JPS6058073A - Dna - Google Patents

Abstract

NEW MATERIAL:DNA having promotor activity of gene to code protein of surface layer of Bacillus brevis. USE:A promoter having high activity. Being inserted into a plasmid capable of multiplying in the cell of Escherichia coli, bacterium belonging to the genus Bacillus, etc. PREPARATION:For example, chromosome DNA of Bacillus brevis 47 mold (FERM-P 7224) is partially hudrolyzed with restricted enzyme Hind III, to give linear DNA having various length. While, plasmid pBR322 is similarly treated, to give linear plasmid pBR322, which is blended with linear DNA, and treated with T4DNA ligase so that both are linked. The linked DNA is transformed with Escherichia coli HB101, cultivated in L-broth medium containing ampicillin, to give a transformed strain having ampicillin resistance. A rabbit antibody to a mixture of protein constituting surface layer structure of Bacillus brevis 47 mold, having 130,000 and 150,000 molecular weight is prepared, and NT-100 strain is obtained from the transformed strain by the use of this antiserum.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to DNA, and in particular to promoter Jf-M.
, Sega-J-V w %I A I+r FilA
→t # J tl +ys k x The present inventors have discovered that Bacillus brevis
Noting that the surface protein of Bacillus plebis is produced in large quantities outside of the bacterial cell, we attempted to isolate its promoter, and finally found I) HA that has the promoter activity of the gene encoding the surface protein of Bacillus plebis. They succeeded in separating it.

That is, E. coli plasmid pBR5 was used as a vector.
22 was selected, and the plasmid pBR522 was digested with restriction enzyme H
1n4 [1], and the 5'-terminal phosphate group was cleaved with bacterial alkaline replacement phosphatase (Bacteria
A linear plasmid pBR522 was prepared by treatment with (aljcaline phosphatase). On the other hand, from cells of 47 Bacillus plevis bacteria, Saito
Miura's method (Bi-oahirn.

Biophys, Acta, 72.619 (19
65) Prepare donor DNA by preparing chromosomal DNA by
Served to A. This chromosomal DNA is digested with restriction enzyme H1n+1l.
Linear DNAs of various lengths were prepared by partial hydrolysis with ll. Next, linear plasmid pER322 and linear DNA
were mixed and the two were ligated with T4DNAIJ gauze. The ligated DNA was transferred to E. coli HB101 by Lehrberg.
, B.M., and Oohen, S.H.

method (J, Bacteriol, 19.107
2-1074 (1974)), and this was transformed into L-broth medium (tryptone (Dirco) 1%,
Yeast extract (Difco) 0.5%, 1 JaOtO
,5%, MgSO4117400,025%r pH7
,2) ampicillin 50μ? /d was added to obtain an ampicillin-resistant transformant.

A clone having a gene encoding the surface structure protein of Bacillus plevis 47 was selected from the transformed strain of C. cholerae by the following method.

The surface structure of Bacillus plebis 47 is composed of major proteins with molecular weights of 130,000 and 150,000. A rabbit antibody against a mixture of these proteins was prepared, and using this antiserum, a clone containing the protein gene constituting the surface structure was isolated from among the above-mentioned transformed strains by Kemp, D.
J, sn6 Qowmtm, A, ? , the method (P
roa, Natl, Acad, Sci, U8A
, 78, 4520-4524 (1981)) to obtain strain NT100.

When this clone was examined for its reactivity with antibodies to proteins with a molecular weight of 150,000 and 150,000, it was found that it produced a protein that reacted only with antibodies to a surface structure protein with a molecular weight of 150,000.

Figure 1 shows plasmid pNT10 contained in the NT100 strain.
0 restriction enzyme map. In the figure, the thick line part is the bus fk
Chromosomal DNA derived from Plebis 47 is shown.

Next, plasmid pNT 100 was injected with various restriction enzymes (Hl
Plasmids of various lengths were prepared by cutting with ndlll, HpaI, BamHI) and religating. E. coli HB101 was transformed using these plasmids by the method described above, and an antigen-antibody reaction was performed in the same manner as above using an antibody against a surface protein with a molecular weight of 150,000 to examine clones that produced a surface protein with a molecular weight of 150,000. , determine the primer site in pNT 100 [also. As a result, it is clear that there are 71 DNA sequences in the b7 fragment shown in Figure 1 that exhibit the promoter activity of a surface protein with a molecular weight of 150,000 bt. In other words, this DNA is Ba
291 bp downstream of the site cleaved by mHI)li
nf I, 156 bp downstream of A
It has a site that is cleaved with lui and a site that is cleaved with HpaI 268 bp downstream thereof.

When the DNA of the present invention is inserted into a plasmid that can proliferate in bacterial cells, such as Escherichia coli or Bacillus flexor microorganisms, it will function as a highly active promoter. That is, by connecting a useful foreign gene downstream of the promoter DNA inserted into a plasmid and introducing the obtained recombinant DNA into the cells of Escherichia coli or Bacillus microorganisms, these microorganisms can absorb useful substances such as proteins. Transformed to produce polypods.

Examples of foreign genes here include those derived from eukaryotes and those derived from prokaryotes. Examples include human genes (eg, inter7erin, insulin, etc.), enzyme protein genes of uE products (eg, tryptophanase, aspartate ammonia lyase, etc.).

Linear DNAs of various lengths were prepared by partial hydrolysis with the limiting enzyme Bind[[. On the other hand, plasmid pBB 522
A linear plasmid pBR522 was prepared by cutting the plasmid with the restriction enzyme H1nLl[l and removing the 5'-end phosphate group by bacterial φ alkaline phosphacose treatment.

These linear DNAs were mixed, and both DNAs were ligated by the action of T and DIIA ligase.

Lederber the ligated DNA into E. coli HB101.
g, H.

M, aaxd 0ohen, 8. N, method (J
, Baateriol. , Su1゜1072~1074
(1974)) and further transformed with I+ −
Ampicillin 50μF in broth medium! -/ld was applied to a medium and cultured to obtain an ampicillin-resistant transformant.

Next, a rabbit antibody against a mixture of proteins with molecular weights of 130,000 and 150,000 that constitutes the surface structure of Bacillus plebis 47 was prepared, and this antiserum was used to construct the surface structure of Bacillus plevis in the above-mentioned transformed strain. Protein m 'm M ff-j 4
-) + --/ Sea v+艷η TT T aM
+4 1'! AWm6YA, F, method (Proc,
Natl, Aoad, Sci, USA, 7
8.4520-4524 (1981)) to obtain the NT100 strain. When we examined the reactivity of this 1JT100 strain with antibodies against proteins with molecular IIk of 130,000 and 150,000, we found that this strain had a molecular weight of 15,000.
It has become clear that the protein that reacts only with antibodies of surface structural proteins of 10,000 yen is produced.

Using this NT100 strain, Birnboin, H.
O,, Doly, ,r.

(Nucleio Ac1ds Rea, 7, 15
15-1525 (1979)) to detect the plasmid and measure its molecular weight. As a result, the molecular weight was 6. This plasmid was named pnTlao. The restriction enzyme cleavage map for pHT100 is shown in FIG.

Plasmid pNT 100 was digested with various restriction enzymes (H1n4n
Seven plasmids of various lengths were prepared by cutting with 1°HpaI, BamHl) and religating, and these plasmids were isolated from E. coli HB by the method described above.
101 and obtained 7 types of clones, with a molecular weight of 1
An antigen-antibody reaction was carried out in the same manner as described above using an antibody against a surface structure protein of 50,000, and the promoter site of a surface structure protein with a molecular weight of 150,000 was determined. The details are shown below.

IindInDNA
Escherichia coli produced a protein that reacted with the antibody of 1,150,000 molecules of protein with only 7 radaments (Fig. 2B). Thursday V'11! +echerichia coli HB
101/pNT 100 is P to Microtechnical Research Institute! ! inM
The plasmid pNT 100 deposited as P-7231 (experiment using Hp&I) was cut with HpaI and religated to generate a plasmid contained in E. coli that produces a protein that reacts with the antibody of the protein molecule fij1.15 million. As a result of the analysis, three types of strains, 0.D and E in Figure 2, were obtained.In other words, the C fragment (Hpai-B
amHl, 400 bp), b fragment (B
amaI-Hpal, 715 bp) and C
7 fragment (1 (paI-Hpai, 215 b
There were various ways of connecting p). In clone D, the method of ligation of fragments B, b, and c is pNT 1
00, and produced a protein that reacted with the antibody of a protein with a molecular weight of 150,000. Furthermore, clone E lacked the C fragment, but like clone D, it produced a protein that reacted with the antibody of a protein with a molecule of 150,000 molecules. Therefore, it is clear that the promoter of a protein with a molecular weight of 150,000 exists in the a, b7 fragment.

Experimental plasmid pliT 100 using both restriction enzymes, BamHI and Hph
a) Clone F (
This bacterium E, coli HB 101 /pNT 152
Fl! ! It has been deposited as BMF-7250. )was gotten. As shown in FIG. 2, clone r lacks the &7 fragment, but produces a protein with a molecular weight of 150,000 that reacts with the antibody. Also, b
Clone G (Honkanmi Jikumi HB 101/pNT
As mentioned above, clones A-(J) produce proteins that are positive in reactivity with the antibody of a protein with a molecular weight of 150,000. Judging comprehensively based on these results, clones A-(J) There is a DNA sequence in fragment b in the figure that shows the promoter activity of a protein with a molecular weight of 150,000.

[Brief explanation of drawings]

FIG. 1 shows a restriction enzyme map of plasmid plJT100, and FIG. 2 shows various plasmids introduced into E. coli. q! Applicant: Shige Uko Takashi Figure 1 = Figure 2 ← B, brevis DNA - One ship, one hit ER322 -
−→Ma HindEeeW Hpa Engineering ψ BamH Engineering (#bf) p) Procedural amendment (spontaneous) October 6, 1980 Commissioner of the Japan Patent Office Kazuo Wakasugi 1, Indication of Case Patent Application 165065 1982 λ Invention Name NA 5 Relationship to the case of the person making the amendment Patent applicant Shigezo Udaka 4, agent 1-1-10 Kyobashi, Chuo-ku, Tokyo 104 & Brief detailed explanation of the invention of the specification subject to amendment & “Birnboin, 11,” on page 7, line 8 of the specification of contents of the amendment.
C5,” to “Birnboim, l(,0,,J
Correct.

Claims (1)

[Scope of Claims] 1. DNA having promoter activity of a gene encoding a surface protein of Bacillus plevis. 2. I) KB&l1) cleavage site in II in HA chain,
The DNA according to claim 1, which has a site cleaved with HLnf I 291 bp downstream thereof, a site cleaved with Alu I in the 1 56 bpT stream, and a site cleaved with Hpa I 268 bp downstream thereof. . & The DNA according to claim 1 or 2, which is inserted into a plasmid that can proliferate in bacterial cells.
. 4. The DNA according to claim 1 or 2, which is introduced into bacterial cells by being inserted into a plasmid that proliferates in bacterial cells.