JP3103881B2 - Plasmid - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a plasmid isolated from a rumen microorganism.

[0002]

2. Description of the Related Art Ruminant animals have four stomachs, of which the stomach into which the feed they eat first enters is called the lumen. The rumen is rich in rumen microorganisms that coexist with ruminant livestock such as bacteria, ciliates and fungi. By the way, in order to carry out gene recombination, a vector having a role of carrying a foreign gene to a gene of a microorganism and a microorganism (host) receiving the vector are required. Therefore, in order to develop a rumen microorganism having a new function by genetic recombination, a method of preparing a host-vector system using a rumen microorganism can be considered.

[0003] Plasmids, phages, transposons, and the like can be used as vectors. Plasmids are the easiest to handle and are easy to develop.
Plasmids are DNAs that are physically independent of the host chromosome, but grow only in specific hosts because their ability to replicate and inherit is highly dependent on the host. Therefore, in order to generate a host-vector system using a rumen microorganism, a plasmid that can grow in the rumen microorganism is required.

[0004]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a plasmid which can be propagated in rumen microorganisms.

[0005]

Means for Solving the Problems The present inventors have conducted intensive studies to solve the above problems, and as a result, have found that a bacterium, Luminobacter amylofilus, which is thought to play an important role in starch degradation in the lumen. amyloph
ilus) to complete the present invention. That is, the present invention is a 2444 bp plasmid having the following restriction enzyme map, which is a plasmid containing a self-replicating sequence derived from Luminobacter amylofilus.

[0006]

Embedded image

[0007] As the above plasmid, the following (a) or
and those comprising the DNA of (b). (a) a DNA consisting of the nucleotide sequence represented by SEQ ID NO: 1; (b) a DNA capable of hybridizing with a DNA consisting of the nucleotide sequence represented by SEQ ID NO: 1 under stringent conditions and capable of self-replication, The present invention will be described in detail.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The plasmid of the present invention contains a self-replicating sequence of Luminobacter amylofilus. In addition, the plasmid of the present invention can be a shuttle vector between Escherichia coli and Luminobacter amylofilus, since the source of the isolation is Luminobacter amylofilus, which is systematically closer than that of Escherichia coli.
Therefore, it is possible to insert a foreign gene into the plasmid of the present invention, transform Escherichia coli, obtain a large amount of a recombinant plasmid, and use the recombinant plasmid to produce a useful substance using Luminobacter amylofilus as a host bacterium. Becomes

[0009] The plasmid of the present invention is prepared, for example, as follows. First, ruminants (eg, cows, sheep,
Collect Luminobacter amylofilus from the rumen of goats. The sampling method is as follows. That is, the rumen solution is diluted, and colonies appear by the anaerobic roll tube method. Bacteria are picked from the colony and bacteria utilizing starch and maltose are selected. The 16S rRNA sequence of the bacteria selected by the PCR method is determined, and the determined sequence is compared with the 16S rRNA (GenBank Accession No.RAY15992) of Luminobacter amylofilus (ATCC 29744) registered in the database, If there is a similar sequence between the two, it is determined (confirmed) that the collected bacteria is Luminobacter amylofilus.

Next, total DNA is obtained from the obtained Luminobacter amylofilus. Total DNA can be prepared by any known method (for example, mini-scale preparation of chromosomal DNA (“Basics of new genetic engineering technology”, Michio Ota, Nae Publishing, pp23-25, 1989)
Year).

[0011] The obtained total DNA is converted to an appropriate restriction enzyme (for example, H
indIII, ScaI) to obtain a partial fragment, which is then pBlues
After insertion into a vector such as cript, E. coli is transformed. Those that became ampicillin resistant and white colonies were selected, and plasmid DNAs were respectively separated from the colonies to prepare a gene library. Suitable primers, for example M13M4
Primer and M13 reverse primer (both TaKaR
PCR is carried out using the above library as a template by (Company a). After electrophoresis of the resulting amplified fragment on an agarose gel,
Blot on a membrane and perform Southern hybridization. In addition, Southern hybridization can be performed according to the manual of Boehringer Mannheim.

A band obtained as a result of Southern hybridization is cut out, and the nucleotide sequence is determined. The nucleotide sequence of the plasmid of the present invention can be determined by a commonly used nucleotide sequence analysis method, for example, the dideoxy method of Sanger et al. [Proc. Natl. Acad. Sci. USA, 74, 5463 (1977)] It can be determined by analysis using an automatic base sequencer. The plasmid thus obtained has a length of 2444 bp (SEQ ID NO: 1), and a self-replicating sequence derived from Luminobacter amylofilus (No. 382 of the base sequence represented by SEQ ID NO: 1).
~ 986).

[0013] However, the plasmid of the present invention has SEQ ID NO: 1.
In addition to those containing DNA consisting of the nucleotide sequence represented by SEQ ID NO: 1 and those containing DNA capable of hybridizing with the DNA consisting of the nucleotide sequence represented by SEQ ID NO: 1 under stringent conditions and capable of self-replication. Included in the plasmid of the invention. Stringent conditions refer to, for example, conditions in which the sodium concentration is 75 to 100 mM, preferably 75 mM, and the temperature is 40 to 42 ° C, preferably 40 ° C. Also,
The plasmid of the present invention has ClaI, BstXI, SacI, PstI and SspI as restriction enzyme sites as described below.

[0014]

Embedded image

The distance between the restriction enzyme sites is BstXI-
888 bp between ClaI, 765 bp between SspI and ClaI, 192 bp between PstI and SspI, 278 bp between SacI and PstP, 3 between SacI and BstXI
21 bp. The plasmid obtained as described above was named "pRAO1", and Escherichia coli (name: E. coli pH21 and E. coli pPR34) containing the plasmid was obtained from the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology (Tsukuba, Ibaraki Prefecture). 1-3 1-3, Higashi-shi
No.), as of May 25, 1998, FERM P-16826 for E. coli pH21 and FERM P-16 for E. coli pPR34.
Deposited as -16825.

E. coli pH21 contains pRAO1 from 1998 to 1
It contains fragments up to 698 (from 1998 to 1698 in the clockwise direction in FIG. 1). Also, pPR34 contains 1 of pRAO1
438 to 2163 (1438 in FIG. 1).
From No. 2163 clockwise), Hind not included in pH 21
It has a fragment of III-Pst I-Hind III (FIG. 1). Therefore, both the plasmid contained in pH21 and the plasmid contained in pPR34 were treated with the restriction enzyme HindIII, and the both were ligated with ligase or the like to obtain the complete plasmid pRd.
AO1 can be obtained.

[0017]

The present invention will be described more specifically with reference to the following examples. However, the technical scope of the present invention is not limited by these examples.

Example 1 Preparation of Plasmid As a bacterial strain, Luminobacter amylofilus NIAH-3 (Animal Health Laboratory) was used. The bacteria were cultured for 14 hours at 37 ° C. using an improved RGCMS medium (a medium containing 10% of rumen solution and 0.025% of starch and maltose as sugar sources). The cells were collected from 15 ml of the culture solution by centrifugation (5000 rpm) and suspended in 50 μl of 0.15 M NaCl-0.1 M EDTA. 400 μl of 0.1 M NaCl-0.1 M Tris HCl (pH 8.
0) A -1% SDS solution was added and lysed by gentle stirring. 450 μl of phenol was added to the obtained lysate,
The mixture was gently stirred, and the aqueous layer was collected by centrifugation. After extracting DNA with an equal amount of phenol / chloroform, ethanol precipitation was performed.

The DNA was dissolved in 30 μl of TE buffer (pH 8.0), mixed with RNase, and subjected to 0.8% agarose gel electrophoresis. Plasmid band (1.5-3.0k with 1kb ladder (BRL))
(near b) was cut out from the gel, and the plasmid DNA was extracted and purified by phenol melting method.

Example 2 Preparation of Restriction Enzyme Map Plasmid DNA was treated with restriction enzymes Hind III and Sac I. As a result, it was revealed that the DNA could be cleaved at least at two or more sites. Therefore, plasmid DNA treated with Hind III
Was ligated to pBluscriptII KS + (Stratagene, USA) digested with the same HindIII and treated with alkaline phosphatase to transform Escherichia coli JM109. Transformants were subjected to selection with ampicillin to select white colonies, and plasmids were extracted from the transformants. Transformant plasmid DNA
Was used as a template to perform PCR using M13-20 primer and M13 reverse primer (Takara). PCR
Using a kit of TaKaRa EX Taq, a reaction solution was prepared according to the instruction manual, and 30 cycles were performed at 94 ° C. for 30 seconds, 55 ° C. for 30 seconds, and 72 ° C. for 60 seconds.

The obtained amplified fragment was electrophoresed on an agarose gel, blotted on a membrane, and subjected to Southern hybridization. In addition, Southern hybridization followed the manual of Boehringer Mannheim. In addition, the probe was prepared by ligating plasmid DNA of Luminobacter amylofilus to DIG High Prime (BoehringerMan
nheim).

The nucleotide sequence of the plasmid is Dye Terminater
In order to determine using a cycle sequencing kit (Perkin Elmer ABI), the above PCR product was used as a template, and a synthesized primer or a commercially available primer (Takara) was allowed to react according to the manual, and analysis was performed using an ABI 373S sequencer.

That is, a positive clone pH21 in which a signal was recognized by Southern hybridization was amplified with the M13 primer set (M13M4 and M13RV), and using the obtained PCR product, about 500 bp on the M13M4 side from the cloned site was used. And the nucleotide sequence of about 500 bp on the M13RV side was determined. The Oligo program (National Biosc
iences, UK) using the following forward primer HM01
HM02 and HM02, and reverse primers HM03 and HM04
Four primers were designed.

HM01: ATCTCTACTGGCGTCTAT (SEQ ID NO: 2) HM02: CAAAWAGGGAACTGGAG (SEQ ID NO: 3) HM03: GCTTCCTTCTGGCGTTCC (SEQ ID NO: 4) HM04: CCCAGGCTCGTTCGTTCT (SEQ ID NO: 5)

A combination of HM01 and HM02 (referred to as HM01-02) or H
PCR by combining M03 and HM04 (referred to as HM03-04)
Was done. PCR was performed using a TaKaRa EX Taq kit (Takara) and 30 cycles of 94 ° C. for 30 seconds, 50 ° C. for 30 seconds and 72 ° C. for 40 seconds as one cycle.

The base sequence of each amplified PCR product is determined,
Furthermore, HM05 was determined from the determined base sequence of the PCR product of HM01-02.
(AAGCAGACATCGCARAAG (SEQ ID NO: 6)) and HM06 (ACTCT
TCGTCCTCTACCA (SEQ ID NO: 7))
Combination of HM02 and HM06 (referred to as HM02-06) or HM0
PCR was performed using the combination of 1 and HM05 (referred to as HM01-05) (using the TaKaRa EX Taq kit; 30 seconds at 94 ° C, 50 minutes).
30 cycles at 30 ° C. and 40 seconds at 72 ° C.).

The nucleotide sequence of the obtained PCR product was determined. As a result, a 2444 bp base sequence represented by SEQ ID NO: 1 was obtained. The obtained base sequence was analyzed using GeneWorks software (IntelliGenetics, USA) to estimate the restriction enzyme cleavage site. Restriction enzymes thought to have only a single cleavage site were confirmed by single or double digestion of the plasmid DNA or PCR product with the restriction enzyme and electrophoresis. As a result, it was found to have the following restriction map.

[0028]

Embedded image

PCR amplified with HM03-04 primer set
The product was cloned into the pCR II vector using the TA Cloning kit. This was named pPR34. pPR34 is pRAO
Hind III-Pst I-Hin not included in pH 21
dIII (Fig. 1).

[0030]

According to the present invention, there is provided a plasmid capable of growing in a rumen microorganism. The plasmid of the present invention is useful in that it can be used as a shuttle vector between Luminobacter amylofilus and Escherichia coli.

[0031]

[Sequence List] SEQUENCE LISTING <110> Yoshihiro Yamashita Director General of National Institute of Animal Industry Ministry of Agriculture, Forestry and Fisheries <120> Plasmid <130> P97-0569 <140> <141> <160> 7 <170> PatentIn ver. 2.0 <210> 1 <211> 2444 <212> DNA <213> Ruminobacter amylophilus <400> 1 aagtaagaat gtaggaattt caagggttta catgtttttt gtgatctttc tcctttattt 60 accaattatg ttggtataaa acaccacaaa catggtatta tgcacggagt cggaagttgg 120 tgagattacc aactgaagtt ggtgagatta ccaactgaag ttggtgagat taccaactga 180 agttggtgag attaccaact tttcaagggt aagaatgtag gaatttcaag ggtttgcacg 240 gatctctctt ctaattctat atatctgatt attttttttt ggtacttttt tatgaaaaac 300 gatgaaaaat taaacaaaat taaacagctt ccaaaaaaca ccgtggaaga aatcgtagag 360 ggcattaatt ccattggcga aatggatatt tctgtaaacg tcgcaagaca aagaataaaa 420 ccagatcagg aatttatttt ttactttctt acaaacatgg attccctgat tagcgatcct 480 gatattacta aacaggatat tgctgttctc atgaaatacg ctgcaaaaat gcaatacggc 540 aatcagattt ccatagctca agcagacatc gcagaagatc ttggaatcga caaatcgaat 600 gtttccaaat cagttagaac acttacgcag aaaggcgttt ttcttaaaga gagaagaagt 660 ctcgttatga attggaagta ccttgccaaa ggtaacttaa cggatttcat caaagcagac 720 aaagaaaaca gcaaactttc aaaatttcaa ttggtagagg acgaagagta atggattgga 780 gtaaacaaga acttgaaaaa atcgaagctg taatgaagca tctcaatgcg attatcagcg 840 tcacaacagg cacttctgta aagctcgatg cggaaaagga gcaggtgcag tttttttccg 900 aaagcggaag aatgtacaag acgatttcag tcgctggaga tagtccatta gctgttgcca 960 aagacgtact caagcagatc gattaataag cgagaagccc ctgcgttgca ggtgcttctc 1020 aagatcaata aaggggggct aaaacccccc tttacccccc tcctggggga ggggcacgta 1080 ccatcttttt cagctactga tgaaaaaatg gtgtaacgtg cttctacacc atttttcaat 1140 tgtgcttgaa aaatgccttc ttttggcgtt accatttttc ctcgtaccaa cttttgttgg 1200 caaggagaaa aaaatggcac atgtaacgca ttatcacagg ggaagcgtgg cagaaattgt 1260 gcaccacaat tcacgaacag aaaattattc aaacagggat atagacgcca gtagaagtaa 1320 gtacaattac agcctttcgg ggcacgagaa tgatttttcg tattacacgc aaaggctctc 1380 ggaagtacac tgcatgaagc gtgcaaacgt ttgtacgctt tctagttggg tcgtgacgct 1440 tccttc tggc gttcctcaag atcaacataa gttgtttttt gctgaaactg taaatttcct 1500 aaaatctcgt tacggagaac agaactgcat aagtgctgat gtacattacg atgaaacaac 1560 tccgcatcta catttcactt ttattcctgt agtttttgac gaaaaaaaac aaagggaaaa 1620 agtctctcgc aaagagcttt ttacgcttaa ggagctttac tccttccatg aagatctcga 1680 aaagcacctg caagaaaagc ttggaaaaaa cataaaaatt cgtaccggta aaacagaaaa 1740 aaatatttct gtaagggaat taaaacaaaa aaccgcacag aattgcgtag aaatcgtcga 1800 gaatgcgaag caggaggcag agagtatctt gagtaaggca aacgaagaga aacgcaatat 1860 agagcaattt tttgcgattg agagcgatgt agcaaaaaaa tacaaattat caaaacagga 1920 atacgacaaa gctgcagagc tggacttcaa agctgtaaac ggtcaactaa agcttccgtg 1980 ggttcctgtc cctaagaagc ttgtatcaaa gctagtgaga gggtacgccc acttacaggc 2040 agtagaggag acaacgaaag caatcggaga aaaagccaaa gaactaaaaa gcactcacta 2100 tgacatggtg cagttggagg ataccctagc gagaaaacac caggagaacg aacgagcctg 2160 ggcacttgtg aaggagctgg aaaagaagct ggaaaaatac gaaccaaagg agctcaaaca 2220 gcaagaaaaa agcaaaaaac gtggattttc ccgataacca aagggggggt aaaggggggt 2280 tttagccccc t tttattaac cttgagaagc acctgcgtgc aggggcttct cgctttatta 2340 atcaaaaaag ttgctaaaat aggcaactga agttgcccta tacgtcaact tttcaaaaaa 2400 gttgccaaaa agggaactgg agttgcctat Artgtial 213 <220> <br> <br> <br> <br> 400> 2 atctctactg gcgtctat 18 <210> 3 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 3 caaawaggga actggag 17 <210> 4 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 4 gcttccttct ggcgttcc 18 <210> 5 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 5 cccaggctcg ttcgttct 18 <210> 6 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 6 aagcagacat cgcaraag 18 <210> 7 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 7 actcttcgtc ctctacca 18

[0032]

[Sequence List Free Text]

 SEQ ID NO: 2: Synthetic DNA SEQ ID NO: 3: Synthetic DNA SEQ ID NO: 4: Synthetic DNA SEQ ID NO: 5: Synthetic DNA SEQ ID NO: 6: Synthetic DNA SEQ ID NO: 7: Synthetic DNA

[Brief description of the drawings]

FIG. 1 is a restriction map of a plasmid of the present invention.

Claims (2)

(57) [Claims] 1. A 2444 bp plasmid having the following restriction map, which contains a self-replicating sequence derived from Luminobacter amylofilus and is capable of growing in a rumen microorganism. Embedded image 2. A plasmid comprising the following DNA (a) or (b): (a) DNA consisting of the nucleotide sequence represented by SEQ ID NO: 1 (b) Hybridizing with the DNA consisting of the nucleotide sequence represented by SEQ ID NO: 1 under stringent conditions, and performing self-replication and growth in a rumen microorganism can do
DNA