KR920009543B1 - Novel beta-urogastrone gene - Google Patents

Abstract

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Description

β-urogastron gene, corresponding recombinant plasmid, corresponding transformant and preparation method thereof, and preparation method of β-urogastron

1 schematically shows the synthesis of oligonucleotides by the solid phase method,

2 shows a method for ligating oligonucleotides A-1 to A-16 to subunit A and introducing into plasmid pBR322 derived from subunit E. coli to obtain recombinant plasmid pUG1,

3 shows a similar method for preparing recombinant plasmid pUG2 by introducing subunit B into plasmid pBR322,

4 shows a method for preparing recombinant plasmid pUG3 from pUG1 and pUG2,

5 shows the results obtained by analyzing the nucleotide sequence of oligonucleotides A-3 by electrophoresis and two-dimensional fractionation of homochromography,

Figure 6 shows the preparation of the recombinant plasmid pGH37,

Figure 7 shows the preparation of the recombinant plasmid pGH35,

8 shows a method for preparing the recombinant plasmid pEK28,

Figure 9 shows the preparation of recombinant plasmids pUG102 to pUG122 and recombinant plasmids pUG103-E and pUG117-E,

10 shows the preparation of recombinant plasmids pBRH02 and pBRH03,

FIG. 11 shows the MboII restriction map of pUG3 containing H fragment (179b.p) containing the β-urogastron gene of the present invention.

12 shows a method for preparing recombinant plasmids pUG2301 to pUG2303,

Figure 13 shows the preparation of recombinant plasmids pUG2101 to pUG2105,

14 shows the preparation of recombinant plasmids pUG2701 to pUG2703,

Figure 15 shows the preparation of the recombinant plasmids pUG1102 and pUG1105,

Figure 16 shows the preparation of the recombinant plasmid pUG1004,

Figure 17 shows the preparation of the recombinant plasmid pUG1201,

Figure 18 shows the preparation of the recombinant plasmid pUG1301,

Photos 1 to 5 show the analysis results of the nucleotide sequences of the recombinant plasmids obtained in the Examples by the Membrane-Gilbert method, respectively.

The present invention relates to novel β-urogastron genes, corresponding recombinant plasmids, corresponding transformants and methods for their preparation and methods for preparing β-urogastron.

β-Eurogastron is a polypeptide hormone synthesized in human salivary glands (eg Heitz et al., Gut, 19 , 408-413 (1978)) and has a primary structure containing 53 amino acids of the following sequence (H Gregory et al., Int. J. Peptide Protein Res., 9 , 107-118 (1977).

In the specification, amino acids are represented by the following symbols.

Asn: Asparagine Ser: Serine

Asp: Aspartic Acid Glu: Glutamic Acid

Cys: Cysteine Pro: Proline

Leu: Leucine His: Histidine

Gly: Glycine Tyr: Tyrosine

Val: Valine Met: Methionine

Ile: Isoleucine Ala: Alanine

Lys: Lysine Gln: Glutamine

Arg: Arginine Trp: Tryptophan

Phe: Phenylalanine

β-Eurogastron has physiological activities such as inhibition of gastric acid secretion and promotion of longitudinal growth (Elder et al., Gut, 16 , 887 to 893 (1975)), and thus are useful for the treatment of ulcers and wounds.

Since β-urogastron is excreted in human urine in small amounts, this compound is currently produced by extracting, separating and purifying urine. However, this method has a problem in that a large amount of the compound cannot be easily obtained because this compound is a small component of human urine.

On the other hand, European Patent Application Publication No. 0046039 describes an attempt to prepare β-urogastron by genetic engineering technique using a synthetic β-urogastron gene. However, the study only describes synthetic genes with specific nucleotide sequences without indicating whether other genes can be expressed by β-urogastron by a similar method or to which genes of a particular nucleotide sequence can be applied.

Multiple nucleotide sequences can encode the amino acid sequence of β-urogastron. Nevertheless, it is difficult to guess whether these genes can express β-urogastron through genetic engineering techniques or whether these genes are suitable for adaptation to genetic engineering techniques. Therefore, many experiments and efforts are made to determine the most suitable nucleotide sequence.

An object of the present invention is to provide a novel β-urogastron gene which is completely different in nucleotide sequence from the gene described in the above publication and which is capable of expressing β-urogastron through genetic engineering techniques.

It is another object of the present invention to provide a gene suitable for expressing β-urogastron by genetic engineering techniques.

Another object of the present invention is to provide novel recombinant plasmids and transformants corresponding to the novel β-urogastron gene.

It is still another object of the present invention to provide a method for mass production of high purity β-urogastron by using a novel gene by genetic engineering techniques.

The above and other objects of the present invention will become more apparent from the following description.

The inventors have conducted a number of experiments to find that gene I having the following nucleotide sequence can fulfill the purpose of the present invention.

Gene I:

Letters represent the purine or pyrimidine bases that form the nucleotide sequence. Where the symbol for base is:

A adenine G guanine

C cytosine T thymine

Gene I itself is novel and unknown and is obtained by determining a particular nucleotide sequence from a very large number of possible nucleotide sequences.

Gene I has the following characteristics.

(1) β-urogastron can be very advantageously expressed by genetic engineering techniques.

(2) The trinucleotide codons constituting gene I can be housed in all host cell, in particular Escherichia coli (E. coli), which can be readily used to safely perform high expression.

(3) Certain restriction enzyme recognition sites can be provided within and at both ends of the gene, which sites can be engineered as desired to facilitate ligation with other genes and insertion into plasmid vectors.

(4) To prepare gene I, the constituent oligonucleotide can be ligated to a block, and this block can be easily ligated to subunits as desired without actually needing ligation.

(5) In expressing β-urogastron as a fusion protein, the desired β-urogastron can be obtained by using a method that can easily remove unnecessary portions.

In fact, when expressing β-urogastron using gene I, the restriction enzyme recognition site is a gene for ligating the promoter necessary for expression, syn-dalgarno sequence (hereinafter abbreviated as "SD sequence"), vector, etc. It may be provided at the front end and / or the rear end of the. Moreover, if desired, start codons and / or stop codons may be provided at the top and bottom of the gene, respectively. The recognition site, starting codon and stop codon are not particularly limited and may be desired.

Shown below is an extended surge comprising a restriction enzyme recognition site arranged at the top of gene I and a start codon and a stop codon and a restriction enzyme recognition site arranged at the bottom of gene I (hereinafter abbreviated as “gene I”). Is an example of a gene with) and these sites and codons are arranged in sequence, and gene II further comprises the following restriction enzyme recognition site.

Gene II:

Symbols representing restriction enzymes in the sequence have the following meanings.

E: EcoRI Ta: TaqI

Bg: BglII S: Sau3AI

Mb: MboII Hf: HinfI

Ba: BamHI Hd: HindIII

Ml: MluI Th: ThaI

The present invention is not limited to genes I and II, but includes nucleotide sequences as other genes that are actually identical to them and capable of expressing β-urogastron.

In the synthetic preparation of gene II, it is advantageous to form gene I or II such that it is divided into a front half and a back half. For example, by separating the middle portion of gene I or II, a subunit having the front half of the nucleotide sequence of gene I or I, and another subunit having the rear half of the nucleotide sequence of gene I or II, is produced and these genes I Or bind two subunits of II together to gene I or II. A subunit having the front half of the nucleotide sequence of gene II may further have a restriction enzyme recognition site at its rear end, and another subunit having a rear half of the nucleotide sequence of gene II may have a restriction enzyme recognition site at its front end. And combining these subunits together yields gene II.

More specifically for explanation, the former subunit contains the front half of gene II and the subunit A with a restriction enzyme (Bam HI) recognition site at its rear end, the latter subunit contains the back half of gene II Subunit B with a restriction enzyme (Hind II) recognition site at its front end. These subunits are shown below.

[Subunit A]

[Subunit B]

Subunits A and B are synthesized, for example, by the following method. Oligonucleotides having 11, 13 or 15 bases are synthesized (A-1 to A-16, and B-1 to B-16 ie 32 oligonucleotides). Next, 4 to 6 of these oligonucleotides are combined and ligated to form a block (blocks 1 to 7, 7 or 7 blocks). These oligonucleotides and blocks are shown below.

Next, the blocks 1 to 3 are ligated together to form subunit A, and the blocks 4 to 7 are ligated together to form subunit B.

The invention is explained in more detail with reference to the accompanying drawings and photographs.

The method itself for forming the gene II of the present invention is known. Oligonucleotides forming gene II may be prepared by known methods, for example solid phase methods (e.g., H, Ito et al., Nucleic Acids Research, 10 , 1755-1769 (1982)), which are briefly described below. .

When using the solid phase method, oligonucleotides are synthesized by continuously binding a mononucleotide or dinucleotide with a nucleoside supported on a polystyrene resin to obtain nucleotides of a designated sequence as shown in FIG.

The resin supporting the nucleotide may be reacted with, for example, N- (chloromethyl) phthalimide with the resin using a partially crosslinked polystyrene resin and the product with hydrazine to obtain an aminomethylated polystyrene resin, Nucleosides having no 5 'hydroxyl group at and having a protected amino group are prepared by ligation using succinic acid as a spacer.

On the other hand, various methods of preparing mononucleotides or dinucleotides are known (e.g. C. Broka et al., Nucleic Acids Research, 8 , 5461-5471 (1980)). For example, a mononucleotide reacts a nucleoside with 0-chlorophenylphosphorodichlorate, triazole and protected 5′-hydroxyl group with a dimethoxytrityl group (DMTr) in the presence of triethylamine The triazolide obtained can be prepared by reacting with β-cyanoethanol in the presence of 1-methylimidazole as a catalyst and then eluting the reaction product with chloroform-ethanol by silica gel column chromatography. This method provides a fully protected mononucleotide.

The dinucleotide is treated with the fully protected mononucleotide obtained above with benzene sulfonic acid or similar acid to give a mononucleotide free of 5'-hydroxyl groups and reacted with these monotriazolides obtained above, followed by reaction The product can be prepared by eluting with chloroform-methanol by silica gel column chromatography.

Solid phase synthesis of oligonucleotides is advantageously performed using a DNA synthesizer (eg, DNA synthesizer of Bachem Inc., USA). The nucleotide support resin obtained above is placed in a reaction vessel, washed with dichloromethane-isopropanol, and then a zinc bromide solution dissolved in dichloromethane-isopropanol is added to the resin to remove the dimethoxy trityl group at the 5 'position. Repeat this method several times until the color of the solution disappears. The resin was washed with dichloromethane-isopropanol, washed with triethyl ammonium acetate solution dissolved in dimethylformamide to remove the remaining Zn ²⁺ , washed with tetrahydrofuran and exposed to nitrogen gas stream for several minutes to dry. Let's do it. Separately, fully protected dinucleotides or mononucleotides are dissolved in pyridine and triethylamine is added, then the resulting solution is shaken, left at room temperature for several hours and then evaporated under reduced pressure. The resulting triethylammonium salt is dissolved in pyridine and azeotropically evaporated several times with pyridine for drying. The salt of nucleotide is dissolved in a solution of mesitylenesulfonyl-5-nitrotriazole (MSNT, condensing agent) dissolved in pyridine. The resulting solution is added to the dried resin and left at room temperature. The liquid portion of the reaction mixture is removed, the resin portion is washed with pyridine and then reacted with acetic anhydride using dimethyl amino pyridine as catalyst in tetrahydrofuran-pyridine to mask unreacted hydroxyl groups. Finally the resin is washed with pyridine to complete one solid phase synthesis. The nucleotide sequence is extended up to one or two chains in length. By repeating the above method, mononucleotides or dinucleotides can be continuously combined with the resin to the desired length, whereby fully protected oligonucleotides can be obtained in a resin supported form.

Tetrimethyl guanidine-2-pyridine aldoximate solution dissolved in pyridine-water is added to the resulting resin and the mixture is left to heat. The resin is filtered off and washed alternately with pyridine and ethanol. The washes and filtrates are combined together and concentrated under reduced pressure. The concentrate is dissolved in an aqueous solution of triethylammonium bicarbonate (TEAB) and washed with ether. This aqueous solution is subjected to Sephadex G-50 column chromatography (eluent: TEAB solution). Fractions are collected and the optical density of each fraction is measured at 260 nm. Concentrate the fraction containing the first eluting peak. The concentrate is purified, for example by high performance liquid chromatography, until a single peak is obtained. Since the oligonucleotide thus obtained was protected at its 5 'end by a dimethoxytrityl group, the product was treated with an aqueous acetic acid solution to remove the protecting group, followed by high performance liquid chromatography or the like to purify until a single peak was obtained. .

The desired oligonucleotides were prepared by the methods described above, and the nucleotide sequences were checked by the two-dimensional fractionation method using electrophoresis and homochromatography and / or the Membrane-Gilbert method, respectively, followed by the preparation of blocks and subunits. Used for

Each nucleotide sequence is checked by a two-dimensional fractionation method for checking the nucleotide sequence, and then used to prepare blocks and subunits.

The two-dimensional fractionation method for checking nucleotide sequences is based on the method of Wu et al. (E. Jay. RA Bambara, R. Padmanabhan and R. Wu, Nucleic Acids Res., 1 , 331 (1974)). Can be performed.

To carry out this method, lyophilized oligonucleotides are dissolved in distilled water to a concentration of about 0.1 μg / μl. A portion of this solution is treated with γ-32P-ATP and T ₄ polynucleotide kinase and the 5 'end is labeled with 32P and partially digested with venom phosphodiesterase from the venom. The product is spotted on a cellulose acetate film and electrophoresis is performed for the primary development to decompose the product according to the difference in base. The developed product is transferred to a diethylaminoethyl cellulose (DEAE cellulose) plate and a second development is performed using a solution of partially hydrolyzed RNA called a homo mixture (this method is called homo chromatography). In this method, oligonucleotides are separated along the chain length. As a result, the nucleotide sequence of the oligo nucleotides is read by autoradiography starting from the 5 'end.

If it is difficult to check the sequence by this method, the Maxsam-Gilbert method can be used if necessary (AM Maxam and W. Gilbert, Proc. Natl. Acad. Sci., USA, 74, 560 (1977), AM). Maxam and W. Gilbert, Methods in Enzymol., Vol 65, p. 499, academic Press 1980).

This method, also called chemical cleavage method, is used for the specific reaction of cleaving oligonucleotides at specific base positions, and the sequence is read from the 5 'or 3' end by a band exhibited by electrophoresis. Base-specific reactions are as follows. Guanine is specifically methylated with dimethylsulfate. Guanine and adenine are depurinated in the presence of acid. Both thymine and cytosine react with hydrazine in low concentrations of salt, but cytosine alone reacts with hydrazine in high concentrations of salt. After completion of the reaction to the tetrabase, each reaction mixture is reacted with piperidine to replace the ring-opening base and catalyze the β-removal of the two phosphates from the sugar, and finally the DNA strand is cleaved at that base. . The resulting reaction mixtures were each subjected to polyacrylamide gel electrophoresis to identify the nucleotide sequences according to the reaction to produce each band.

The nucleotides are then ligated using T ₄ DNA ligase as shown in FIG. For correct ligation, 16 oligonucleotides A-1 to A-16 corresponding to subunit A were set in three sets, as shown in Figure 2, namely A-1, A-2, A-3, A-14, A- Block 1 containing 15 and A-16, A-4, A-5, A-6, A-11, A-12 and Block 2 containing A-12, A-7, A-8, A- Ligation is divided into blocks 3 containing 9 and A-10. By electrophoresis, blocks 1 to 3 having the correct sequence are obtained and further ligated to give subunit A.

More specifically, some of the 5'-terminus of 16 oligonucleotides A-1 to A-16 are labeled with 32P using γ-32P-ATP and T ₄ polynucleotide kinase and the remaining 5'-terminus. The hydroxyl group is labeled with ATP and the remaining 5'-terminal hydroxyl group is phosphorylated with ATP. To form each triblock, oligonucleotides are collected and ligated using T ₄ DNA ligase, and the product is electrophoresed on a polyacrylamide gel to separate the desired block. The three blocks thus obtained are ligated using T4 DNA ligase to prepare subunit A. While this structure can be prepared in the ligation reaction, subunit A can be readily obtained by cleavage with the restriction enzymes EcoRI and BamHI. Subsequently, as shown in FIG. 3, the known plasmid vector pBR322 derived from E. coli and EcoRI and BamHI are cleaved, and subunit A is inserted into the vector to obtain a recombinant plasmid pUG1.

The same method is also carried out on subunit B. As in the case of subunit A, 16 oligos and nucleotides B-1 to B-16 are ligated into four sets as shown in FIG. 3, and the blocks are ligated with each other to prepare subunit B. If dimers are prepared, cleavage with the restriction enzymes HindIII and BamHI yields subunit B. Plasmid vector pBR322 is cleaved with HindIII and BamHI and subunit B is inserted into the vector to obtain recombinant plasmid pUG2 as shown in FIG.

In addition, as shown in FIG. 4, the recombinant plasmid having pβ1-reduced gastron structural gene (gene II) by cutting pUG1 with restriction enzymes HindIII and SalI, inserting a fragment removed from pUG2 by the same restriction enzyme into pUG1. Prepare pUG3.

pUG1, pUG2 and pUG3 are recombinant plasmids containing subunit A, the front half of the pBR322 and β-urogastron structural genes, subunit B, the rear half of the gene, or the entire structural gene, respectively. These recombinant plasmids are known to the host and transformed into a host such as strain HB101 of E. coli which can be readily used according to the calcium method (E. Lederberg and S. Cohen, J. Bacteriol., 119 1072 (1974)). It can be propagated in large quantities by introduction.

Whether pUG1, pUG2 and pUG3 can be present in a host such as strain HB101 of E. coli can be checked by the following method. After obtaining the plasmid by alkali extraction method, it is checked whether the BglII recognition site which does not exist in vector pBR322 exists in pUG1 and pUG2. Likewise, it is checked whether the MluI cleavage site which is not present in pBR322 exists in pUG2 and pUG3.

According to the alkali extraction method, E. coli containing the plasmid is incubated to collect the cells, and the cell wall is lysed by lysozyme. The cells are disrupted using a mixture of sodium hydroxide and sodium lauryl sulfate, the DNA is denatured, and then neutralized with sodium acetate buffer. The chromosomal DNA remains denatured, but the plasmid, the DNA of the chromosome, initially retains the double-stranded form. These properties are used to collect plasmids. This plasmid is purified by density gradient ultracentrifugation using cesium chloride and ethidium bromide and passed through a Biogel A 50m column to remove RNA. Thereby, a high purity plasmid can be obtained in large quantities. In this way the β-urogastron gene (gene II) of the present invention can be obtained.

Next, a method of introducing the β-urogastron gene into the host cell will be described.

The host cell used in the present invention is not particularly limited, and for example, known ones such as E. coli, Bacillus, Pseudomonas, yeast and the like are used, of which E. coli cells are preferred.

Methods of transforming the β-urogastron gene using E. coli include those that directly express β-urogastron, and those that are expressed with a protein fused with β-lactamase or other protein. .

In order to directly express the β-urogastron gene, a promoter and SD sequence must be introduced on top of the β-urogastron gene of the recombinant plasmid. The promoter is not particularly limited, and a preferred promoter is one capable of performing high expression such as λP _L , a left protective promoter of λ phage, lac UV5, etc. present on the β-galactosidase gene of E. coli. . When λP _L is used as a promoter, the SD sequence is not particularly limited, and it is preferable to use the 4-base sequence of AGGA. In addition, when using lac UV5 as a promoter, it is preferable to use an SD sequence or a chemically synthesized sequence under the lac UV5 promoter.

The system for directly expressing the β-urogastron gene will be described with reference to the case of using the λP _L -SD sequence-β-urogastron gene.

λP _L is a good promoter (J. Hedgpeth et al., Molecular and General Genetics, 163, 197-203 (1978)), but a fully activated λP _L promoter is not lethal because it has a lethal effect on host E. coli cells. after growth the cells under conditions which also acts, it is necessary to effect the P _L. On the other hand, CI857, which is a gene in λ phage, is one of the mutation genes of the CI repressor acting on the λP _L operator. At low temperatures (below about 30 ° C.), the CI857 repressor binds to the operator and completely inhibits the action of λ P _L as a promoter to grow E. coli. Therefore, after propagating host cells in this state, λ P _L is acted by raising to high temperature (37 ° C. or higher). Moreover, plasmids such as pBR322, which are known and commercially available and have strict replication mechanisms, are not difficult to be compatible with each other but can coexist in the same E. coli cells.

Thus, a recombinant plasmid pGH37 inserted with the tetracycline-resistant plasmid vector pSC101 (showing a lac UV5 promoter on top of it for effective expression of CI857) was formed as shown in FIG. It is appropriate to introduce a plasmid into E. coli (HB101 strain) to prepare a transformant (ECI-2 strain) that can be used as a vector for host to express β-urogastron under the control of the λP _L promoter. .

According to the invention, the λP _L -SD sequence-β-urogastron gene is introduced into eg pBR322 to obtain β-urogastron expression vector, which is used for transformation of strain ECI-2. There is provided a so-called 2-plasmid system in which two useful plasmids coexist in E. coli cells.

Using this system, when the cells are incubated at 30 ° C., for example, a CI857 repressor encoding pGH37 is coupled to an operator for the λP _L promoter of the second plasmid to grow the cells. After sufficient growth of the cells in this state, when the temperature is raised to 40 ° C., for example, the CI857 refresher dissociates from the operator, indicating the activity of the λP _L promoter for expression of β-urogastron.

per et al, Nature, 289, 555∼559(1981)).A similar concept to this case can be applied to the expression of fibroblast interferon, SV-40 small t antigens, etc., but lambda ribogen is used as a host to introduce the DNA of lambda phage carrying the CI857 gene into the host chromosome (R. Derynck et al. Nature, 287, 193-197 (1980), C. Derom et al, Gene, 17, 45-54 (1982), K. K

per et al, Nature, 289, 555-559 (1981).

However, using the system of the present invention, the CI857 gene is introduced into another tetracycline resistant plasmid. Therefore, the system of the present invention has no advantage of inducing proliferation of lambda phage introduced into the host chromosome, and has the advantage that the strain can be easily controlled. Of course, the 2-plasmid system is first used as a system for expressing β-urogastron.

According to another system, portions of other protein genes, such as the β-lactamase gene, are ligated to the β-urogastron gene to express the β-urogastron gene as a fusion protein. This method has the advantage of protecting β-urogastron because the fusion protein is less likely to be degraded by proteases in E. coli. Another advantage is that the fusion protein migrates and accumulates in the E. coli cells in the outer envelope (SJ Chan et al, Proc. Natl. Acad. Sci., USA, 78, 5401-5405 (1981)), making it easy to isolate and purify. It is.

More particularly, a gene encoding two basic amino acids that can be cleaved with an enzyme to provide a cleavage site for taking β-urogastron from the fusion protein is inserted into the β-lactamase gene at the appropriate restriction enzyme cleavage site and β-urogastron gene is ligated to β-lactamase gene.

Preferred acid sequences of the two basic amino acids are -Lys-Arg or -Arg-Lys. Examples of enzymes that recognize amino acid sequences for cleaving β-urogastron from fusion proteins are kallikrein, trypsin and the like. Examples of restriction enzymes for cleaving the β-lactamase gene are XmnI, HincII, ScaI, PvuI, PstI, BglI, BanI and the like.

The β-lactamase-β-urogastron recombinant plasmid thus prepared can express a large amount of the fusion protein in E. coli. [Beta] -urogastron can be obtained by treating the resulting fusion protein with Kali Crane or the like.

The transfection system analyzes the nucleotide sequence of the gene directly by the Membrane-Gilbert method, thereby inserting the gene by mini-preparation or guidance method (HC Birnboim et al., Nucleic Acids Research, 7, 1513-1523 (1979)). Or by checking its orientation, or by radioimmunoassay of β-urogastron.

By culturing the transformant of the present invention thus obtained in a conventional manner, a large amount of high purity β-urogastron can be collected.

The invention is described in more detail with reference to the following examples, which do not limit the invention.

EXAMPLE

1) Preparation of Resin Supporting Nucleotide

Various nucleotide-supported resins are prepared by the following method.

1% by weight of cross-linked polystyrene resin ＂S-XI＂ (200-400 mesh), US Biorad Laboratory, 2.41 g of N- (chloromethyl) phthalimide, 0.22 ml of trifluoromethanesulfonic acid and 50 ml of dichloro Mix with methane by stirring at room temperature for 2 hours. After completion of the reaction, the resin was filtered, washed successively with dichloromethane, ethanol and methanol, dried under reduced pressure and refluxed overnight with heating to 50 ml of a 5% by weight solution of hydrazine dissolved in ethanol. The resin is filtered off, washed successively with ethanol, dichloromethane and methanol and dried under reduced pressure. Amino methylated polystyrene resin (2.5 g) obtained in the above method, mono succinic acid ester of 0.75 mM 5'-o-dimethoxytrityl nucleotide, 1.23 mM dicyclohexyl carbodiimide and 1 mM dicyclo To a mixture containing hexyl carbodiimide and 1 mM dimethylamino pyridine, add 30 ml of dichloromethane and leave at room temperature overnight. The resin is filtered, washed successively with dichloromethane, methanol and pyridine, immersed in pyridine-acetic anhydride (90:10 volume ratio), and left at room temperature for 30 minutes. The resin supporting the obtained nucleoside is filtered, washed with pyridine and dichloromethane, dried under reduced pressure and used for the solid phase synthesis reaction.

2) Synthesis of Dinucleotides

As an example the synthesis of fully protected dinucleotides having the base sequence of TA is described. Adenosine (13.14 g) and 6.34 g of triazole having 5 'hydroxyl group protected with dimethoxytrityl (DMTr) group and amino group protected with benzoyl group are dissolved in anhydrous dioxane. 8.35 ml of triethylamim are added to the solution while ice-cooling, 6.86 g of o-chlorophenylphosphoro dichlorate is added dropwise to the mixture over 10 minutes, and the resulting mixture is stirred at room temperature for 2.5 hours.

The resulting triethylamine hydrochloride was filtered off and the filtrate was concentrated to about 2/3 of its volume, followed by 3.6 g of β-cyano ethanol and 4.8 g of 1-methyl imidazole together with the concentrate at room temperature. Mix by stirring for 3 hours. The reaction mixture is concentrated under reduced pressure. The residue is dissolved in ethyl acetate, washed three times with aqueous 0.1 M sodium phosphate dibasic solution and twice with water, then concentrated under reduced pressure to afford 19.96 g of crude product. The product is purified by silica gel column chromatography (eluent: chloroform-methanol (98: 2)). The purification process is repeated to yield 15.12 g of fully protected adenosine mono nucleotides.

The adenosine mononucleotide (7.81 g) thus obtained was added to a 2 wt% solution of benzene sulfonic acid dissolved in chloroform-methanol (70: 30 vol. Ratio), the mixture was stirred with ice cooling for 20 minutes, and then neutralized with an aqueous sodium hydrogen carbonate solution. . The separated chloroform layer is washed with water and concentrated under reduced pressure to yield 7.11 g of crude product. The product was subjected to silica gel column chromatography and eluted with chloroform-methanol (97: 3 by volume) to yield 4.31 g of adenosine mononucleotide without 5 'hydroxyl group.

Thymidine (1.64 g) and 0.95 g of triazole whose 5 'hydroxy group is protected with a dimethoxytrityl group are dissolved in 21 ml of dioxane anhydrous and added to 1.25 ml of triethylamine solution, followed by 0.69 ml of o-. Chlorophenylphosphorodichlorate is added dropwise to the mixture over 5 minutes with stirring and ice cooling. The mixture is stirred at rt for 1 h. Triethylamine hydrochloride produced in the reaction was filtered off, and the filtrate was stirred with 1.1 ml of pyridine (1M) aqueous solution for 10 minutes. To this solution is added a dioxane solution (10 ml) of adenosine mononucleotide (1.17 g) free from 5 'hydroxyl group prepared above and 0.72 ml of 1-methyl imidazole, and the mixture is stirred at room temperature for 3 hours. The reaction mixture obtained is concentrated under reduced pressure, and the residue is dissolved in ethyl acetate, and then the solution is washed with this basic aqueous sodium phosphate solution (0.1 M) and water, and concentrated under reduced pressure to give 2.39 g of crude product. The product is subjected to silica gel column chromatography and eluted with chloroform-methanol (98: 2 vol. Ratio) to give 2.39 g of fully protected dinucleotide TA.

Various nucleotides are prepared by the same method as described above.

3) Synthesis of Oligonucleotides

Solid phase synthesis of A-1 of oligonucleotides, namely undeka nucleotide AATTCGAAGAT, is described.

The nucleotide T-supported resin (40 mg) prepared in Method 1) was placed in a reaction vessel, washed three times with dichloromethane-isopropanol (85: 15 vol. Ratio), and then zinc bromide (dissolved in dichloromethane-isopropanol) 1M) solution to remove the dimethoxytrityl group at the 5 'position. Repeat this method several times until the color of the solution disappears. The resin was washed with dichloromethane and washed with a solution of triethylammonium acetate (0.5M) dissolved in dimethylformamide to remove the remaining Zn ²⁺ , followed by further washing with tetrahydrofuran and for several minutes through the reaction vessel. Pass through nitrogen gas to dry.

The fully protected dinucleotide GA (50 mg) prepared in Method 2) is dissolved in 1 ml of pyridine, shaken with 1 ml of triethylamine and left at room temperature for several hours. The solution is evaporated under reduced pressure. The residue is azeotropically evaporated several times with pyridine to convert the nucleotides to triethylammonium salts. This salt is dissolved in 0.3 ml of a solution of mesitylene-sulfonyl-5-nitrotriazole (0.3M) dissolved in pyridine. The solution is added to the dried resin and allowed to react at room temperature for 60 minutes. The liquid portion was filtered out of the reaction mixture, the solid portion was washed with pyridine and then left in a mixture of 0.8 ml of a solution of methylamino-pyridine (0.1M) dissolved in 0.2 ml of acetic anhydride and tetrahydrofuran-pyridine. To protect the reactive hydroxyl groups. Finally, one solid phase synthesis process is completed by washing the resin with pyridine. At one time the nucleotide chain extends to two bases in length. By repeating the same method as described above, the fully protected undeca nucleotide AATTCGAAGAT is prepared in a resin-supported form by combining dinucleotides AA, CG, TT and AA with the resulting nucleotides by condensation.

The resulting resin (20 mg) was allowed to stand at 40 ° C for 1 hour with 0.6 ml of a tetramethyl guanidine-2pyridine aldoxin mate (0.5M) solution dissolved in pyridine-water (90:10 volume ratio). The resin is passed through a Pasteur pipette covered with cotton and filtered. The resin is washed repeatedly with pyridine and ethanol. The washes and filtrates are combined and concentrated at 40 ° C. under reduced pressure. The residue is dissolved in 2 ml of triethyl ammonium bicarbonate (TEAB, 10 mM). The solution is washed three times with ether. The aqueous phase is placed in a Sephadex G-50 column (2 x 100 cm) and eluted with 10 mM TEAB solution. The fractions are checked for absorbance at 260 nm. The fraction containing the first eluting peak is concentrated. The residue is subjected to high performance liquid chromatography (pump: model 6000A, detector: model 440, from Waters Associates) to obtain purified fractions with a single peak. The column used for high performance liquid chromatography was μ-bondapark C ₁₈ (Water Associates, USA), and an aqueous solution of acetonitrile-triethyl ammonium acetate (0.1M) was used as the eluent for the gradient elution (5 to 40% by volume). use. This purified undeca nucleotide is still protected with a dimethoxy trityl group at its 5 'end, so that the compound is treated with 80% by volume aqueous acetic acid solution for 15 minutes to remove the dimethoxytrityl group and a high-speed liquid until a single peak is obtained. Purify again by chromatography. The column is the same as described above, and acetonitrile-triethylammonium acetate (0.1 M) aqueous solution is used for the gradient elution (5 → 25% by volume).

Oligonucleotides A-2 to A-16 and B-1 to B-16 are synthesized in the same manner as above.

Table 1 shows the yield of each oligonucleotide measured by cleaving the oligonucleotide from the resin using 20 mg of the resin produced by the solid phase method and performing protecting group removal and purification.

The yield is calculated by measuring the absorbance of the final purified product at 260 nm, and the sum of absorbances evaluates the nucleotide base.

TABLE 1

4) Checking Oligonucleotide Nucleotide Sequences

The sequence is checked according to the above mentioned homochromatography and two-dimensional fractionation by electrophoresis.

It is found that oligonucleotides A-1 to A-16 and B-1 to B-16 each have the required nucleotide sequence. 5 shows the results obtained by analysis with oligonucleotide A-3, where A-3 has the sequence of GATTCTGAGTG from the 5 'end.

In addition, the nucleotide sequence of each oligonucleotide is checked by the above-mentioned Samsam-Gilbert method.

It is confirmed that oligonucleotides A-1 to A-16 and B-1 to B-16 each have a sequence of the required nucleotides.

5) formation of oligonucleotide blocks and subunits

Blocks and subunits are prepared by the method shown in FIG. 2 described in detail below.

First, a solution having a concentration of about 0.1 μg / μl by dissolving about 5 μg of each oligonucleotide A-1, A-2, A-3, A-14, A-15 and A-16 in distilled water (50 μl) To obtain. Each of these six aqueous solutions is filled in six different eppendorf tubes, each 10 μl (1 μg calculated by DNA). A mixed solution (6 μl) containing 250 mM Tris-HCI (pH 7.6), 50 mM magnesium chloride, 10 mM spimidine and 50 mM DTT was added to each tube and 0.5 μl of γ-32-P-ATP aqueous solution (Amersham International LTD., UK. Product), 0.5 μl of T ₄ polynucleotide kinase (manufactured by Takarashu Corporation, Japan) and 13 μl of distilled water are added to obtain a mixture of 30 μl. The mixture is reacted at 37 ° C. for 30 minutes, and further reacted for 30 minutes while adding 1 μl of 30 mM ATP aqueous solution. The reaction is terminated by heating at 100 ° C. for 2 minutes. The reaction mixture is cooled rapidly with ice. This puts 5 'terminal phosphorylated oligonucleotides A-1, A-2, A-3, A-14, A-15 and A-16 into another 1.5 ml Eppendorf tube, 10 μl each. . 40 μl of 250 mM Tris-HCI demand (pH 7.6), 40 μl of 50 mM magnesium chloride and 35 μl of distilled water are added to make the total volume 175 μl. The mixture is heated at 90 ° C. for 2 minutes and gradually cooled to room temperature. 10 μl 200 mM DTT aqueous solution, 10 μl 20 mM ATP aqueous solution and 5 μl (100 units) of T ₄ DNA ligase (manufactured by Nippon Tong Gene) were reacted overnight at 4 ° C. to raise oligonucleotides A-1, A-2 Prepare Block 1, which is ligated with A-3, A-14, A-15 and A-16.

Block 2 and Block 3 by ligation of A-4, A-5, A-6, A-11, A-12 and A-13 and ligation of A-7, A-8, A-9 and A-10 To form.

Two times the volume of ethanol was added to the reaction mixture of the block thus prepared, and the mixture was allowed to stand at -80 ° C for 30 minutes to precipitate DNA, followed by electrophoresis and autoradiography on 12.5% by weight polyacrylamide gel. do. Block 1 is the band at positions 72b.p. (base pair) and 36b.p., block 2 is the band at position 36b.p. and block 3 is 48b.p. And bands at positions 24b.p. Each band is cut and added to it is a mixture of 10 mM Tris-HCI (pH 7.6) and 10 mM EDTA aqueous solution (Tris-EDTA). The mixture is extracted by standing overnight at room temperature. The resulting mixture is centrifuged to separate the supernatant, and the supernatant is sufficiently shaken with Tris-EDTA saturated phenol and then centrifuged to remove the lower layer. Repeat the same process twice with Tris-EDTA saturated phenol. Finally, the upper layer is passed through a column 1 cm in diameter and 20 cm long and packed with Sephadex G-50 to remove phenol and acrylamide. The eluate is concentrated to 200 μl, and then allowed to stand for 30 min at −80 ° C. with twice the volume of ethanol to concentrate the precipitate.

Combine the three blocks obtained together. To the mixture is added 50 mM Tris-HCI (pH 7.6), 10 mM magnesium chloride, 20 mM DTT, 1 mM ATP and 5 μl (100 units) of T ₄ DNA ligase. The resulting mixture is left to lignate at 4 ° C. overnight. A double volume of ethanol was added to the mixture, and the mixture was allowed to stand for 30 minutes at −80 ° C. to precipitate DNA, followed by electrophoresis and autoradiography on 8% by weight polyacryl amide gel. And bands at 192b.p. Each band is cut and Tris-EDTA is added thereto, and the mixture is extracted by standing overnight at room temperature. The mixture is centrifuged to separate the supernatant, the supernatant is sufficiently shaken with Tris-EDTA saturated phenol, and then the lower layer is discarded. This process is repeated twice with further addition of Tris-EDTA saturated phenol. The upper layer is passed through a Sephadex G-50 column, the eluate is concentrated, ethanol is added to the concentrate at twice the volume, and the DNA is allowed to settle for 30 minutes at -80 ° C. Cleavage with the products EcoRI and BamHI yields subunit A.

This method is repeated as in Figure 3 oligonucleotides B-1, B-2, B-15 and B-16 ligated to block 4, oligonucleotides B-3, B-4, B-13 And B-14 is ligated to block 5, oligonucleotides B-5, B-6, B-11 and B-12 are ligated to block 6, and oligonucleotides B-7, B-8, B-9 and B -10 ligates to block 7. 26b.p. And 208b.p., followed by cleavage with HindIII and BamHI to obtain subunit B.

6) Cloning of subunits and analysis of recombinant plasmids

Referring to FIG. 2, pBR322 is cleaved with EcoRI and BamHI and phosphate groups are removed from the 5 'end using alkaline phosphatase (manufactured by Takarashu Co., Ltd.) in order not to return to the original state. The cleaved and dephosphorylated pBR322 and subunit A were ligated overnight at 4 ° C. in a mixture of 50 mM Tris-HCI (pH 7.6), 10 mM magnesium chloride, 20 mM DTT and 1 mM ATP with 5 μl of T ₄ DNA ligase. Let's do it. Its double volume of ethanol is added to the reaction mixture, and the mixture is allowed to settle for 30 minutes at -80 ° C. The mixture is centrifuged, the precipitate is dried and dissolved in 100 μl of distilled water to give plasmid pUG1 with subunit A inserted in pBR322.

This plasmid pUG1 is used to transform E. coli strain HB101 by the calcium method.

Strain HB101, which is used as a host, is incubated at 37 ° C. in 50 ml of LB culture medium (1 wt% bactotrypton, 0.5 wt% yeast extract and 0.5 wt% sodium chloride). When the absorbance reached 0.25 at 610 nm, 40 ml of culture broth was transferred to a centrifuge tube and 6000r. p. Centrifuge for 10 minutes at m and 4 ° C. The supernatant is removed and the precipitate is suspended in 20 ml of ice cold 0.1 M magnesium chloride, then the suspension is centrifuged again under the same conditions and the supernatant is removed. The precipitate is suspended in 020 ml of an ice cold solution of 0.1 M calcium chloride and 0.05 M magnesium chloride and ice cooled for 1 hour. After the suspension is centrifuged and the supernatant is removed, the precipitate is suspended in 2 ml of an ice cold solution of 0.1 M calcium chloride and 0.05 M magnesium chloride. 10 μl of an aqueous solution of pUG1 is added to 200 μl of the suspension and the mixture is ice cooled for 1 hour and then heated in a water bath at 43.5 ° C. for 30 seconds. Then 2.8 ml of LB culture medium is added to the mixture and incubated at 37 ° C. for 1 hour. Cultures are spread out on LB plates containing 50 μg / ml ampicillin and in an amount of 200 μl / dish and incubated at 37 ° C. overnight. Growing colonies are checked by further transplantation into LB plates containing 50 μg / ml of ampicillin and into LB plates containing 20 μg / ml of tetracycline and incubated at 37 ° C. overnight. Only colonies resistant to ampicillin are isolated to obtain transformed cells.

Plasmids are collected on a small scale from the cells by an alkaline extraction method and the presence of the BglII cleavage site is checked. One of the cells containing the plasmid with the BglII cleavage site was cultured in large quantities to obtain plasmid pUG1 purified by alkaline extraction method.

The nucleotide sequence of subunit A inserted into the resulting pUG1 is analyzed for two strands by the above-mentioned Mam-Gilbert method.

Photos 1 and 2 show the analysis results. Lanes 1 to 4 are electrophoretic results of EcoRI-SalI fragments, and lanes 5 to 8 are electrophoretic results of BamHI-PstI fragments. Lanes 1 and 5 represent reaction products using guanine, lanes 2 and 6 represent reaction products using guanine + adenine, lanes 3 and 7 represent reaction products using thymine + cytosine, and lanes 4 and 8 represent cytosine The reaction product used is shown. Photograph 2 shows the results obtained by the sample as described above, where the portion on the high molecular weight side (corresponding to the upper portion of photo1) is enlarged. In this way, the nucleotide sequence of subunit A is identified.

Referring to FIG. 3, plasmid pBR322 is cleaved with HindIII and BamHI, and larger fragments are separated by electrophoresis and then ligated to subunit B. Thus, as in the case of pUG1, plasmid pUG2 having subunit B introduced into pBR322 is obtained. The resulting plasmid pUG2 is used to transform strain HB101 and select only the colonies that are resistant to ampicillin. Plasmids are harvested from the colony and the presence of the BglII cleavage site and the MluI cleavage site is checked. Cells containing plasmids with two sites are selected. One of the selected cells is incubated in bulk to obtain purified plasmid pUG2. The nucleotide sequence of subunit B in pUG2 is analyzed in two strands by the Maksam-Gilbert method.

Photo 3 shows the results of the analysis. Lanes 1 to 4 show the results obtained by the HindIII-SalI fragment. Lanes 5 to 8 are the results obtained by the same sample, in which the portion on the high molecular weight side (corresponding to the upper portion of lanes 1 to 4) is enlarged. Each lane represents the same as the corresponding reaction product in photo 1. Thus, the nucleotide sequence of subunit B is confirmed.

Referring to FIG. 4, pUG1 is cleaved with HindIII and SalI, and larger fragments are separated through a biogel 1.5 cm column. pUG2 is cleaved with HindIII and SalI and electrophoresed to obtain smaller fragments. The two fragments are combined with each other and treated with T ₄ DNA ligase for ligation to obtain plasmid pUG3 with subunit A + B, ie, β-urogastron gene inserted in pBR322. This plasmid pUG3 is used to transform E. coli strain HB101. This transformant has been deposited with the Budapest Convention, an international treaty on deposit with the accession number of FERM BP-543, to the Japan Institute of Trade, Industry and Technology, Microbiological Technology Research Institute since June 22, 1984. In addition, this strain was deposited with KFCC-10155 accession no.

Also in this case, cells containing plasmid pUG3 that are resistant to ampicillin only and have a MluI cleavage site are selected. One of the selected cells is incubated in bulk to obtain purified plasmid pUG3. The nucleotide sequence of the β-urogastron gene in pUG3 is analyzed in two strands by the Maksam-Gilbert method.

Photo 4 shows the analysis results. Lanes 1-4 show the results obtained with the BamHI-PstI fragment. Lanes 5 to 8 show the results obtained with the same sample, in which the portion on the high molecular weight side (corresponding to the upper portion of lanes 1 to 4) is enlarged. The reaction product of lanes is the same as the corresponding product in photo 1. The analysis identifies the nucleotide sequence of the β-urogastron gene.

7) Expression vector with λP _L promoter inserted

The λP _L promoter, which is the left promoter of λ phage, is used for the expression of β-urogastron described below.

First, the preparation of strain ECI-2 derived from E. coli strain HB101 will be described. ECI-2 is used as a host for λP _L transgenic plasmid. Cloning of the DNA fragment containing the λP _L promoter from the DNA of λCI857S ₇ , which is a mutant strain of λ phage, and the formation of the expression plasmid from the cloning DNA will be described. It also describes the expression of the β-urogastron gene by the λP _L promoter in the host ECI-2 strain.

7-1) Formation of Strain ECI-2

Strain ECI-2 is E. coli HB101 containing plasmid pGH37 for expressing the CI857 gene.

pGH37 is produced by the method shown in FIG. First, the DNA of λCI857S ₇ is cleaved with BgIII. The sticky end of the cleavage site is digested with S1 nuclease. 1 μg of λCI857S ₇ digested with BglII is reacted for 30 minutes at 20 ° C. with 200 units of SI nuclease in 100 μl of an aqueous solution containing 200 mM sodium chloride, 300 mM sodium acetate and 5 mM zinc sulfate (pH 4.5). DNA fragments with blunt ends thus obtained were subjected to 1.0% by weight agarose gel electrophoresis to separate 2385b.p. Fragments having a total CI857 structural gene therefrom. This fragment is inserted into the PvuII cleavage site of plasmid PGL101 to form plasmid pGH36 which expresses the CI857 gene under the control of promoter lac UV5. Subsequently, pGH36 is cleaved with two restriction enzymes EcoRI and PstI to obtain a fragment of 1193b.p., Which is inserted between the EcoRI and PstI cleavage sites of plasmid pSC101 to prepare plasmid pGH37.

Strain HB101 of E. coli is transformed using pGH37 by the calcium method described above. One of the resulting strains is named ECI-2. Strain ECI-2 has been deposited with the Budapest Convention, an international treaty on deposit with the accession number of FERM BP-542, to the Japan Institute of Commerce, Industry and Technology, Microbiological Technology Research Institute since June 22, 1984. In addition, this strain was deposited with KFCC-10154, Accession No., of August 22, 1985 to the Korean spawn association. This strain is resistant to tetracycline and expresses the CI857 gene, allowing for the association of other plasmids derived from pBR322, for example via transformation. Strain ECI-2 is therefore used as a host for λP _L expressing plasmids.

7-2) Cloning of λP _L Promoter and Preparation of Expression Plasmids

As shown in FIG. 7, pGH35 is first formed. DNA of λCI857S7 was digested with EcoRI and SalI to produce 592b.p. containing λP _L promoter and CI857 gene as well as λP _R promoter. Obtain a fragment of This fragment is inserted between the EcoRI and SalI cleavage sites of plasmid pBR322 to form plasmid pGH25.

Next, pGH25 is cleaved with BamHI and ligated into pBR322 likewise cleaved with BamHI to obtain pGH34.

pGH34 was cleaved with AvaI and BalII, and then treated with SI nuclease to yield about 4500b.p. A fragment having a blunt end of is obtained. This fragment is cyclized with T ₄ DNA ligase to form plasmid pGH35.

Next, pEK28 is formed as shown in FIG. Synthetic oligonucleotides C-1-1 and C-1-2 comprising the SD sequence as adapter and having the nucleotide sequence shown below are ligated to the fragment obtained by cleaving pGH35 with HpaI. It is further ligated to plasmid pMC1403 digested with BamHI to give plasmid pEG2. pEG2 is a two ampicillin resistance gene and expresses the β-galactosidase gene derived from pMC1403 by using the included SD sequence of the adapter and the starting codon under the λP _L promoter control.

Fragments C-1-1 and C-1-2 have the following nucleotide sequences.

Miller's method is used to confirm the expression of β-galactosidase in host ECI-2 containing plasmid pEG2 (Miller. J. (1972) “Experiments in Molecular Genetics” New York, Cold Spring Harbor Laboratory). pp 352-355). This method is to react the β-galactosidase with the synthetic substrate ONPG (o-nitrophenylgalactoside) to release the yellow compound o-nitrophenol. Miller's method is explained in more detail. Permeability of the bacterial sample measured at 610 nm is measured. Toluene is evaporated off with aspirator. While adding 0.2 ml of ONPG solution (400 mg of ONPG solution dissolved in 100 ml of assay buffer), the mixture is incubated at 30 ° C. until yellow develops, and 0.5 ml of 1M sodium carbonate is added to stop the enzyme reaction. The absorbance of the reaction mixture is measured at 420 nm and 550 nm.

The activity of β-galactosidase is defined in units of 1 ml of liquid culture drug according to the following equation, and the absorbance at 610 nm is calculated as 1.0.

activity (unit) of β-galactosidase =

t: incubation time (min)

v: amount of sample added to the reaction system (0.1 ml)

OD ₆₁₀ : absorbance at ₆₁₀ nm of the sample.

The following results appear when performing the above method. When the ECI-2 strain containing pEG2 was incubated at 30 ° C, the β-galactosidase activity was 98 units. However, when the incubation is further performed at 42 ° C. for 1 hour, the λP _L promoter is activated, resulting in 9637 units of β-galactosidase activity. This indicates that the sequences from the λP _L promoter to β-galactosidase are in the expected order.

pEG2 contains two BglII cleavage sites, but only the BglII site that exists immediately after the SD sequence of β-galactosidase is needed, and other sites are unnecessary. Thus, the plasmid is cleaved with BamHI and ligated again to remove fragments with about 770 b.p. The formation of pEK28, the expression plasmid using the λP _L promoter, is completed.

7-3) Expression of the fusion gene of the anterior half of β-urogastron and β-galactosidase

9 is a schematic representation of a series of processes described below.

The plasmid pUG101 having the front half of β-urogastron and the fusion gene of β-galactosidase was formed by the following method. More specifically pMC1403 with pUG101 and β-galactosidase genes, which are the first half of the β-urogastron gene, is cleaved with BamHI and ligated to form pUG101. This plasmid ligates the front half of the β-urogastron gene and the β-galactosidase gene within the same frame. Thus, this plasmid expresses two amino acid sequences as fusion proteins.

To express this plasmid under the control of the λP _L promoter, pUG101 and pEK28 are cleaved with BglII, respectively.

형의 돌출된 5' 말단을 갖는 DNA 단편이 생성된다. 그러나, DNA 단편을 4종의 데옥시리보뉴클레오티드 트리포스페이트 dGTP, dATP, dTTP 및 dCTP의 존재하에 이.콜리 DNA 폴리머라제 I의 큰 단편(클레노우 단편)과 반응시킬 때, 상응하는 뉴클레오티드가 채워짐으로써

형의 무딘 말단이 수득된다. 뉴클레오티드 성분으로서 dGTP만을 가하면, 반응의 종결로 인해

의 말단이 수득된다. 다음 S1 뉴클레아제를 사용하여 나머지 단일 가닥을 분해할 때,

형의 무딘 말단이 수득된다. 마찬가지로 dGTP 및 dATP를 클레노우 반응에 가하고, S1 뉴클레아제로 분해하면,

가 수득된다. dGTP, dATP 및 dTTP를 가하여 클레노우 반응을 수행하고, S1 뉴클레아제로 분해할 때,

가 수득된다. 또한 클레노우 반응을 수행하지 않고 S1 뉴클레아제로 분해할 때,

가 수득된다. 그러므로,

의 말단을 갖는 DNA 단편을 다른 뉴클레오티드를 사용한 클레노우 반응 및/또는 S1 뉴클레아제 반응시킬 때, 한 염기쌍 길이에 의해 서로 다른 5종의 무단 말단이 수득된다.Cutting with BalII

DNA fragments with raised 5 'ends of the form are produced. However, when a DNA fragment is reacted with a large fragment of E. coli DNA polymerase I (cleno fragment) in the presence of four deoxyribonucleotide triphosphates dGTP, dATP, dTTP and dCTP, the corresponding nucleotides are filled by

A blunt end of the form is obtained. If only dGTP is added as the nucleotide component,

The terminal of is obtained. When the next single strand is cleaved using the S1 nuclease,

A blunt end of the form is obtained. Similarly, when dGTP and dATP are added to the Klenow reaction and digested with S1 nuclease,

Is obtained. When the klenow reaction is performed by adding dGTP, dATP and dTTP and digested with S1 nuclease,

Is obtained. In addition, when digested with S1 nuclease without performing the cleno reaction,

Is obtained. therefore,

When a DNA fragment having the ends of is subjected to a Klenow reaction and / or an S1 nuclease reaction using other nucleotides, five different endless ends are obtained by one base pair length.

The cleno reaction and the S1 nuclease reaction are carried out under the following conditions.

* Klenow reaction:

1 μg of DNA as substrate was subjected to 1 unit of enzyme (cleno fragment) and 1 mM in 50 μl of reaction medium containing 40 mM potassium phosphate buffer (pH 7.4), 1 mM β-mercapto-ethanol and 10 mM magnesium chloride. React with 1 mM of each deoxyribonucleotitriphosphate at 12 ° C. for 30 minutes in the presence of ATP.

* S1 nuclease reaction:

As a substrate, 1 μg of DNA and 200 units of enzyme are reacted for 30 minutes at 20 ° C. in 100 μl of a reaction medium containing 200 mM sodium chloride, 30 mM sodium acetate, and 5 mM zinc sulfate (pH 4.5).

pEK28 and pUG101 are respectively cleaved with BglII and combined with various Klenow reactions and S1 nuclease reactions to obtain fragments with five blunt ends.

When two kinds of these fragments are combined with each other, as shown in Table 2, 21 combinations having different nucleotide sequences and numbers between the SD sequence of the fusion protein and the codon for codon are provided.

Indeed, the DNA fragment thus obtained separates fragments containing genes encoding the fusion proteins of β-urogastron anterior half and β-galactosidase from pUG101, and fragments containing the P _L promoter from pEK28. . These fragments are ligated using T ₄ DNA ligase in the combinations shown in Table 2.

As a result, the recombinant plasmid of Table 3 is obtained. Β-galactosidase activity of the expressed fusion protein is measured by Miller's method. Table 3 shows that all plasmids express relatively high levels of β-galactosidase activity. In particular pUG103, pUG104 and pUG117 show significant results.

TABLE 2a

TABLE 2b

TABLE 3

Next, the expression vector of β-urogastron is prepared from pUG103 or pUG117. Plasmids (pUG103 or pUG117) were cleaved with HindIII and PvuII to obtain 1.2 kb fragments containing portions of the front half of the β-urogastron gene from the λP _L promoter. pUG2 is cleaved with EcoRI to fill with Klenow fragment and cleaved with HindIII to yield a 4.1 kb fragment. Two fragments were ligated with T ₄ DNA ligase and plasmid pUG103-E for transforming E. coli strain ECI-2 by the calcium method to express the entire β-urogastron under the control of the λP _L promoter or Obtain a recombinant plasmid containing pUG17-E.

8) Vector for the expression of the fusion protein

The β-lactamase gene β-urogastron gene of the plasmid pBR322 is ligated to express β-urogastron as a fusion protein described below.

8-1) Donor of β-lactamase gene

pBR322 is obtained by cleaving pBR322 with AvaI and PvuII and ligation with Klenow reaction and T ₄ DNA ligase. This plasmid has genes for ampicillin resistance (AP ^R ) and tetracycline resistance (TC ^R ) as markers. pBR322 was cleaved with AvaI and HindIII, clenou reaction and ligation yielded pBRH03, which plasmid has Ap ^R and chloram phenicol resistance (Cm ^R ) as markers. 10 shows these plasmids.

8-2) Donor of β-Eurogastron Gene

PUG3 prepared as described above was cleaved with MboII to obtain 13 DNA fragments, named A-M in size order as shown in FIG. Among these DNA fragments, it was found that the H fragment consists of 179b.p., starting with the nucleotide encoding the asparagine at the N-terminus of β-urogastron and ending with the lower 16 base of the stop codon. The fragment has the total structural gene of β-urogastron. To separate the H fragment, the fragment is electrophoresed by 6% by weight polyacrylamide gel and the fragment is purified.

8-3) Adapter

Oligonucleotides described in Table 4 were prepared in the above-described manner as adapters. These adapters are designed to code basic amino acid pairs of Lys-Arg or Arg-Lys to enzymatically cleave β-urogastron from the expressed fusion protein.

TABLE 4

8-4) Expression system of fusion protein of β-lactamase and β-urogastron linked by Lys-Arg

The expression vector of the β-lactamase-β-urogastron fusion protein is prepared to create a restriction enzyme recognition sequence in the portion containing the adapter.

8-4-a) Formation of pUG2301 to pUG2303

Perform the method shown in FIG.

Plasmid pBRH02 is cut completely with XmnI over 3 hours at 37 ° C. About 0.1 μg of a β-urogastron fragment of 3 μg of vector, 179b.p., and about 1 μg of E-1 and E-2 (non-phosphorylated 5 ′ end) acting as an adapter were added at once at 12 ° C. over 15 hours. Ligation yields a plasmid for the expression vector. This plasmid is used to transform strain HB101 by the calcium method.

Of the 499Tc ^R colonies obtained, 168 colonies (33.7%) were Ap ⁵ . These colonies are checked by mini-manufacturing for the size of the plasmid DNA. The 13 plasmids are about 200 b.p. larger than the vector, with the β-urogastron gene inserted. All of these with MluI sites are cleaved with HinfI and the direction of β-urogastron gene insertion is checked by 1.5% by weight agarose gel electrophoresis. Three of the checked ones are about 1050b.p. And 800b.p. This indicates that the β-urogastron gene was inserted in the same direction as the β-lactamase. These three plasmids are named pUG2301 to pUG2303.

8-4-b) Formation of pUG2101 to pUG2105

Perform the method shown in FIG.

Plasmid pBR322 with the unique PvuI site of the β-lactamase gene is used as the plasmid vector.

According to the method described in 8-4-a), the expression vector is formed using E-1 and E-5 as adapters.

When the 1626Tc ^R colony obtained was tested for Ap sensitivity, 31 colonies (1.9%) were Ap ^s . Mini- formulations are performed on the 22 Tc ^R and Ap ⁵ communities and the insertion of β-urogastron gene into the plasmid is checked by cleavage with MluI. It was found that 20 plasmids had MluI sites. The direction is checked by cutting with HinfI or BamHI. It is found that plasmids pUG2101 to pUG2105 have a β-urogastron gene inserted in the same direction as the β-lactamase gene.

8-4-c) Formation of pUG2701 to pUG2703

As shown in FIG. 14, the expression plasmid is formed by a process such as 8-4-a) using vector pBR322 with vector and E-7 and E-8 with adapter.

Of the 217Tc ^R colonies obtained, 106 colonies (48.8%) are Ap ^S. Mini-manufacturing is performed for 25 of these communities. 8 plasmids are about 200b.p. Since the β-urogastron gene is fragmented, these 8 plasmids are cleaved with BamHI and the direction of the gene is checked. As a result, it is found that the 3 plasmids have the β-urogastron gene inserted in the same direction as the β-lactamase, which are named pUG2701 to 2703.

Plasmid pUG2301 obtained by the above method 8-4-a) produces a fusion protein of β-lactamase and β-urogastron moiety. The expected amino acid sequences and corresponding nucleotide sequences are shown below.

Likewise, plasmid pUG2101 obtained by method 8-4-b) produces a fusion protein having the following primary structure.

Likewise, plasmid pUG2701 obtained by method 8-4-c) produces a fusion protein having the following primary structure.

Nucleotide sequences encoding fusion proteins to plasmids pUG2101, pUG2301 and pUG2701 are analyzed by the Membrane-Gilbert method.

Photo 5 shows the results of the analysis. In photo 5, lanes 1-4 show the results obtained with the MluI-PstI fragment (224b.p.) of pUG2101, and lanes 5-8 show the results obtained with the MluI-EcoRI fragment (721b.p.) of pUG2101. Lanes 9-12 show the results obtained with the MluI-BamHI fragment (452b.p.) of pUG2301, and lanes 13-16 show the results obtained with the MluI-PstI fragment (335b.p.) of pUG2701. , Lanes 17 to 20 show the results obtained with the MluI-EcoRI fragment (610b.p.) of pUG2701. Lanes 1,5,9,13 and 17 represent reaction products using guanine, lanes 2,6,10,14 and 18 represent reaction products using guanine + adenine, lanes 3,7,11,15 and 19 Denotes the reaction product using thymine + cytosine and lanes 4,8,12,16 and 20 represent the reaction product using cytosine. The part marked} is the adapter.

8-5) Expression system of fusion protein of β-lactamase and β-urogastron linked by Arg-Lys.

8-5-a) Preparation of pUG1102 and pUG1105

The β-urogastron gene is inserted into the unique PvuI restriction site of the β-lactamase gene of pBR322 to obtain the transfection vector of the fusion protein of β-lactamase and β-urogastron as shown in FIG. 15.

Plasmid pBR322 is cleaved with PvuI at 37 ° C. over 3 hours. Several plasmids were checked by 1% by weight agarose gel electrophoresis to confirm that they were completely cleaved. Adapters D-1-3 and D-3-2 are ligated to the fragments over 15 hours at 12 ° C., and the ligation products are separated by 1% by weight agarose gel electrophoresis to separate DNA fragments. The β-urogastron fragment and the vector are then mixed together in a molar ratio of about 5: 1 and ligated at 12 ° C. over 15 hours. After ligation, strain HB101 is transformed with the resulting plasmid, referring to Tc ^R and selecting colonies.

71 Tc ^R transformed colonies are obtained and Ap sensitivity is checked. Plasmid DNA is prepared from 20A ^S colonies (28.2%) and the presence of the MluI restriction site is checked to confirm the insertion of the β-urogastron gene. It is found that 5 out of 20 plasmids have the MluI site of the β-urogastron gene. The DNA was cut with HinfI, and 1.5 wt% agarose gel electrophoresis was used to check the insertion direction. It is found that two plasmids, pUG1102 and pUG1105, carry genes in the proper direction.

8-5-b) Preparation of pUG1004, pUG1201 and pUG1301

Repeating Method 8-5-a) using PstI, HincII and XmnI instead of PvuI yields pUG1004, pUG1201 and pUG1301 shown in Figures 16, 17 and 18 degrees, respectively.

9) Confirmation of Expression of β-Eurogastron

E. coli HB101 or ECI-2 was transformed using the thus formed expression plasmid, and the cells were cultured by the following method, and then expression was confirmed by extraction and radioimmunoassay.

9-1) Culture of Recombinant Microorganism with β-Eurogastron Gene and Protein Extraction

9-1-a) Expression system using λP _L promoter

Strain ECI-2 containing the expression plasmid pUG103-E and strain ECI-2 containing the plasmid pUG117-E are each incubated at 25 ° C. in two flasks containing 1 L of LB culture medium. The flask was heat-sensitized at 42 ° C. for 1 hour when one culture showed an absorbance of 0.3 at 660 nm. Cultures of the other flasks continue to incubate at 25 ° C. until the absorbance reaches 0.4. Cells were collected in each flask, washed with PBS buffer (137 mM sodium chloride, 2.7 mM monobasic sodium phosphate, pH 7.0), resuspended in 3 volume% PBS buffer of the original culture, and then cooled by sonication (model 5202). Crushing (30 seconds, 100 times at 100 W, 3 times) by Oda Kei Work Co., Ltd. The supernatant separated from the cell lysate by centrifugation (40000 g for 1 hour) was dialyzed in a 0.01 N acetic acid aqueous solution and dialysate. After freeze drying, radioimmunoassay (hereinafter abbreviated as “RIA”) is performed.

9-1-b) Expression system for fusion proteins

E. coli strain HV101 containing plasmids pUG1004, 1301, 2101, 2303 or 2703 was pre-incubated at 37 ° C. in a culture medium containing 50 μg.ml of tetracycline and diluted in a volume ratio of 1: 100 with the same medium. After that, the cells are incubated until the absorbance at 660 nm is 0.4. The cells are harvested, washed with PBS buffer, resuspended in 3% by volume PBS buffer of the original culture, and then sonically crushed (30 seconds at 100 W, 3 times) with an acoustic wave crusher with ice cooling. The supernatant separated from the cell lysates by centrifugation (1 hour at 40000 g) is dialyzed in an aqueous 0.01 N acetic acid solution, lyophilized and RIA analyzed.

In order to confirm the accumulation of fusion proteins in the foreign matter, the methods described in the literature Chan, S. J. et al., Proc. Natl, Acad. Foreign material fractions are prepared according to Sci., USA, 78, 5401-5405 (1981). A portion of the culture was taken from fresh E culture medium (10 g sodium phosphate dibasic, 3.5 g sodium ammonium hydrogen phosphate, 0.2 g magnesium sulfate heptahydrate, 2 g citric acid, 2 g glucose, 0.23 g L-proline, Aqueous solution containing 39.5 mg of L-leucine, 16.85 mg of thiamin and 20 mg of tetracycline hydrochloride (diluted in 1: 1 by volume, incubated at 37 ° C. until absorbance at 660 nm is 0.4 The cells were harvested (6000 r.pm, 10 min) and washed twice with 10 mM Tris-HCI (pH 8.0) and 30 mM sodium chloride Cells (1 g) were again washed with 80 ml of 20% by weight sucrose-30 mM Tris-HCI. (pH 8.0) and EDTA are added to the suspension at a concentration of 1 mM The mixture is shaken by rotary shaker at 180 r.pm for 10 minutes (25 ° C) and centrifuged (13000 g, 10 minutes) to collect the cells. Then resuspend in 80 ml of distilled water. Allow to stand for about 10 minutes, centrifugation (13000 g, 10 minutes), and collect the supernatant as an aliquot (O-Sup) The pellet is suspended in a mixture of 10 mM Tris-HCI (pH 8.0 and 30 mM sodium chloride), Treatment with a sonic crusher as above yields a cytoplasmic fraction (O-Ppt) These samples are analyzed by RIA.

9-2) Radiation Immunoassay

9-2-a) Setting of RIA System

Antiserum is obtained by immunizing rabbits using purified human β-urogastron as an antigen. β-Eurogastron (300 μg) is dissolved in 0.2 ml of distilled water, 1.5 ml of 50% polyvinylpyrrolidone solution is added to the solution, and the mixture is stirred at room temperature for 2 hours. A complete Freund's adjuvant (2.0 ml) is added to the mixture to give an emulsion, which is injected subcutaneously in the chest of three rabbits. After four immunizations are repeated every two weeks, 50 μg of the antigen is further injected intravenously, three days later the total serum is collected to separate serum.

Next, the following RIA conditions were determined in view of a titration curve for measuring the dilution of the assay antiserum, incubation time for optimizing the assay conditions, and method for separating radiolabeled antigens bound thereto from unlabeled antigens. do.

The dilution solution used is phosphate buffer (10 mM, pH 7.4) containing 0.5% by weight bovine serum albumin (BSA), 140 mM sodium chloride and 25 mM disodium EDTA. Dilution solution (400 μl), 100 μl sample or standard human β-urogastron and 100 μl of anti human β-urogastron serum are mixed together. After incubating the mixture for 24 hours at 4 ° C., 100 μl of ^{12 =} I-labeled human β-urogastron solution (about 5000 cpm) is added to the mixture. After further incubating the mixture for 48 hours at 4 ° C., 100 μl of the second antibody (anti-rabbit β-globulin goat serum) (diluted 20 times with PBS buffer), and 900 μl of 10 mM PBS buffer containing 5 wt% polyethylene glycol were added. Add to the resulting mixture and incubate further at 4 ° C. for 3 hours. The culture solution is centrifuged at 3000 rpm for 30 minutes, the supernatant is removed, and the precipitate is counted. The content of the immunoreactive substance as human β-urogastron in the sample is determined from standard curves obtained using standard human β-urogastron.

9-2-b) Confirmation of β-Eurogastron Productivity of Recombinant Microorganisms

Table 5 shows the results of RIA performed on the transfection system using the λP _L promoter.

TABLE 5

Table 6 shows the results of RIA performed on the fusion protein expression system.

TABLE 6

Table 7 shows the localization of the expressed fusion protein.

TABLE 7

Tables 5 and 6 show that the expression system of the compounds as λP _L promoter system and fusion protein for direct expression of β-urogastron both express β-urogastron immune responsiveness in E. coli. Table 7 shows that for fusion proteins, the expressed β-urogastron immune responsiveness is almost localized in the foreign vagina.

Claims (11)

Β, which is characterized by culturing a transformant containing a host cell having a recombinant plasmid capable of expressing the β-urogastron gene having the following nucleotide sequence, and collecting the expressed β-urogastron Eurogastron manufacturing method.

The method of claim 1, wherein the β-urogastron gene has a restriction enzyme recognition site attached to the front and / or back ends of the gene. The method of claim 2, wherein the β-urogastron gene has a restriction enzyme recognition site provided at the top of the gene and a start codon and / or a stop codon and a restriction enzyme recognition site provided at the bottom of the gene, and these codons and recognition The site is arranged in the order mentioned. The method of claim 3, wherein the β-urogastron gene has the following nucleotide sequence.

The method of claim 1, wherein the recombinant plasmid is inserted into the β-urogastron gene according to claim 4, the SD sequence linked to the promoter and promoter for regulating the expression of the gene inserted on top of the β- eurogastron gene A method containing a plasmid vector having. The recombinant plasmid of claim 1, wherein the recombinant plasmid has a sequence of the fourth and subsequent base pairs of the β-urogastron gene according to claim 4, wherein the promoter, SD sequence and A method containing a plasmid vector with a combination of different genes. The method of claim 6, wherein the other gene is a β-lactamase gene. The method of claim 5 or 6, wherein the promoter is λP _L or lac UV5. 7. The method of claim 5 or 6, wherein the plasmid vector is pBR322. The method of claim 1, wherein the host cell is E. coli. The method of claim 1, wherein the host cell is transformed with a recombinant plasmid having a Tc ^R gene and a CI857 gene. [Photo 1]

[Photo 2]

[Photo 3]

[Photo 4]

[Photo 5]

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