Recombinant human follicle stimulating hormone and preparation method thereof

A human follicle-stimulating hormone and ovarian cell technology, applied in the field of bioengineering, can solve the problems of low biological activity and poor purification effect of recombinant human follicle-stimulating hormone, and achieve the effects of stable protein quality, increased sialic acid content and improved safety.

Pending Publication Date: 2021-01-08
北京双鹭生物技术有限公司 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] For this reason, the present invention provides a recombinant human follicle-stimulating hormone and its preparation method to solve the problems of poor purification effect and low biological activity of recombinant human follicle-stimulating hormone (rhFSH) in the prior art

Method used

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  • Recombinant human follicle stimulating hormone and preparation method thereof
  • Recombinant human follicle stimulating hormone and preparation method thereof
  • Recombinant human follicle stimulating hormone and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1, construction of rhFSHα subunit gene expression vector

[0044] The gene sequence of the coding region of the FSHα subunit of the present invention is shown in SEQ ID NO: 1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO: 10. The amplification primers of the FSHα subunit gene of the present invention are to add NheI and NotI enzyme cutting sites on the primers at both ends of the gene of the FSH-α subunit, and the designed primers are shown in Table 1.

[0045] Table 1

[0046]

[0047] The above primers I and II were used to amplify the rhFSHα subunit gene using human 293 cell genomic DNA as a template, and Pfu DNA polymerase was used for PCR amplification.

[0048] The PCR amplification conditions are: 95°C for 5min, 94°C for 30s, 60°C for 30s, 72°C for 2min, a total of 30 cycles of amplification, and 72°C for 5min to amplify the DNA fragment of the FSHα subunit gene. Recover on 1% agarose gel, then digest the DNA fragment o...

Embodiment 2

[0050] Embodiment 2, construction of rhFSHβ-CTP subunit gene expression vector

[0051] The C-terminus of the FSHβ subunit of the present invention is linked with the gene sequence of the coding region of CTP as shown in SEQ ID NO: 2, and the amino acid sequence of the encoded protein is shown in SEQ ID NO: 11. The primers at both ends of the rhFSH β-CTP subunit gene of the present invention are added with Nhe I and Xho I restriction sites, and the primer sequences are designed with the rhFSH β-CTP subunit gene. The primer sequences from primer I to primer V are shown in Table 2 .

[0052] Table 2

[0053]

[0054] Using the DNA fragment of the FSHβ gene as a template, primer I (SEQ ID NO: 3) and primer II (SEQ ID NO: 4) were used to amplify, and the DNA polymerase used pfuDNA polymerase from TIANGEN Company.

[0055] PCR amplification conditions are: 95°C for 4min, 94°C for 30s, 60°C for 30s, 72°C for 100s, amplifying for 30 cycles, and extending at 72°C for 5min to obta...

Embodiment 3

[0060] Example 3, Cell Transfection and Screening of Highly Expressed Cell Line rhFSH-CTP

[0061] Transform the recombinant expression vectors pcDNA15.6-FSHα and pcDNA16.1-FSHβ-CTP into Escherichia coli DH5α competent cells, rejuvenate the transformed bacteria with LB liquid selection medium containing ampicillin and expand to 100mL for mass culture, shaking at 37°C After culturing overnight, extract and purify the plasmid, adjust the concentration of the purified plasmid DNA to 1 μg / μL, and mix the recombinant expression plasmids pcDNA15.6-FSHα and pcDNA16.1-FSHβ-CTP at a molar ratio of 1:1.

[0062] One day before transfection, the host cell CHO-S cells were divided into 0.5×10 6 The seeding density of cells / mL was subcultured to 20 mL of fresh medium. On the day of electroporation, three groups of cells in the logarithmic growth phase were collected in parallel, and the number of cells in each group was 1×10 7 After removing the medium by centrifugation, wash the cells w...

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Abstract

The invention discloses a recombinant human follicle stimulating hormone and a preparation method thereof. The preparation method comprises the following steps: jointly transfecting a constructed expression vector containing an FSHalpha subunit and an expression vector containing an FSHbeta subunit into Chinese hamster ovary cells, screening out stably expressed cell strains, and purifying and culturing the cell strains to obtain a culture solution, thereby obtaining the recombinant human follicle stimulating hormone, wherein the C-terminus of the FSHbeta subunit is connected with CTP. According to the preparation method of the recombinant human follicle stimulating hormone, hFSHalpha and hFSHbeta sequences comprising introns are cloned from human embryonic kidney 293 cell genomes respectively, CTP sequences are connected to the terminus of the beta subunit in a primer extension mode to obtain hFSHbeta-CTP, then the two subunit sequences are transfected to CHO-S cells, rhFSH-CTP is obtained through expression, the half-life period of the rhFSH-CTP obtained by expression in vivo is longer, the final half-life period is 2-3 times that of recombinant FSH, and the biological activity is higher.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a recombinant human follicle-stimulating hormone and a preparation method thereof. Background technique [0002] FSH is a heterodimeric glycoprotein with a molecular weight of about 31KD, composed of non-covalently bonded α subunits and β subunits, of which the relative molecular weight of the α subunit is about 14KD, and consists of 92 amino acid residues. Contains 2 N sugar chains and 5 disulfide bonds; the relative molecular mass of the β subunit is about 17KD, consisting of 111 amino acid residues, containing 2 N sugar chains and 6 disulfide bonds. Both FSHα subunit and FSHβ subunit undergo glycosylation after transcription, and this protein glycosylation is very important for its biological activity and is the key to its activity. [0003] Long-acting recombinant human follicle-stimulating hormone is a follicle-stimulating hormone with a longer half-life. The drug m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/66C12N15/16C12P21/02C07K14/59C07K1/22C07K1/20C07K1/18C07K1/16C07K1/34C07K1/36
CPCC12N15/85C12N15/66C07K14/59C12N2800/107
Inventor 高丽婷杨仲璠鲍朋朱红雨张龙章许可王丹丹徐明波
Owner 北京双鹭生物技术有限公司
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