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Recombinant human follicle-stimulating hormone and preparation method and drug application thereof

A technology of human follicle-stimulating hormone and gene sequence, which is applied in the fields of molecular biology and medicine, can solve the problems of difficult purification, low activity in vivo, and low expression of rhFSH, and achieve the effects of high yield, high purity, and simple process

Pending Publication Date: 2020-06-30
ZONHON BIOPHARMA INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Solve the problems of low rhFSH expression, difficult purification and low in vivo activity in the prior art

Method used

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  • Recombinant human follicle-stimulating hormone and preparation method and drug application thereof
  • Recombinant human follicle-stimulating hormone and preparation method and drug application thereof
  • Recombinant human follicle-stimulating hormone and preparation method and drug application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1 rhFSH β and α chain codon optimization

[0062] Step 1: rhFSH β chain codon optimization

[0063] According to the gene sequence in the DNA sequence of Homo sapiens follicle stimulating hormone betasubunit published by NCBI (NCBI accession number: NM_000510.2), the inventor obtained the gene of the present invention after codon optimization as shown in SEQ ID No: 2. rhFSH beta chain gene, the nucleotide sequence is shown in SEQ ID No: 1, the amino acid sequence is shown in SEQ ID No: 3, and the signal peptide of the above rhFSH beta chain gene is screened and codon optimized to obtain the rhFSH beta chain of the present invention in The signal peptide sequence is secreted and highly expressed in CHO, its nucleotide sequence is shown in SEQ ID No: 4, and its amino acid sequence is shown in SEQ ID No: 5.

[0064] The following is a comparison of the parameters before and after rhFSHβ chain codon optimization:

[0065] 1. Codon Adaptation Index (CAI)

[0066...

Embodiment 2

[0080] Example 2: Co-expression plasmid construction of rhFSH β chain and α chain

[0081] Step 1: Construction of rhFSHβ chain single expression plasmid

[0082] The optimized signal peptide was directly fused with the rhFSHβ chain, and the AvrII restriction site sequence was introduced at the 5' end, and the BstZ7I restriction site sequence was introduced at the 3' end, and the whole gene synthesis was carried out, and the synthetic gene fragment was constructed Into the pUC57 plasmid, a long-term storage plasmid was obtained, which was designated as the pUC57-rhFSHβ plasmid. The pUC57-rhFSHβ plasmid was used as a template for PCR amplification, and the primer sequences used were as follows:

[0083] Upstream primers:

[0084] M13 F: CGC CAG GGT TTT CCC AGT CAC GAC

[0085] Downstream primers:

[0086] M13 R: AGC GGA TAA CAA TTT CAC ACA GGA

[0087] The total reaction volume was 50 μL, in which 2.5 μL was added to each primer with a concentration of 10 μmol / L, and 1 μL ...

Embodiment 3

[0096] Example 3: Pressurized screening of rhFSH stably expressing cell lines

[0097] Puromycin is an aminoglycoside antibiotic that blocks protein synthesis in mammalian cells by interfering with ribosome function. The pac gene from Streptomyces has the effect of detoxifying Puromycin. The pCHO1.0 vector contains the pac gene, so Puromycin can be used as a screening antibiotic for pCHO1.0 as the expression vector. MTX is a folic acid antagonist, which can inhibit the activity of DHFR after conversion in cells, inhibit nucleic acid synthesis, and cause cytotoxicity. pCHO1.0 contains the DHFR gene, and MTX can be used as a screening reagent. The transfected cells contain Puromycin and MTX resistance genes, and the concentration of the screening reagent is continuously increased to increase the copy number of the target gene in the positive cells to increase the expression level.

[0098] The correctly sequenced pCHO1.0-rhFSH plasmid was linearized with NruI restriction end...

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Abstract

The present invention relates to recombinant human follicle-stimulating hormone and a preparation method and a drug application thereof. The recombinant human follicle-stimulating hormone is rhFSH heterodimer containing alpha and beta chains; for a mammalian cell CHO expression system, optimized design of coding genes is performed; and expression plasmids are constructed in a targeted manner, so that expression and correct combination of the alpha and beta chains of the hFSH in mammalian cells CHO are better facilitated. The rhFSH has the advantages of simple process, high target product yield, high purity, high in vivo activity, convenience for intermediate process control, easiness for large-scale production and the like.

Description

technical field [0001] The invention relates to the fields of molecular biology and medicine. More specifically, the present invention relates to a recombinant human follicle-stimulating hormone and its preparation method and pharmaceutical application. Background technique [0002] According to the World Health Organization (WHO) report: about 1 of every 7 couples in the world has reproductive disorders, and infertility affects about 10% to 15% of the world's population. The incidence of infertility in my country is increasing year by year, and in some economically developed coastal areas, due to environmental pollution, work pressure and other factors, more than 20% of infertility patients are affected, especially in those with In the representative Guangdong area, more than two and a half couples out of 10 suffer from infertility due to various reasons. Statistics show that among the causes of female infertility, follicular dysplasia syndrome caused by endocrine dysfunct...

Claims

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Application Information

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IPC IPC(8): C07K14/59C12N15/85C12N5/10C07K1/36C07K1/34C07K1/22
CPCC07K14/59C12N15/85
Inventor 马永王安良
Owner ZONHON BIOPHARMA INST
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