there is no need to leave unnecessary parts for stable replication in the cell. So, after confirming the stability using the necessary part for stable replication, 2.3 kb PstI-SpeI fragment was selected and inserted into pGEM5zf (+) to construct the vector pPS (FIG. 3).

Example 3 Confirmation of Expression of Recombinant Gene Using Constructed Vector

Expression vector pJSN was constructed using the smallest and most stable pPS and α-amylase promoter-signal sequence (FIG. 4). Β-glucosidase gene (β-glucosidase gene; 1.5 kb) under the control of the alpha-amylase promoter-signal sequence to locate the beta-glucosidase gene (β-glucosidase gene) gene) was amplified by PCR from pET24-Glu1 and cloned into BamH I and Sal I positions of p8A1 to construct p8A1-α25. Forward primer (forward primer; 5'- GGATCC GAAT TCGCGCTTGAAAGT C-3 ') is for the easy cloning was designed to contain a BamH I position (shown in underline), a nucleic acid C (underlined), the alpha-amylase signal It was designed for in-frame fusion between the α-amylase signal sequence and the N-terminus of the β-glucosidase mature gene (FIG. 5). Reverse Bang ?? The reverse primer (5'-CTGCAG CTCGAGTCATCA CGCGGT-3 ') is designed to include the Xho I position (underlined) and any TGA stop codons (underlined). p8A1-α25 was digested with EcoR I- Hind III and the 2.5 kb-fragment containing alpha-amylase promoter-signal sequence-β-glucosidase was pKS-α25 Subclones were constructed with pBluescript II KS (+) at the same location. Finally, pKS-α25 was cleaved with Sac II- Apa I and then inserted into the same position of the pPS vector to construct a pJSN expression vector.

β-glucosidase activity was investigated by measuring p-nitrophenol (p-NP) liberated from paranitrophenyl-beta-di-glucopyranoside (pNPG). Transformed B. subtilis DB104 constructing the pJSN vector was precultured overnight in 3 ml of LB medium containing 10 μg / ml of chloroamphenicol. 3 ml of preculture medium was then transferred to 50 ml of CPGY medium in a 250 ml flask. And shaking culture at 200 rpm at different growth temperatures of 37 ℃, 30 ℃ and 25 ℃. 1 ml of culture was centrifuged at 15000 rpm for 20 minutes. 50 µl of the supernatant of the culture was mixed in 20 mM potassium phosphate buffer in which 450 µl of 10 mM pNPG solution was dissolved. (KH ₂ PO ₄ , pH 5.5). The reaction was carried out at 37 ° C. for 15 minutes and stopped by adding a stop solution (2 M Na ₂ CO ₃ solution). Free pNP was measured by reading A ₄₀₅ . One unit was defined as the amount of enzyme that hydrolyzes 1 nmol of pNP per minute at 37 ° C.

The profile of the protein in the media supernatant was analyzed by SDS-PAGE. Proteins in the medium were precipitated using trichloroacetic acid with a final concentration of 10% (w / v). Precipitation was stopped in 50 μl of 0.1 N NaOH. After addition of 10 μl of 5 × sample buffer, the samples were boiled for 5 minutes and loaded onto 10% SDA-polyacrylamide gel.

Because pPS is stable and smallest (6.8 kb), pPS was chosen for the construction of secretion vectors, pPS was selected from the commercial cloning vectors pGEM5zf (+) (Promega, USA) and pMMH1 from E. coli and It is a hybrid of the cat (chloramphenicol acetyltransferase) gene. Thus, pPS acts in both E. coli and B. subtilis .

For expression and secretion, the alpha-amylase promoter-signal sequence system (1 kb) of Bacillus subtilis was used. When oats β-glucosidase gene was inserted, beta Glucosidase was successfully secreted. The activity of beta-glucosidase in the supernatant increased steadily after 15 hours and peaked at 27 hours (FIGS. 6, 7). In SDS-PAGE analysis of the proteins in the supernatant, beta-glucosidase with an expected size of about 60 kD was found only from transformations containing expression / secretion vectors. This confirms the secretion of beta-glucosidase (β-glucosidase) (FIG. 8).

As described above, the present invention can supplement foods with physiologically useful substances such as vitamins and proteins that are easily lacking in foods by using an edible vector pPS that is stable and easy to produce foreign genes. It is a very useful invention in the pharmaceutical industry.

Claims (2)

A vector comprising a 2.3Kb fragment obtained by cleaving pMMMH1 derived from Bacillus mesentericus (KCTC 0750BP) by restriction enzymes PstI and restriction enzyme SpeI. Vector pPS prepared by inserting a 2.3Kb fragment obtained by cleaving pMMMH1 derived from Bacillus mesentericus (KCTC 0750BP) by restriction enzymes PstI and restriction enzyme SpeI into pGEM5zf (+).