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Nucleotide sequence, expression vector containing the same, zooblast inverted from said vector and method for producing human fsh using the zooblast

A nucleotide sequence and expression vector technology, applied in the fields of a nucleotide sequence, an expression vector containing the sequence, animal cells transformed by the vector, and the production of human FSH by using the cells, can solve the problem of decreased expression rate and no way Comparing different laboratories, analytical methods not specific for FSH, etc.

Active Publication Date: 2008-08-27
LG CHEM LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the biological assays developed early have the following disadvantages: first, there is no specific analytical method for FSH; second, there is no way to compare the results of different laboratories due to the difficulty of quantification due to lack of standardization
However, the expression rate of recombinant FSH is significantly reduced without the use of animal-derived serum in the culture
The present invention relates to a method of using an animal cell expression vector containing a new gene nucleotide sequence to overcome the problem of low human FSH expression rate caused by the use of serum-free medium

Method used

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  • Nucleotide sequence, expression vector containing the same, zooblast inverted from said vector and method for producing human fsh using the zooblast
  • Nucleotide sequence, expression vector containing the same, zooblast inverted from said vector and method for producing human fsh using the zooblast
  • Nucleotide sequence, expression vector containing the same, zooblast inverted from said vector and method for producing human fsh using the zooblast

Examples

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Effect test

example 1

[0043] Example 1: Preparation of human FSH gene

[0044] The protein sequence was predicted by the information of the cDNA known to encode the α-subunit and β-subunit of natural human FSH (Fiddes and Goodman, (1979) Isolation, cloning, and sequence analysis of the cDNA for the α-subunit of human chorionic gonadotropin, Nature 281:351-356; Fiddes and Goodman, (1981) Thegene encoding the common α-subunit of the four human glycoprotein hormones, J.Mo l.Appl.Genet.1:13-18; Watkins et al., (1987) DNAsequence and regional assignment of the human follicle stimulating hormone β-subunit gene to the short arm of human chromosome 11, DNA6: 205-211). On this basis, a protein sequence that encodes the same protein sequence as the natural human FSH composed of α subunit (CGα) and β subunit (FSHβ) and can promote the protein synthesis efficiency is used to make the protein in animal cells Novel genes with maximized expression levels. Both the prepared α subunit and β subunit have different...

example 2

[0084] Example 2: Construction of animal cell secreted expression vector

[0085] The pLCED expression vector is a variant of pCDM8. In pLCED, a bicistronic expression vector, the M13 origin has been eliminated and the IRES and DHFR genes have been inserted. The construction process of the rhFSH expression vector is outlined below, and expressed as figure 1 .

[0086] Construction process of rhFSH expression vector

[0087] 1. Construction of pLCED

[0088] The M13 origin in pCDM8 vector (Invitrogen, Cat. No. V308-20) was removed with restriction enzymes NHE I and Sac II to construct pCDMBS. Then the sarcoma virus 40 (SV40) intron / poly A signal sequence was replaced with the EMCV / DHFR / poly A signal sequence in the pED vector by restriction enzymes Xba I and Bam II to construct pLECD.

[0089] 2. Construction of BSYH-CGα-DHFR

[0090] The CGα DNA sequence was synthesized by digestion with Hind III and Xba I, and then inserted into pLECD.

[0091] The resulting recombinan...

example 3

[0096] Example 3: Preparation of animal cells transformed with expression vectors

[0097] A permanent cell line (ATCC CRL 9096) derived from Chinese hamster ovary cells knocked out of dihydrofolate reductase (CHO / dhfr-) was used as host cells. The cell line was purchased from the American Type Culture Collection (ATCC, American Type Culture Collection). The growth of the cell line is dependent on proline and has a typical fibroblast morphology.

[0098] The animal cell expression vector expressing the α subunit and the animal cell expression vector expressing the β subunit are constructed independently, but they have the same structure except for the difference in the coding region. Transformed cell lines expressing FSH were prepared as follows.

[0099] The host cell CHO / dhfr-used for transformation is prepared with 10% FBs (GibcoCat.#16000-044, Lot.#1105269) and 1xHT mixed salt (HT supplement, Cat.#111067-030, Lot.#1120062) IMDM medium at 37 °C with 5% CO 2 Cultured in a...

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Abstract

A nucleotide sequence is disclosed, comprising a gene capable of coding protein sequence which is identical to natural human FSH comprising alpha subunit and beta subunit, and capable of improving expression level of the protein sequence. The invention also discloses a recombinant expression carrier comprising the nucleotide sequence, animal cells transformed by the recombinant expression carrier, and a method for using the transformed animal cells to produce secretion type human FSH. In accordance with the invention, expression carrier BSYH / CG alpha-DHFR and BSYH / FSH beta-DHFR are designed to express the animal cell secretion type human FSH, the expressed secretion type recombinant human FSH protein includes the amino acid sequence same as the natural FSH, and includes the sufficient value for clinical usage, therefore the invention is suitable for commercialization and able to be used for effective treatment of sterility.

Description

technical field [0001] The present invention relates to a method for producing secreted human follicle-stimulating hormone (folliclestimulating hormone, hereinafter referred to as FSH) protein by using animal cells. Specifically, the present invention relates to a nucleotide sequence comprising a new gene, a nucleotide sequence comprising the nucleus A recombinant expression vector of a nucleotide sequence, an animal cell transformed by the recombinant expression vector, and a method for producing secretory human FSH using the transformed animal cell, wherein the new gene can encode and contain an α subunit (CG α ) and β subunit (FSHβ) of the same protein sequence of natural human FSH, and can increase the expression level of the protein sequence. Background technique [0002] FSH, luteinizing hormone (hereinafter referred to as LH), and human chorionic gonadotropin (hereinafter referred to as hCG) are reproduction-related hormones that play an important role in reproductive...

Claims

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Application Information

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IPC IPC(8): C12N15/16C12N15/63C12N5/10C07K14/59A61K38/24A61P15/08
Inventor 李承远李泰昊金元谦金炯徹
Owner LG CHEM LTD
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