Recombinant equine chorionic gonadotropin fusion protein as well as preparation method and application thereof

A chorionic gonadotropin and recombinant protein technology, applied in the field of biomedicine and animal reproduction, can solve problems such as mare abortion, PMSG animal husbandry production restrictions, and brutal methods

Inactive Publication Date: 2019-02-15
BEIJING VJT BIO CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The currently commercially available PMSG products are extracted from the serum of mares at 40-120 days of pregnancy. Generally, metaphosphoric acid is used to remove impurities in the serum, and then ethanol is used to precipitate them in stages. The production titer of eCG obtained by this treatment method is low. The potency of high-quality PMSG is about 1000IU / mg, so PMSG is greatly restricted in animal husbandry production, and the market urgently needs a large amount of PMSG products
At the same time, the FSH / LH ratio of different batches of PMSG varies greatly, resulting in a large difference in the effect of PMSG
Serum is collected from pregnant mares to prepare PMSG. Excessive blood collection often leads to miscarriage of mares and death of fetal horses. The method is brutal and inhumane

Method used

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  • Recombinant equine chorionic gonadotropin fusion protein as well as preparation method and application thereof
  • Recombinant equine chorionic gonadotropin fusion protein as well as preparation method and application thereof
  • Recombinant equine chorionic gonadotropin fusion protein as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0108] Example 1. Obtaining of recombinant reCG and reCG-Fc fusion protein

[0109] In order to obtain active recombinant eCG protein products, reCG and reCG-Fc fusion proteins were simultaneously designed: search eCGα (GenBank NM_001099763.1), β (GenBank NM_001197093.1) and human Fc (GenBank AH005273.2) in GenBank The gene sequence of the α subunit, β subunit and β-Fc subunit gene sequence was optimized according to the mammalian codon bias, and the optimized α subunit nucleotide sequence is shown in SEQ ID NO: 2 in the sequence table, The optimized β subunit nucleotide sequence is shown in SEQ ID NO:4 in the sequence listing, and the optimized β-Fc subunit nucleotide sequence is shown in SEQ ID NO:6 in the sequence listing.

[0110] The nucleotide sequences shown in SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:6 in the artificially synthesized sequence listing.

[0111] The artificially synthesized above-mentioned nucleotide sequences, that is, the nucleotide coding sequences of ...

Embodiment 2

[0118] Example 2 Fermentation and purification of recombinant reCG-Fc fusion protein

[0119] The reCG-Fc stably transfected cell line prepared in Example 1 was cultured in a shake flask, and the cell culture conditions were optimized. The optimized conditions showed that 100 μM Cu was added to the basal medium 2+ , adding 2mM N-acetyl-D-mannosamine to the feed medium can increase the degree of glycosylation of recombinant reCG-Fc and increase the content of sialic acid by about 20%. After the culture conditions of the shake flask cells were optimized successfully, they were scaled up in a 7L bioreactor and cultured to express the recombinant reCG-Fc fusion protein.

[0120] Cultivate the expressed reCG-Fc fusion protein cell culture fluid, remove cells and cell debris through a two-stage deep filter membrane bag, and then filter with a 0.22 μm filter membrane to obtain a clarified fermentation broth. The clarified fermentation broth was first purified by weak cation exchange...

Embodiment 3

[0121] Example 3 Activity Assay of Recombinant reCG-Fc Fusion Protein

[0122] The biological activity of recombinant reCG-Fc was measured by the rat ovary weight gain method (Steelman-Pohley method), and the drug activity was tested by referring to the "blood gonadotropin bioassay method" in the "Quality Standards for Veterinary Drugs", and the commercially available PMSG was used as the standard. . The specific implementation is as follows: Recombinant reCG-Fc (estimated specific activity 10000U / mg) and PMSG are formulated into three doses of 40IU, 20IU and 10IU, high, medium and low. Female SD (Sprague Dawley) rats aged 21-23 days and weighing 40-55 g were randomly divided into 6 groups, 6 rats in each group. Each rat was subcutaneously injected with 0.5ml of the corresponding drug, and after 6 days, the rat was killed, weighed, dissected, the ovary was removed, weighed, and converted into the weight of the ovary per 100g body weight. The specific activity of reCG-Fc calc...

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Abstract

The invention provides a recombinant equine chorionic gonadotropin fusion protein as well as a preparation method and application thereof. The recombinant equine chorionic gonadotropin fusion proteinspecifically comprises a reCG-Fc fusion protein, the reCG-Fc fusion protein contains an alpha subunit and a beta-FC subunit, the beta-FC subunit is formed through fusion of an eCG beta subunit and animmune globulin Fc segment, and the alpha subunit is connected with the beta-FC subunit through Van der Waals' force. The reCG-Fc fusion protein is expressed by using a mammal eukaryotic expression system. The reCG-Fc fusion protein prepared in the invention is used in the field of animal breeding, which comprises estrus synchronization and superovulation of dams; and compared with Pregnant Mare Serum Gonadotropin (PMSG) extracted from serum of pregnant mare at present, the recombinant equine chorionic gonadotropin fusion protein is more stable in quality, higher in purity, less in impuritiesand better in pesticide effect, and the fusion protein can be applied in animal breeding instead of PMSG.

Description

technical field [0001] The invention belongs to the technical fields of biomedicine and animal reproduction, and specifically relates to a recombinant horse chorionic gonadotropin fusion protein, a preparation method thereof and an application thereof. Background technique [0002] Equine Chorionie Gonadotropin (eCG) is a glycoprotein hormone secreted by the endometrial cup tissue of pregnant horses, donkeys or zebras. It mainly exists in the serum of pregnant horses, so it is also called pregnant horse serum. Gonadotropin (Pregnant Mare Serum Gonadotropin, PMSG). eCG contains two subunits, α and β. The α subunit consists of 96 amino acid residues and contains two N-glycosylation sites, located at N56 and N82 respectively. The β subunit consists of 149 amino acid residues and contains 1 N-glycosylation site and 12 O-glycosylation sites, of which the N-glycosylation site is located at N33 and the O-glycosylation site The points are N118, N123, N127, N128, N129, N130, N131, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C07K1/20C07K1/18C12N15/62C12N15/74A61K38/24A61K47/68A61P15/00
CPCA61K38/00A61K47/6811A61P15/00C07K14/59C07K2319/30C12N15/74
Inventor 罗昊澍师磊韩国
Owner BEIJING VJT BIO CO LTD
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