KR101531286B1 - Japanese eel, Anguilla japonica Single Chain Follicle-Stimulating Hormone, and Use Thereof - Google Patents

Abstract

The present invention relates to Anguilla Japonica single chain follicle-stimulating hormone, and use thereof. The Anguilla Japonica single chain follicle-stimulating hormone contains peptides having an amino acid sequence of SEQ ID NO.1.

Description

(Japanese Eel, Anguilla japonica Single Chain Follicle-Stimulating Hormone, and Use Thereof)

The present invention relates to a follicle-stimulating hormone, a freshwater eel monolith, and its use.

Follicle-stimulating hormone (FSH) and leutinizing hormone (LH) -related genes have been cloned in various animals and fishes. In particular, FSH among glycoprotein hormones has been secreted mainly by early maturation LH is known to be involved mainly in final maturation and ovulation.

Freshwater eel ( Anguilla japonica ) is inadequate in the ability to synthesize gonadotropin (GTH) in the pituitary gland in an anthropogenic environment and thus requires gonadal administration for the induction of artificial maturation. Therefore, mass production of GTH is urgent.

Hormones used to induce an artificial maturity of freshwater eel induce mature through high concentration and repeated administration of pituitary gland (salmon, Oncorhynchus keta ) pituitary gland, but problems such as chromosome abnormality, poor quality of egg and fertilization, To solve these problems, mass production of freshwater eel GTH using molecular biologic methods and improvement of hormone zero are required.

Although it is currently being harvested and processed from salmon pituitary gland, which is a sexually inducing substance of freshwater eel imported from Japan, it is necessary to mass-produce it from recombinant form using animal cells and to secure a stable fertilized egg by inducing sexual maturity when necessary .

In addition, the securing of sex-related genes and proteins in freshwater eel can lead to industrial economic benefits through the enhancement of the base of freshwater eel aquaculture and improvement of productivity through the import substitution effect and activation of bio-industry related to fisheries through their industrial applications. The need is growing.

Korean Patent Registration No. 10-0742115

Therefore, the inventors of the present invention have conducted intensive studies to obtain a protein capable of measuring the expression of follicular stimulating hormone in the freshwater eel, and found that the follicle stimulating hormone gene is cloned from the pituitary gland of freshwater eel, The present invention has been completed on the basis thereof.

SUMMARY OF THE INVENTION The present invention provides a follicle-stimulating hormone polypeptide having a single chain of an alpha group and a beta group of a freshwater eel follicle stimulating hormone and a freshwater eel monolith.

Another object of the present invention is to provide a nucleic acid molecule having a nucleotide sequence encoding said polypeptide.

Another object of the present invention is to provide a vector comprising the nucleic acid molecule.

Another object of the present invention is to provide a method for producing a follicular stimulating hormone polypeptide, which is a single element of freshwater eel.

Another object of the present invention is to provide a hormone composition for inducing sexual maturation of freshwater eel, which comprises the follicle-stimulating hormone polypeptide as the active ingredient.

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To this end, A single chain of alpha group and beta group of freshwater eel follicle stimulating hormone, SEQ ID NO. 1 < / RTI > amino acid sequence of SEQ ID < RTI ID = 0.0 > 1, < / RTI >

The present invention also provides a nucleic acid molecule having a nucleotide sequence encoding said polypeptide.

The present invention also provides a vector comprising the nucleic acid molecule.

The present invention also relates to a method for producing a follicular stimulating hormone polypeptide, a freshwater eel monolith, comprising introducing the nucleic acid molecule into a cell and culturing the cell under conditions such that the encapsulated freshwater eel monolayer follicle stimulating hormone polypeptide is expressed ≪ / RTI >

The present invention also provides a hormone composition for inducing sexual maturation of freshwater eel containing the follicular stimulating hormone polypeptide as the active ingredient.

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According to the present invention, it is possible to replace salmon pituitary gland, which is a sex-inducing substance of freshwater eel, which is imported and used all over Japan by using follicle stimulating hormone polypeptide, a freshwater eel monolith capable of mass production, .

FIG. 1 is a PCR-amplified electrophoresis image of beta and alpha complexes of freshwater eel follicle stimulating hormone.
2 is a schematic diagram for constructing a single chain of freshwater eel follicle stimulating hormone.
3 is a schematic diagram showing a gene that is a monolithic follicle stimulating hormone monolith.
Fig. 4 shows the gene base sequence of follicle stimulating hormone.
Figure 5 shows the amino acid sequence of follicle stimulating hormone.
6 is a map of a follicle stimulating hormone expression vector for Escherichia coli of the present invention.
FIG. 7 is a photograph of SDS-PAGE results of three clones among the transformants of the present invention subjected to confirmation of protein production and Ni-TAE cepharose purification.
Figure 8 is a photograph of SDS-PAGE results to confirm the production of follicle stimulating hormone after final purification.
9 is a photograph showing the expression vector of the follicle stimulating hormone for animal cells.

Hereinafter, the present invention will be described in more detail.

In the present invention, a follicle stimulating hormone polypeptide having a single chain of an alpha group and a beta group of a follicle stimulating hormone of Anguilla japonica was prepared.

For this purpose, in order to clone the FSH gene derived from freshwater eel pituitary gland, the FSH gene of the freshwater eel, which has been found in the past, is cloned into NCBI (National Center for Biotechnological Information, http://www.ncbi.nlm.nih.gov/blast) A primer was constructed to amplify the freshwater eel by searching the data.

Using the above primer, PCR (Polymerase Chain Reaction) was performed on cDNA synthesized from total RNA of freshwater eel pituitary gland, and thus a DNA fragment estimated by FSH was obtained. The PCR method used herein can be variously changed depending on the characteristics of the biological DNA and the primer, and in some cases, it is also necessary to establish the most suitable conditions through several additional attempts.

The nucleotide sequence of the amplified DNA was analyzed in the same manner as described above, and the nucleotide sequence analyzed was NCBI data, and it was confirmed that the gene was actually a freshwater eel FSH gene.

In order to link the alpha and beta genes to single strand of the amplified DNA by the above method, an overlapping PCR method was used to connect the single FSH having no alpha signal portion on the beta termini.

The present invention relates to a polynucleotide comprising a nucleotide sequence of SEQ ID NO. 1 < / RTI > amino acid sequence of the follicular stimulating hormone polypeptide.

The polypeptide may be produced by any method known in the art, including expression from a host cell encoding a nucleic acid molecule encoding it, into a host animal, and / or from a nucleic acid molecule encoding it in vitro . Expression hosts include human cell lines and transgenic animals. Coli, yeast, plants, insect cells and mammalian cells. Expression hosts may differ in the level of protein production as well as the types of post-translational modifications present in the expressed protein. The expression host can be selected based on these factors and other factors, such as control and safety considerations, production costs and the need for purification and purification methods.

Expression in eukaryotic hosts can be controlled by insect cells such as yeast cells such as Saccharomyces cerevisae and Pichia Pastoria, Drosophila cells and lepidopteran cells, Cells, and plant and plant cells such as tobacco, corn, rice, seaweed, and lemna. Eukaryotic cells for expression also include mammalian cell lines such as Chinese hamster ovary (CHO) cells. Eukaryotic expression hosts also include production in transgenic animals, including, for example, production in milk and eggs.

Many expression vectors are available for expression of the follicular stimulating hormone polypeptide of the present invention, the freshwater eel monolith. The choice of expression vector is influenced by the selection of the host expression system. In general, the expression vector may include a transcriptional promoter and optionally an enhancer, a transcriptional signal, and a transcriptional and transcriptional termination signal. Expression vectors used for stable transformation typically have selectable markers that allow selection and maintenance of the transformed cells. In some cases, the copy origin can be used to amplify the copy number of the vector.

The purification of the polypeptide from the host cell depends on the host cell and the expression system. In the case of secretory molecules, the protein is generally purified from the culture medium after removal of the cells. For intracellular expression, the cells can be lysed and the protein purified from the extract. Transgenic organisms, such as transgenic plants and animals, are used for expression, and tissues or organs can be used as a starting material for preparing dissolved cell extracts.

Additionally, transgenic animal production may involve the production of polypeptides in milk or eggs that can be harvested and additional proteins may be extracted and further purified using standard methods in the art, if desired have.

The freshwater eel monolithic follicle stimulating hormone polypeptide can be purified using standard protein tablets known in the art including SDS-PAGE, size fractionation and size exclusion chromatography, ammonium sulfate precipitation and ion exchange chromatography. In addition, the affinity purification technique can be used to improve the efficiency and purity of the formulation.

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In yet another example, the invention provides a composition comprising the amino acid sequence of SEQ ID NO. The present invention provides a hormone composition for inducing sexual maturation of freshwater eel which comprises as an active ingredient a follicle stimulating hormone polypeptide, a freshwater eel monolith comprising a peptide consisting of an amino acid sequence of SEQ ID NO: 1.

The composition of the present invention can be used to induce the maturation of freshwater eel in place of the salmon pituitary gland, which has been dependent on conventional imports.

Hereinafter, the present invention will be described in more detail with reference to examples. These embodiments are only for illustrating the present invention, and thus the scope of the present invention is not construed as being limited by these embodiments.

Example  One: FSH  Isolation and sequencing of genes

The pituitary gland of freshwater eel was recovered and the total RNA was isolated according to the method of Sambrook et al., Molecular cloning: A Laboratory manual, 2 eds Vol.1 pp. 101-104, Cold Spring Harbor Laboratory Press, After separation, 1st strand cDNA was synthesized. In order to clone the FSH gene from the synthesized cDNA, a PCR primer was synthesized as described below, and the FSH gene was amplified by PCR using the primer. Synthesis of PCR primers FSHb F (5'-T GAA TTC ATG CAT CTG GCT GTC ACA GCG-3 ') and FSHb R (5'-GTT GTT GGG ATA CTG GGT CAG ACA GCC TGA-3') having the following sequences Respectively. Synthesis of FSHa F (5'-TGT CTG ACC CAC TAT CCC AAC AAC GAA ATG-3 ') and FSHa R (5'-AC GTC GAC TTA AAA TTT GTG GTA GTA GTA-3' Respectively.

According to the PCR method, a beta 381 bp, alpha 279 bp fragment of the FSH gene was obtained from the fresh-caught eel-derived cDNA using the above primer (Fig. 1).

In order to construct the single chain of the FSH single chain shown in FIG. 2, the beta and alpha fragments obtained and the FSHb F primer and the FSHa R primer PCR was performed to construct a 674 bp FSHb / a single chain (Fig. 3). And the nucleotide sequence and amino acid sequence of the unilateral follicle stimulating hormone (Figs. 4 and 5).

Example  2: Recombination for microorganisms FSH  Construction of Expression Vectors

An FSH gene expression vector was constructed to produce a recombinant protein using the FSH gene identified in Example 1 above.

The 5'-portion of the FSH gene was cloned into a fresh-water eel FSH NdeI primer (5'-AGT CAT ATG TGT GGT CTC GCC AAC-3 ') and a fresh-water eel FSH XhoI primer (5'- CTC GGA TTA AAA TTT GTG GTA GTA GCA-3 ') was synthesized. DNA fragments amplified by PCR were obtained by using the primer and DNA as a freshwater eel FSH monolith, and the obtained fragment was inserted into E. coli expression vector pREST (Invitrogen USA, USA) to construct the FSH recombinant expression vector. The map of the FSH recombinant expression vector thus produced is shown in FIG.

Example  3: Produced from microorganisms Recombinant FSH Purification of

The expression vector prepared in Example 2 was transformed into Escherichia coli E. coli ), and a stable clone was selected therefrom.

For selection of stable clones among the transformed E. coli, LB medium supplemented with ampicillin (50 ug / ml) was inoculated and the surviving cells were selected by incubating at 37 ° C for one day in an incubator.

In order to confirm the protein production, three clones of the transformants prepared above were mass-cultured using a clone producing the most protein among them, and the protein was purified through a Ni-NTA sepharose purification method 7).

The electrophoresis results of the finally purified recombinant single chain freshwater eel FSH protein are shown in FIG.

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Example 4 Production of Recombinant FSH Protein in Freshwater Eel Monolith in Animal Cells

In order to produce a recombinant protein in animal cells using the FSH gene identified in Example 1, a single FSH expression vector was prepared.

Since the EcoRI restriction enzyme site and the SalI site were added at the 3 'end of the alpha at the 5' end of the beta gene in the above Example 1, the FSH gene and the animal cell expression vector pcDNA3 (Invitrogen, USA) To the EcoRI / SalI restriction enzyme recognition site to construct an FSH recombinant expression vector (Fig. 9).

Animal cells (CHO) were transformed using the expression vector prepared above. After 24 hours, the culture medium was changed, and the cells were further cultured for 48 hours. Three days after the transformation, 300 cells were separated on a 10 cm ² culture dish and cultured in G418 (Geneticin; Gibco, USA) culture medium (800ug / ml) in order to isolate only the cells into which the expression vector was inserted.

After 2 weeks, the cells in which the expression vector was inserted had a small colony formed. The colonies were separated, transferred to a 6-well plate, and then the colonies were separated. For the production of FSH protein, the upper layer was recovered by culturing in a serum-free medium CHO-S-SFMII (Gibco, USA) for 2-3 days when grown from 75 cm ² to 70%, and then centrifuged using Centricon (Millipore, USA) And concentrated to 3-4 times at -6,000 rpm.

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<110> Republic of Korea (National Fisheries and Development Institute)          Hankyong Industry Academic Cooperation Center <120> Japanese eel, Anguilla japonica Single Chain Follicle-Stimulating          Hormone, Antibody for Detecting it and Use Thereof <130> DP140189 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 220 <212> PRT <213> Artificial Sequence <220> <223> Single Chain Follicle-Stimulating Hormone Polypeptide <400> 1 Met His Leu Ala Val Thr Ala Leu Cys Leu Thr Leu Ala Pro Ile Leu   1 5 10 15 Ala Arg Ala Ser Thr Ser Cys Gly Leu Ala Asn Ile Ser Ile Ser Val              20 25 30 Glu Asn Glu Glu Cys Gly Gly Cys Val Thr Phe Asn Thr Thr Ala Cys          35 40 45 Ala Gly Leu Cys Phe Thr Gln Asp Ser Val Tyr Lys Ser Ser Leu Lys      50 55 60 Pro Tyr Pro Gln Gln Ala Cys Asn Phe Arg Asp Val Val Tyr Glu Thr  65 70 75 80 Val His Leu Pro Gly Cys Pro Ser Gly Met Asp Leu His Phe Thr Tyr                  85 90 95 Pro Val Ala Leu Ser Cys Glu Cys Ser Lys Cys Asn Thr Asp Ser Thr             100 105 110 Asp Cys Gly Pro Leu Asn Thr Glu Val Ser Gly Cys Leu Thr His Tyr         115 120 125 Pro Asn Asn Glu Met Ala Arg Gly Gly Cys Asp Glu Cys Arg Leu Gln     130 135 140 Glu Asn Asn Ile Phe Ser Lys Pro Ser Ala Pro Ile Phe Gln Cys Val 145 150 155 160 Gly Cys Cys Phe Ser Arg Ala Tyr Pro Thr Pro Leu Arg Ser Lys Lys                 165 170 175 Thr Met Leu Val Pro Lys Asn Ile Thr Ser Glu Ala Thr Cys Cys Val             180 185 190 Ala Arg Glu Val Thr Arg Leu Asp Asn Met Lys Leu Glu Asn His Thr         195 200 205 Asp Cys His Cys Ser Thr Cys Tyr Tyr His Lys Phe     210 215 220

Claims (9)

A single chain of alpha group and beta group of freshwater eel follicle stimulating hormone, SEQ ID NO. 1 &lt; / RTI &gt; amino acid sequence of SEQ ID NO: &lt; RTI ID = 0.0 &gt; 1. &lt; / RTI &gt; 10. A nucleic acid molecule having a nucleotide sequence encoding a follicle stimulating hormone polypeptide of claim 1, delete delete delete A method for producing a follicular stimulating hormone polypeptide of freshwater eel, which comprises introducing a nucleic acid molecule according to claim 2 into a cell and culturing the cell under conditions such that the encapsulated freshwater eel monolayer follicle stimulating hormone polypeptide is expressed. Way. A hormone composition for inducing sexual maturation of freshwater eel, which comprises the monolithic freshwater eel follicle stimulating hormone polypeptide according to claim 1 as an active ingredient. delete delete

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