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Application of long-acting recombinant FSH fusion proteins in batch production of sows

A fusion protein and sow technology, which is applied in the direction of medical preparations containing active ingredients, 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. Achieve the effects of improving reproductive efficiency and breeding level, shortening average litter size, and high titer and 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
[0021] More importantly, there are no relevant reports on the application of long-acting recombinant FSH fusion proteins in batch production of sows

Method used

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  • Application of long-acting recombinant FSH fusion proteins in batch production of sows
  • Application of long-acting recombinant FSH fusion proteins in batch production of sows
  • Application of long-acting recombinant FSH fusion proteins in batch production of sows

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Embodiment 1 Preparation of recombinant FSH-Fc fusion protein

[0073] 1.1 Construction of gene expression vector encoding recombinant hFSH-Fc fusion protein

[0074] 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.

[0075] 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

[0090] Preparation of embodiment 2 recombinant FSH-CTP fusion protein

[0091] 2.1 Construction of gene expression vector encoding recombinant hFSH-CTP fusion protein

[0092] 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.

[0093] 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

[0110] The pharmacokinetic determination of embodiment 3 recombinant FSH-Fc fusion protein

[0111] 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.

[0112] 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 present invention discloses an application of long-acting recombinant FSH fusion proteins in batch production of sows. The long-acting recombinant FSH fusion proteins comprise human follicle stimulating hormone Fc fusion protein (hFSH-Fc), porcine follicle stimulating hormone Fc fusion protein (pFSH-Fc), human follicle stimulating hormone CTP fusion protein (hFSH-CTP), and porcine follicle stimulating hormone CTP fusion protein (pFSH-CTP). The recombinant FSH fusion proteins can significantly prolong an in-vivo half-life period of the proteins and can induce simultaneous development of follicles when used in multiparous sows and replacement sows. The long-acting recombinant FSH fusion proteins can significantly promote simultaneous estrus of the sows, can also significantly improve anestrus rate, a total conception rate, a total delivery rate, and a litter size of the replacement and multiparous sows, and significantly improves reproductive performances of the sows after used in the batch production of 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 the application of long-acting recombinant FSH fusion protein in batch production of sows. Background technique [0002] With the development of China's economy and the improvement of people's living standards, the demand for meat is also increasing, and the aquaculture industry has developed rapidly. 34.27 million sows in stock. [0003] Due to factors such as inconsistent reproductive cycles of sows, breeding factories usually need a large number of breeding sheds and facilities as well as manpower and material resources to ensure the reproductive needs, resulting in high breeding costs, which in turn limits the large-scale intensive breeding of animal husbandry. At least 50 years ago, researchers tried to regulate the estrus cycle of pigs in order to maximize the use of facilities and personnel for pig breeding. Es...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/85C07K1/22A61K38/24A61P15/08
CPCA61K38/24A61P15/08C07K14/59C07K2319/02C07K2319/30C12N15/85
Inventor 雷瑶侯永敏李梅陈成涌黄虎张玉杰
Owner GUANGZHOU VBIO PHARM CO LTD
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