KR100204736B1 - Process for the preparation of korean rockfish growth hormone in e. coli - Google Patents

Abstract

The present invention relates to a method for producing zosterbolac growth hormone in E. coli, and to isolate the nucleotides and amino acid sequence of the zosterbolac growth hormone gene which has not been reported so far, to produce an E. coli expression vector containing the gene By cloning it to E. coli strains, it is possible to genetically produce Zhibolanac growth hormone, thereby greatly contributing to improving food conversion efficiency and promoting growth of cultured fish.

Description

Preparation method of Zopibolac growth hormone in E. coli

The present invention relates to a method for producing Korean rockfish (Sebastes schlegeli) growth hormone in Escherichia coli, and more specifically, after the separation of the gene for Zobicvolac growth hormone, reveals its nucleotide sequence and amino acid sequence, E. coli expression carrier The present invention relates to a method for producing Zopibolac growth hormone by recombination.

Due to the limited amount of fish harvested in the natural state, artificial farming has recently increased. Recently, studies using growth hormone as a way to increase the feed conversion efficiency of fish have been conducted. It is ongoing.

The growth hormone of fish was first analyzed by caffeine such as salmon growth hormone (Skeine, S., et al., Proc. Natl. Acad. Sci. USA, 82, 4306-4310 (1985)). Ron and Chen analyzed cDNA of rainbow trout growth hormone (Agellon, LB and Chen, TT, DNA, 5, 463-471 (1986)), and cDNA of eel growth hormone was identified by Saito et al. Expression of growth hormone was first reported (Saito, N., et al., Gene, 72, 545-552 (1988)). In addition, Cyto et al. Published cDNA sequence of tuna growth hormone (Saito, N., et al., Biochem. Biophys. Acta., 9 49, 35-42 (1988)), and Kimura, a recombinant tuna growth hormone Expression (Kimura, A., Crit. Rev. Biotech, 11, 113-127 (1991)). In particular, Wataiki et al. Identified cDNA sequence and amino acid sequence of flounder growth hormone (Masanori Watahiki, et al., J. Biol. Chem, 264, 312-316 (1989)) and expressed recombinant flounder growth hormone in Escherichia coli. After separation and purification and administration to rainbow trout, it has been reported to have a great effect on growth promotion (Masanori Watahiki, et al., Biosci. Biotech. Biochem, 56, 1012-1016 (1992)).

The inventors of the present invention have continued to research the production of zophybolac growth hormone, which has been widely farmed in Korea but whose nucleotide sequence has not yet been reported, by genetic engineering method, and the phytoplank pituitary cDNA phage using the flounder growth hormone gene probe. The search for a phage library has resulted in the successful isolation of the Zopibolac growth hormone gene and recombination into the E. coli expression carrier, leading to the completion of the present invention.

Accordingly, an object of the present invention is to contribute to the improvement of food conversion efficiency and growth promotion of cultured fish by supplying a large amount of Zofibolac growth hormone inexpensively genetically.

1 shows the nucleotide sequence and the amino acid sequence of Zopibolac growth hormone,

2 is a comparison of the amino acid sequences of Zofibolac growth hormone and other fish growth hormones,

FIG. 3 shows the process of cloning the Zopibolac growth hormone gene into an E. coli expression vector.

Figure 4 is the result of confirming the expression of Zopibolac growth hormone by SDS polyacrylamide gel electrophoresis.

In order to achieve the above object, the present invention provides a Zopibolac growth hormone comprising an amino acid sequence as shown in FIG. 1, a gene encoding the same, an expression vector comprising the gene, and E. coli transformed with the expression vector. do.

The present invention also provides a method for producing Zopibolac growth hormone comprising the step of culturing the transformed Escherichia coli under conditions suitable for the expression of Zopibolac growth hormone, and separating and purifying the produced Zopibolac growth hormone.

Hereinafter, the present invention will be described in detail.

First, RNA is extracted from the pituitary gland of Zopibolac and the mRNA to be used as a template for cDNA synthesis is isolated using an oligod (T) cellulose column. From this, double-stranded cDNA was synthesized, cloned into phage vectors, and then packaged to create a Zofibolac pituitary cDNA phage library.

Next, the phage library is searched using the flounder growth hormone gene of the applicant's Korean Patent Application No. 95-41475 as a probe to select a clone having a positive reaction. Recombinant plasmids containing cDNA fragments were obtained from the selected positive clones, and then the base sequence of the cDNA fragments was confirmed by Sanger et al. (Sanger, F. et al., PNAS USA, 74, 5463 (1977)). The amino acid sequence was estimated from the nucleotide sequence and compared with the amino acid sequence of other known fish growth hormones. The similarity was 89.3% with tuna, 61.5% with salmon, and 63.6% with flatfish.

On the other hand, polymerase chain reaction (hereinafter referred to as polymerase chain reaction) corresponding to the 5 'end and the 3' end of the Zopibolac growth hormone gene encoding the Zopibolac growth hormone protein from the base sequence information of the Zopibolac growth hormone identified as described above Primers for PCR) are designed and synthesized respectively. The 5 'primer allows the restriction enzyme NdeI recognition site to exist to facilitate cloning into an E. coli expression vector, and the corresponding 3' primer inserts an end codon and a restriction enzyme XhoI recognition site to synthesize an artificial protein from the start codon. Complete the protein synthesis in the codon inserted into the codon. Using these primers and the Zobicbolac Growth Hormone cDNA obtained above as a template, amplified the Zovolvolac Growth Hormone gene by PCR reaction.

The gene fragment obtained by cleaving the amplified Zobicbolac growth hormone gene with a restriction enzyme was then cloned into an appropriate expression vector, for example, E. coli expression vector ptrp3H BGHRAN (KCTC 0143BP, Korean Patent Application No. 94-40025). The restriction enzyme NdeI recognition site near the site (ori) is removed by site-specific mutagen esis, and then replaced with the BGHRAN gene to prepare an E. coli expression vector containing a Zibobolac growth hormone gene.

A suitable host microorganism, for example, E. coli strain W3110 (ATCC 37339), is transformed with the Zopibolac growth hormone expression vector thus prepared, followed by culturing the transformed E. coli under conditions suitable for expression of the present gene. Whether the protein is produced can be usually confirmed by SDS-polyacrylamide gel electrophoresis, and the resulting protein can be separated and purified from the cultured Escherichia coli by a known method.

Hereinafter, the present invention will be described in more detail by the following examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.

Example 1

Isolation and Sequencing of Zofibolac Growth Hormone Gene

(Step 1) Preparation of cDNA Library from Zofibolac Pituitary Gland

RNA was extracted from the Zopibolac pituitary gland following the guanidium thiocyanate method of Cologinski et al. (Cholozynski, P. et al., Anal. Biochem., 162, 156 (1987)) and the oligo d (T) cellulose column MRNA was isolated using. The cDNA library was prepared using Zap-cDNA synthesis kit (Stratagene, Cat No. 200400) using mRNA as a template for the obtained pipinakac pituitary gland, and gigapack II gold packaging extract (Gigapack II gold packaging extract; Cat No. 200214). The obtained Zopibolac pituitary cDNA phage library was amplified in Escherichia coli XL I -Blue MRF cells (Stratagene).

(Step 2) Exploration of Zofibolac pituitary cDNA Haji library

Clones showing the Applicant of the Republic of Korea Patent Application No. 95-41475 favor flounder growth hormone gene was labeled with ³² P isotope as a fragment search for jopi bolnak pituitary cDNA library phage obtained in Step 1 out with this probe were selected by positive . Hybridization was carried out at 50 ° C. according to Maniatis et al. (Maniatis, T. et al., Molecular cloning (1982)), filter washing twice at 30 ° C. at 1 × SSC, 0.1% SDS to 50 ° C., and 0.5 × SSC. , 15% once at 55 ° C. in 0.1% SDS. Recombinant pBluescript plasmids containing cDNA fragments were obtained from E. coli XL I-Blue cells, ExAssist helper phage, and E. coli SOLR cells (Stratagene) from selected positive clones.

(Step 3) zophybolac growth hormone gene sequence confirmed

The base sequence of the cDNA fragment cloned into the recombinant pBluescript plasmid obtained in step 2 was subjected to a die cycle sequencing system according to Sanger et al. (Sanger, F. et al., PNAS USA, 74, 5463 (1977)). (Applied Biosystems, USA) and analyzed using ABI 377 DNA sequencer (Applied Biosystems). The amino acid sequence was estimated from the base sequence thus analyzed. Figure 1 shows the base sequence and amino acid sequence of Zopibolac growth hormone.

Figure 2 compares the amino acid sequences of Geoffbolac Growth Hormone with other fish growth hormones, where krGH is Zofbolac Growth Hormone, tGH is Tuna Growth Hormone, and yGH is the Yellowtail Growth Hormone. , sGH refers to salmon growth hormone, fGH refers to flounder growth hormone. As shown in FIG. 2, the amino acid sequence of Zhibolvolak growth hormone showed similarity with 89.3% with tuna, 61.5% with salmon and 63.6% with flatfish. From this, it can be confirmed that the positive clone obtained from the Zopibolac pituitary cDNA phage library is Zopibolac growth hormone cDNA.

Zofibolac growth hormone, which includes a signal sequence, consists of 204 amino acids. When compared with the amino acid sequences of other fish growth hormones, 17 amino acids at the amino terminus are assumed to be signal sequences. After translation, it is processed and truncated, so that Zopibolac growth hormone, which does not contain a signal sequence, is composed of 187 amino acids.

Example 2

Expression of Zopibolac Growth Hormone in Escherichia Coli

(Step 1) Amplification of Zopibolac Growth Hormone Gene

The following primers were synthesized from the nucleotide sequence information of Zofibolac growth hormone obtained in step 3 of Example 1.

Precursor NdekrGH:

5'-CTCTGCATATGCAGCCAATCACAGACGGCCAGCG-3 'corresponds to the 5' end of Zobolbolac growth hormone, has a restriction enzyme NdeI recognition site, and is designed to include 23 bases after the signal sequence of the gene of Zovolvolac growth hormone;

Precursor XhokrGH:

5'-TTCTCGTCGACTACAGGGTGCAGTTGGCTTCTGG-3 'corresponds to the 3' end of Zobolbolac growth hormone and is designed to have an end codon and a restriction enzyme XhoI recognition site so that translation is terminated.

2 μg of the primer NdekrGH and 2 μg of the primer XhokrGH obtained above were added to the reaction tube, and as a template, 1 ng of a recombinant pBluscript plasmid containing the zodibolac growth hormone cDNA fragment whose base sequence was confirmed in step 3 of Example 1 was added. 10 μl of 10-fold polymerization buffer (500 mM KCl, 100 mM Tris-HCl, pH 9.0, 1.0% Triton X-100), 10 μl of 2 mM dNTP (2 mM dGTP, 2 mM dCTP, 2 mM dATP, 2 mM dTTP), 2.5 Taq polymerase of the unit was added and distilled water was added to make the total volume 100 μl, followed by 1 min at 95 ° C. (modification step), 40 sec. At 55 ° C. (immersion step), and 2 min. At 72 ° C. (polymerization step). PCR was carried out 25 times. PCR products obtained therefrom were separated on 7% polyacrylamide gel and confirmed that about 580 base pairs of DNA were amplified. Hereinafter, this DNA fragment is called "fragment krGH".

(Step 2) Preparation of Expression Vector of Zofibolac Growth Hormone

Restriction enzyme NdeI recognition site near the origin of replication (ori) of ptrp3H BGHRAN (KCTC 0143BP, Dec. 26, 1994), the E. coli expression vector of the applicant's Korean Patent Application No. 94-40025, was removed by site-specific mutation. 2 μg of the plasmid was digested completely with restriction enzymes NdeI and SalI under conditions of NEB buffer 3 (10 mM Tris-HCl, 10 mM MgCl ₂ , 100 mM NaCl, 1 mM DTT, pH 7.0) and then in 0.7% agarose gel. The fragments of about 2.4 kb were separated and purified by electrophoresis. Hereinafter, this cross section will be referred to as 'cross section ptrp 3HI -N / L'.

Meanwhile, 'fragment KrGH' obtained in step 1 was completely cleaved with restriction enzymes NdeI and XhoI under the conditions of NEB buffer solution 3 and then separated on 7% polyacrylamide gel to separate and purify the fragment of about 570 base pairs. This fragment is called 'fragment KrGH-N / X'.

Put 100ng of 'fragment ptrp 3HI-N / L' and 100ng of 'fragment KrGH-N / X' into the conjugation reaction tube and add 2μl of 10-fold conjugation solution (0.5M Tris-HCl, 0.1M MgCl ₂ , 0.2 MD TT, 10 mM ATP, 0.5 mg / ml bovine serum albumin, PH 7.8) and 10 units of T4 DNA ligase were added, and distilled water was added so that the total volume was 20 μl and reacted at 16 ° C. for 12 hours. After the conjugation reaction, E. coli W3110 (ATCC 37339) was transformed, and the E. coli expression vector ptrp 3HI KrGH containing the fragment KrGH was obtained from the transformed cells. Figure 3 shows the procedure for cloning the E. coli expression vector of the Zopibolac growth hormone gene.

Escherichia coli strain W3 110 / ptrp3HI KrGH transformed with the Zopibolac growth hormone expression vector of the present invention was deposited in the Korea Gene Bank as Accession No. KCTC 02 83BP dated November 11, 1996.

(Step 3) Confirmation of the base sequence of the amplified Zobiculnak growth hormone gene

The nucleotide sequence of the amplified gene cloned in the recombinant ptrp 3HI KrGH plasmid obtained in step 2 is a die cycle sequencing system according to Sanger et al. (Sanger, F. et al., PNAS USA, 74, 5463 (1977)). The Dye Cycle Sequencing System (Applied Biosystems, USA) was used and analyzed by ABI 377 DNA sequencer (Applied Biosystems).

(Step 4) Expression of Zopibolac Growth Hormone in Escherichia Coli

The recombinant E. coli cells obtained in step 2 were shaken for 12 hours in liquid Luria medium (6% bactotrypton, 0.5% yeast extract, 1% sodium chloride) containing 50 μg / ml of empicillin. 3 ml of the culture was added to 300 ml of M9 medium (40 mM K ₂ HPO ₄ , 22 mM KH ₂ PO ₄ , 8.3 mM NaCl, 18.7 mM NH ₄ Cl, 1% glucose, 0.1 mM MgSO ₄ , 0.1 mM CaCl ₂ , 0.4% casamino acid , 10µg / ml Vit.B1, 40µg / ml Empicillin) and shake culture at 37 ° C. for about 4 hours, and the final concentration of indoleacrylic acid (IAA) was about 50µg at 650nm. / ml was added. About 4 hours after the addition of IAA, the absorbance of the cell culture was measured, and E. coli cell precipitates were collected by centrifugation at 11,000 rpm for 25 minutes using a centrifuge (Beckman J2-21, JA14 roter). The harvested cell precipitate was confirmed by expression of 15% polyacrylamide gel electrophoresis in the presence of SDS according to the method of Lamli (Laemmli, Nature, 227, 680 (1970)). Figure 4 is the result of confirming the expression of Zopibolac growth hormone by SDS polyacrylamide gel electrophoresis. Here, the first column is E. coli culture cell lysate containing no expression vector, the second column is E. coli culture cell lysate containing the expression vector ptrp 3HI KrGH of the present invention, and column M is a standard molecular weight labeling protein. .

As described above, in the present invention, the phytoplank growth hormone gene, which has not been reported so far, is first isolated, the nucleotide sequence and the amino acid sequence are identified, the E. coli expression vector containing the gene is prepared, and then cloned into the E. coli strain. By culturing, it is possible to genetically produce Zhibolbolac Growth Hormone, which can contribute to improving food conversion efficiency and promoting growth of cultured fish.

Claims (8)

Zofibolac growth hormone protein comprising the following amino acid sequence. The gene encoding the Jopibolac growth hormone protein of claim 1. The gene according to claim 2, which has a nucleotide sequence shown in FIG. An expression vector comprising the gene of claim 2. The plasmid ptrp3HI KrGH according to claim 4. E. coli transformed with the expression vector of claim 4. E. coli W3110 / ptrp3HI KrGH (KCTC 0283BP). E. coli is cultured in a condition suitable for the expression of zolbolac growth hormone, and the method for producing zolvolac growth hormone comprising the step of separating and purifying the produced zolbolac growth hormone.