SU1708847A1 - Recombinant plasmid dna pbghtrp3, encoding cattle growth hormone, and the strain of bacteria escherichia coli - a producer of cattle growth hormone - Google Patents

Abstract

This invention relates to the microbiological industry, genetic engineering and biotechnology. The aim of the invention is to increase the yield of the target product. The plasmid DNA pBGHtrp3, containing the gene encoding cattle growth hormone (GRS cattle), under the control of the promoter of the trp operon E.soM, and a fragment of the bacterial 153 element located after the gene HRS cattle was constructed. The producer strain was obtained by transformation of the plasmid pBGHtrp3 strain B.soI DN1. The presence of a 133-element fragment in the plasmid stabilizes the productivity of the producer strain and allows for a high level of synthesis of the target product, not an induction to induction, of at least 50-60 µg / ml of culture. 2 sec. f-ly. • ^ • ^ Ё

Description

This invention relates to the microbiological industry of genetic engineering and biotechnology.

The purpose of the invention is to increase the yield of the target product.

Recombinant plasmid DNA pBGHtrp3 was constructed to encode bovine growth hormone (GR) under the control of the E. coli trp operon promoter and containing a fragment of a bacterial 183 element located after the genome of the BGG 9 cattle, E. plasmid transformed E. coli strain E.coI DH1. The presence in the plasmid of a fragment of the 153 element stabilizes the productivity of the producer strain and makes it possible to achieve a high level of synthesis of the target product, without recourse to induction. The level of synthesis of cattle GR is about

20% of the total cell protein or 50-60 mcg per 1 ml of overnight culture.

The recombinant plasmid rVCHHr3 contains the replicon and genes for resistance to ampicillin and tetracycline plasmid rBr322, the promoter of the E. coli trp operon, the fragment encoding GRS cattle, and the fragment IS3 of the bacterium consists of the following elements:

EcoRi-HInd III - a fragment of the PSTH2191 plasmid of 5300 bp in size, including the origin of replication, the genes for resistance to ampicillin (Na) and tetracycline (tet) H promoter of the trp operon E. col:

EcoRl - Htnd ill - a fragment encoding a KnCi GR of 601 bp in size:

Hind III - fragment of IS3 element, 916 bp in size

The size of the plasmid pBGHtrpS is 6819 bp. The plasmid copy number is about 20 molecules per cell.

The plasmid DNA pBGHtrp3 contains unique restriction sites EcoRI, Hpal, Smal, BamH I, SalGI. EcoRV, 2 Hind III restriction sites and 3 Pstl restriction sites.

Strain producing GRS cattle obtained by transformation of E. col DH1 cells with the plasmid pBGHtrpS.

The strain is characterized by the following features.

Morphological features: straight, rod-shaped cells, Gram-negative, unborponeous.

Cultural characteristics: cells grow well on commonly used nutrient media. When grown on Difco nutrient agar, the colonies are smooth, round and shiny gray. Kra colonies are smooth. With growth in liquid media YT. LB, M9 form a smooth intensive dregs.

Physiological and biochemical characteristics: optimum cultivation temperature of 37 ° C. optimum pH from 6.8 to 7.5, as

AATTCTATGTTCCCAGCTATGTCTCTATCTGGTCTATTGCTAACG

e / s /

Iiiill

Vivii. Vim

/ / h ((

() (.1Ii

GATACAAGGGtCGATACAGAGATAGACCAGATAAGCGATTGCGACAAG CTGTTCTTCGGGAGAGCATCTTCATCAGGTGA

-) ((IV

IX X fi.-j /

AAGCCCGGGTCGTAGAAGTAGTCGACTTCGA

The structure of the oligonucleotides is chosen so that the synthetic DNA fragment has sticky ends for EcoRl and Hind III restriction enzymes. The initiating ATC-kodon is located in front of the codon of the 1st amino acid of the mature GR of cattle-phenylalanine. In the region corresponding to the codons, there is a restriction enzyme site PVU il, which allows stitching the synthetic DNA fragment with the fragment encoding the rest of the GH molecule (residues 24-190).

Phosphorylation and oligonucleotide cross-linking is carried out as follows.

5 μg of each of the 10 oligonucleotides are incubated at 37 ° C for 15 min in 20 μl of an incubation mixture containing 50 mM

/ 1The carbon source uses many carbohydrates, alcohols, organic acids. As a source of nitrogen, both mineral salts in ammonium or nitrate forms are used, as well as organic compounds — peptone, tryptone, amino acids.

Antibiotic resistance: exhibits ampicillin resistance due to the presence of a plasmid. Show

resistance to tetracycline when the concentration of the latter is not more than 5-7 mg / l.

Productivity. The level of GH synthesis according to enzyme immunoassay is 50-60 µg per ml of overnight culture.

The strain is deposited in the All-Union Collection of Microorganisms at the Institute of Biomedical Physics of the Academy of Sciences of the USSR under the number VKMSR-325D.

PRI me R 1. Construction of recombinant plasmid DNA pBCHtrp3.

BUT). Construction of plasmid syn / pBR327.

To obtain a DNA fragment encoding the N-terminal part of GR of cattle (amino acids 1-23), synthetic

oligonucleotides.

-) Hind III

V

TrisNC, pH 7.5, 5 mM MgCI., 5 mM dithiuteutol (DTT), 5 mKCI Y - P-ATP (800 C1 / tM), 4 units. phage T4 polynucleotide kinase. Then, 2 µl of 10 mM solution of non-labeled ATP is added and incubated for another 30 minutes at 37 ° C. The incubation mixtures are combined, 50 units are added. Phage T4 ligase DNA and incubated for 12 h at 4 ° C. The mixture is heated for 10 minutes at 70 ° C.

35 5 M NaC solution to a concentration of 0.1 mM, 50, units, restrictase EcoRI, 50 units, restrictase Hind III and incubated at 37 ° C for 5 hours. The incubation mixture is passed through a COLUMN with Sephadex G-50, fractionated

40 by electrophoresis in 8% polyacrylamide gel, cut out the gel band corresponding to a fragment of about

75 bp, and the DNA is eluted from the gel, the DNA after re-precipitation with alcohol is dissolved with 120 µl of water.

5 μg of the plasmid pBR327 are hydrolyzed with restriction enzymes EcoRI and Hind 111 in an incubation mixture of the following composition: 50 Mtris HCl, pH 7.5.10 tM NaCl, 10 tM MgCla, 1 tM DDT, 10 u, EcoRI and 10 u. Hind HI for 2 hours at 37 ° C. Mix the mixture in 1% agar gel and elute the linear plasmid. using lightweight agarose from BRI. The DNA is reprecipitated with alcohol and dissolved in 50 µl of water.

The ligation of the DNA fragment and the plasmid is carried out in a mixture of 20 µl containing 1 µl of the fragment solution, 1 µl of the plasmid solution, 20 mM TrisNA, pH 7.5, 10 mM DTT, -10 mM MgCi2, 1 mM ATP, 5 units. DNA ligase phase T4, at 4 ° C for 12 hours. To 12 µl of the incubation mixture, add 48 µl of TE buffer (50 M trisN, pH 8.0, 1 tM EDTA). The DNA solution transform cells of the strain E. coli HB101. Clone is selected for antibiotic resistance. The insert with the desired orientation is determined by sequencing. As a result, a plasmid designated syn / r BR327 was selected.

B. Construction of plasmid pBGHI190.

10 μg of plasmid pb GH18 DNA is hydrolyzed in an incubation mixture with a volume of 50 μl 30 units. EcoRI restictase for 1.5 hours at 37 ° C, as previously described, the DNA from the mixture is precipitated with alcohol, dried and dissolved in water. Hydrolysis of the linearized plasmid pBGH18 with the Pvu II restriction enzyme prog was carried out in 100 µl of the incubation mixture in medium salt buffer for 10 min at 37 ° C. 10 μg of DNA take 30 units. enzyme. The reaction is stopped by adding a solution of EDTA to a concentration of 20 mM. The reaction mixture is extracted with an equal volume of phenol: chloroform. DNA from the aqueous phase is precipitated with alcohol, dried, dissolved in water - fractionated in 1% agarose gel. The desired DNA fragment (Pvu II - EcoRI fragment of 680 bp) is eluted from low-melting agarose using a standard procedure, precipitated with alcohol, dried and dissolved in water.

0.5 μg Pvu II - EcoRI fragment is incubated in 10 μl srrdnesolevy buffer with 5 units. p Hind III strictase for 1 h at 37 ° C. The DNA from the reaction mixture is precipitated with alcohol, dried and dissolved in water.

To obtain a vector from the syn / pBR327 plasmid, 4 µg of DNA is hydrolyzed in 20 µl of an incubation mixture based on a medium salt buffer containing 15 units. Pvu II and 20 units. Hind III, for 1.5 hours at

37 ° C. The reaction mixture is fractionated on a 1% agarose gel and the vector is eluted,

5 using low melting point agarose. The DN K is precipitated with alcohol, dried and dissolved in water.

Ligation of the Pvu II - Hind 111 fragment to the DNA of HRS cattle with the Pvu II - Hind IU vector (syn / pBR327) was performed in 20 µl of the incubation mixture of the following composition: 50 tM TrisHa, pH 7.6, 10 tM MgCl2, 5 TM DDT. 0.1 M spermidine, 0.05 mM EDTA. 1 tm ltr. Value vector: insert

5 (mol) was 1: 2. The reaction mixture is incubated with 10 units. PhageT4 ligase DNA overnight at 8 ° C. Ligase mixture transform cells of the strain E.coI HB101. Plasmids from the clones obtained are analyzed by restriction enzyme digestion. The correctness of the structure is confirmed by sequencing using the Maxim-Gilbert method. As a result, the plasmid of the desired structure was selected, designated

pBGHl-190.

B. Construction of plasmid pBGHBa112.

To remove the 3-untranslated region of the cattle GR cDNA, 10 µg of the plasmid pBGHI0 190 is digested with the restriction enzyme Wat H I in 20 µl of the incubation mixture based on medium salt buffer for 1 hour at 37 ° C. The linear form of the plasmid is treated with the nucleus Ba131. Next, the obtained Klenow DNA polymerase 1 fragment is processed in the presence of dNTP and the synthetic Hind Illinker 5-CCAAGCTTGG-3 is flashed. The mixture is heated for 5 minutes at 70 ° C, 10 units are added.

0 restrictase EcoRI and 30 units. restrictase Hind III and incubated for 3 hours at 37 ° C. The mixture of the obtained fragments was fractionated on a 1% agarose gel, and fragments 580-650 p. After

5 ethanol re-precipitation of DNA is dissolved in 20 µl of water. 5 μg of the plasmid pV CIS are hydrolyzed with EcoBI and Hind III restrictases. To 100 ng pV C18. digested with EcoRI and Hind 111, add 5 µl of the fragment solution

0 DNA isolated from agarose and incubated with DNA ligase of phage T4. Ligase mixture transform the strain TC1. Cells are plated on indicator plates with, X-gal and IPTG containing ampicillin.

5 Conduct selection of white colonies. Plasmids were isolated from the clones obtained, the nucleotide sequence of the 3-terminal part of the GRS cattle GR cDNA was determined by the Sanger method, the plasmid containing

0 EcoRI-Hind III - a fragment with a gene of GR of cattle size 601 p. O., designated pBGHBa112.

G. Construction of plasmids pBGHtrpl.

Plasmd DIC pSTH2191 containing trp-npoMOTOp, hydrolus and memory are restrictase EcoRI and Hind III. Vector DNA was isolated from 1% agaros gel. The pBGHBa112 hydro plasmid DNA was digested with EcoRi and Hind III restriction enzymes and a fragment of 601 GKO was isolated from an agarose gel, the fragment and the vector were treated with DNA of phage T4 ligase under standard conditions. Ligase mixture transform the cells of E.coI HB101. Plasmids from the obtained clones were analyzed by dt, gmdrolysis with EcoRI and Hind ill. As a result, a plasmid designated pBGHtrpl was selected. The obtained plasmid transform the strains of E. soSh K802 and DH1. The content of HRS of cattle in the strains obtained is analyzed by the method of enzyme immunoassay and by electrophoresis of proteins using polyacrylamide gel with sodium dodecyl sulfate sodium. The highest yield is observed in the Q case of the DHI / pBGHtrpi strain, however, it decreases rapidly with culture stored.

D. Construction of the Plasmdy rVOI / RZ.

G | las.m. bottom 0 pOGHtrpll DNA is hydrolyzed sequentially with restrictase EcoRi and Smal. The DNA vector fraction was made by electrophoresis in 1% -iiOM agro-re / i. The pBGHtrpl plasmid DNA was also hydrolyzed, with Angle restrictases, and a 427 bp fragment was isolated. Conducted ciij-shku fragment and the vector D1- | K leagues of phage T4 and the transformation of the cell with the strain E.E.S.P. DH ligase mixture. Clones that synthesize GR of cattle are selected. Etc; The plasmid structure confirms the sequencing of the 1 insertion. As a result, a plasmid designated pBGHtrpS was selected.

G p and measures 2, Obtaining elaim E. coli DHI - producer of HR cattle,

Plasmid pBGHtrp3 transform the strain E.co.DHi on / growling strain producing GRS cattle.

The strain is grown overnight in 3 ml of UT medium (5 g / l) baktotryptoma, 8 g / p yeast extract ... 5 g / l NaC, 25 mg / l ampicillin). Kegkl precipitated by centrifugation and suspended in 100 ml of buffer containing 100 gpM trisNS (pH 8.0, 1 fiM EDTA and 1% SDS. Suspension: about warming up; for 10 minutes at boiling water ba. E, cooled to ( o 4 with a temperature and centrifuged. 10 µl of the supernatant was taken, 10 µl of buffer containing 20% glycerol, 0% mercaptoethanol was added, 0.2 M trisNA, pH 7.0, 8% SDS, 0.025% bromophenol blue. min at 100 ° C and subjected to polyacrylamide gel electrophoresis. The gel was scanned with a laser dynitometer. The content of HR cattle in cells of the strain is It needs 20-25% of the total protein of the cells.

The content of the hormone in the biomass of the strain is determined by lysing the bacteria and measuring the hormone in the lysate by enzyme immunoassay. The accumulation of GR of cattle in biomass reaches 50-60 mg per 1 liter of night culture.

Claims (1)

1. Recombinant plasmid DNA pBGHtrp 3, encoding growth hormone croup: cattle, 6.9 kb in size, containing: EcoRI - Hind III - a fragment of the plasmid p5 „TH2191 with a size of 5300 bp; EcoRI - Hind in DNA fragment of plasmid pBGH18. encoding growth hormone in cattle, size 601 p. o .; Hind And - DNA fragment of the plasmid pOGH trpll, including the 153-element, the size of 916 p. O .; one EcoRI restriction enzyme cleavage site:. one Srnal restriction enzyme cleavage site located at a distance of 427 bp. from the EcoRi site; three Pst restriction enzyme cleavage sites located at a distance of 300, 1353, and 5095 bp. from the EcoRI site; Yes, the Hind 111 restriction enzyme cleavage site located at a distance of 601 g. and 1517 bp from EcoRI site: one EcoRV restriction enzyme cleavage site located at a distance of 1673 bp. from the EcoRI site; one restriction enzyme 8amHI cleavage site located at a distance of 1863 bp. from EcoRi site: one restriction enzyme SalGl cleavage site located at a distance of 2138 bp. from EcoRI site: one restriction enzyme cleavage site ilpal, located at a distance of 6784 bp. from EcoRI site: Z - genetic markers: Na-gene providing resistance to ampicillin, located in the area from up to 2100 p. Anti-clockwise from the NRA site: a tetracycline resistance tag gene located in the region from 1500 to 2500 bp, clockwise from the EcoRI site: 9170884710 before the sequence encoding2. The bacterial strain Escherlchlacoll VKM bovine growth hormone, paradise-CH325D - the producer of growth hormone is a large-lying promoter region of trypto-horned cattle. new operon.