Long-acting recombinant FSH fusion protein, preparation method thereof and application of long-acting recombinant FSH fusion protein to timing semen deposition of sows

A fusion protein and composition technology, which is applied in biochemical equipment and methods, fusion polypeptides, drug combinations, etc., can solve the problems of no long-acting recombinant FSH fusion protein, low expression of recombinant hFSH, and complicated preparation process, and achieves the promotion of Simultaneous development of sow follicles, improving sow estrus rate, high titer and high purity

Inactive Publication Date: 2019-07-23
GUANGZHOU VBIO PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, recombinant hFSH (Human follicle-stimulating hormone, referred to as hFSH), a human drug produced by CHO cells, is on the market, but there are still the following problems: first, the expression level of recombinant hFSH produced by the existing method is too low, and the preparation process is complicated. The cost is too high; secondly, its half-life is short, requiring frequent injections
[0019] More importantly, there are no relevant reports on the application of long-acting recombinant FSH fusion protein in timed insemination of sows

Method used

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  • Long-acting recombinant FSH fusion protein, preparation method thereof and application of long-acting recombinant FSH fusion protein to timing semen deposition of sows
  • Long-acting recombinant FSH fusion protein, preparation method thereof and application of long-acting recombinant FSH fusion protein to timing semen deposition of sows
  • Long-acting recombinant FSH fusion protein, preparation method thereof and application of long-acting recombinant FSH fusion protein to timing semen deposition of sows

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Embodiment 1 Preparation of recombinant FSH-Fc fusion protein

[0078] 1.1 Construction of gene expression vector encoding recombinant hFSH-Fc fusion protein

[0079] The gene sequence design was optimized based on the preferred codons of CHO cells, and the optimized fusion gene encoding the signal peptide and hFSH protein β-chain mature peptide, CTP and hFSH protein α-chain mature peptide was synthesized by artificial synthesis. The synthesized DNA fragment is inserted between the EcoRV restriction sites in the transfer vector such as pUC57 to obtain the hFSH plasmid (ie phFSH), and the correctness of the inserted sequence is verified by DNA sequencing method.

[0080] The expression vector was transformed according to pcDNA3.0. First remove the original NeoR / KanR (neomycin / kanamycin) of pcDNA3.0 and replace it with the anti-apoptotic gene BIRC2. BIRC2 gene binds to activated Caspase-3 / 7 and relies on E3 ubiquitin ligase Activity, mediate Caspase-3 / 7 degradation, the...

Embodiment 2

[0095] Preparation of embodiment 2 recombinant FSH-CTP fusion protein

[0096] 2.1 Construction of gene expression vector encoding recombinant hFSH-CTP fusion protein

[0097] The gene sequence design is optimized based on the preferred codons of CHO cells, and the optimized DNA fragment encoding hFSH protein β chain mature peptide, CTP, bGH termination signal, CMV promoter and hFSH α chain mature peptide is synthesized by artificial synthesis , with SpeI and EcoRI restriction sites at both ends. The synthesized DNA fragment was inserted between the EcoRV restriction sites in a transfer vector such as pUC57 to obtain a hFSH-CTP plasmid (ie, phFSH-CTP), and the correctness of the inserted sequence was verified by DNA sequencing.

[0098] The expression vector was transformed according to pcDNA3.0. First remove the original NeoR / KanR (neomycin / kanamycin) of pcDNA3.0 and replace it with the anti-apoptotic gene BIRC2. BIRC2 gene binds to activated Caspase-3 / 7 and relies on E3 ub...

Embodiment 3

[0115] The pharmacokinetic determination of embodiment 3 recombinant FSH-Fc fusion protein

[0116] The test is divided into the recombinant hFSH-vIgG2-hFc, hFSH-vIgG1-hFc, pFSH-vIgG2-hFc, pFSH-vIgG1-hFc, pFSH-pFc fusion protein administration group provided by the present invention and porcine pituitary FSH (commercial product) administration group. For the medicine group, 5 male SD rats with a body weight of 220 ± 10 g were selected for each group, and were injected subcutaneously at a dose of 30 IU / kg respectively.

[0117] Pig pituitary FSH group took blood at 1h, 2h, 4h, 6h, 8h, 12h, 24h, 36h, 60h after administration, recombinant hFSH-vIgG2-hFc, hFSH-vIgG1-hFc, pFSH-vIgG2-hFc, pFSH-vIgG1 -hFc, pFSH-pFc fusion protein were collected at 0h, 2h, 6h, 12h, 24h, 32h, 48h, 56h, 72h, 80h, 96h, 104h, 120h after administration, and centrifuged at 4°C and 3000rpm After 5 minutes, the serum was aspirated and stored at -20°C. ELISA kit (BIOCHECK, USA) was used to measure FSH immune...

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Abstract

The invention discloses long-acting recombinant FSH fusion protein, a preparation method thereof and an application of the long-acting recombinant FSH fusion protein to timing semen deposition of sows. The long-acting recombinant FSH fusion protein comprises human follicle-stimulating hormone Fc fusion protein (hFSH-Fc), porcine follicle-stimulating hormone Fc fusion protein (pFSH-Fc), human follicle-stimulating hormone follicle hormone CTP fusion protein(hFSH-CTP) and porcine follicle-stimulating hormone CTP fusion protein (pFSH-CTP). The long-acting recombinant FSH fusion protein can notablyprolong the half-life in vivo of protein, can notably induce synchronous development of ovarian follicle after being used for multiparous sows and replacement sows, and can promote synchronous estrusof the sows when being used in timing semen deposition procedure of the replacement sows and the multiparous sows, and can also notably increase the estrus rate, the total conception rate and the number born per litter for the sows.

Description

technical field [0001] The invention relates to the fields of molecular biology and veterinary medicine. More specifically, the present invention relates to a long-acting recombinant FSH fusion protein, its preparation method and its application in timed insemination of sows. Background technique [0002] Animal reproduction technology plays an important role in improving the breeding level of our country, but compared with the developed countries, there are still problems such as low reproductive efficiency. The main reason is that the breeding management mode of the existing pig farms in our country has led to the problems of low reproductive efficiency and difficult disease prevention and control, which have also become the bottleneck of the development of the pig industry. [0003] In animal husbandry production, batch breeding is a very important breeding management method. It can truly achieve all-in-all-out, effectively prevent and control diseases, improve the utili...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/85C12N15/62A61K38/24A61K47/68A61P15/08
CPCA61K38/00A61P15/08C07K14/59C07K2319/02C07K2319/30C12N15/625C12N15/85C12N2800/107
Inventor 侯永敏谢显泰吴俊辉孙艺飞吴茂柏陈学敏
Owner GUANGZHOU VBIO PHARM CO LTD
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