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Recombinant pig FSH-CTP fusion protein as well as preparation method and application thereof

A technology of FSH-CTP and fusion protein, which is applied in the field of recombinant pig FSH-CTP fusion protein and its preparation, can solve the problems of large product batch differences, abortion of mares, and limited sources, so as to achieve uniform quality and improve litter performance , the effect of high protein purity

Active Publication Date: 2018-10-19
BEIJING VJT BIO CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, PMSG is mainly extracted from the serum of pregnant mares. The source is limited and the product batches vary greatly. Excessive blood collection will lead to miscarriage of mares and death of fetal horses.

Method used

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  • Recombinant pig FSH-CTP fusion protein as well as preparation method and application thereof
  • Recombinant pig FSH-CTP fusion protein as well as preparation method and application thereof
  • Recombinant pig FSH-CTP fusion protein as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1 Preparation of pFSH-hCTP and pFSH-eCTP proteins

[0050] The gene sequences of porcine FSHα (GenBank NM-214446.1), porcine FSHβ (GenBank NM-213875.1), human CGβ (GenBank NM-000737.3), and horse CGβ (GenBank NM-001197093.1) were searched in the gene bank. Codon optimization: pFSHα nucleotide sequence, as shown in SEQ ID NO:2; pFSHβ-hCTP sequence, as shown in SEQ ID NO:4; pFSHβ-eCTP sequence, as shown in SEQ ID NO:6.

[0051] The artificially synthesized pFSHα, pFSHβ-hCTP and pFSHβ-eCTP genes were respectively cloned into the vector pcDNA3.1. The recombinant vectors of pFSHα and pFSHβ-hCTP, pFSHα and pFSHβ-eCTP were electroporated into 293 cells to express pFSH-hCTP and pFSH-eCTP, and the transiently expressed proteins were purified to verify their activity. After the activity, the recombinant vectors of pFSHα and pFSHβ-hCTP, pFSHα and pFSHβ-eCTP were linearized and then electroporated into CHO cells to obtain pFSH-hCTP and pFSH-eCTP stably transfected cell lin...

Embodiment 2

[0053] Example 2 Activity detection of pFSH-hCTP and pFSH-eCTP proteins

[0054] The activities of pFSH-hCTP and pFSH-eCTP were determined by rat ovary weight gain method (Steelman-Pohley method). The product of the present invention can replace PMSG and be used in the field of animal reproduction. Therefore, the activity of the drug is tested with reference to the "blood gonadotropin bioassay method", and PMSG is used as a standard product. The specific implementation is as follows: pFSH-hCTP (estimated specific activity 10000U / mg), pFSH-eCTP (estimated specific activity 10000U / mg) and PMSG are prepared 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 9 groups with 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 ...

Embodiment 3

[0055] Example 3 Pharmacokinetic study of pFSH-hCTP and pFSH-eCTP proteins

[0056] Ten female SD rats of about 40 g were selected and randomly divided into two groups: pFSH-hCTP group and pFSH-eCTP group. Subcutaneously inject 20 IU / kg body weight of the corresponding drug, take 100 μl of blood at 0, 1, 2, 4, 8, 12, 24, 48, 72, 96, 120, and 144 hours after administration, and centrifuge at 3000 rpm to get serum and freeze it at -80°C live. The contents of pFSH-hCTP and pFSH-eCTP in serum were detected by FSH ELISA kit, and each blood sample was analyzed three times. Using Pksolver software to calculate the half-life of pFSH-hCTP is 25.7h, and the half-life of pFSH-eCTP is 36.4h, which is higher than that of porcine pituitary FSH (the half-life of pFSH in rats is about 5h).

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Abstract

The invention provides a recombinant pig FSH-CTP fusion protein. The fusion protein means that a beta subunit of pig FSH is directly or indirectly linked to a beta subunit carboxy terminal peptide CTPof human, primate or equine mammalian chorionic gonadotropin, and an alpha subunit of pig FSH is bound to the beta subunit of pig FSH through Van der Waals force. The fusion protein can be prepared by using a eukaryotic expression system on the basis of a genetic engineering technology. Compared with pig pituitary FSH, the pig FSH-CTP fusion protein provided by the invention has a longer half-life and a better drug effect, and can replace pig pituitary FSH and pregnant horse serum gonadotropin in the current market in the field of animal propagation.

Description

technical field [0001] The invention relates to the technical fields of biomedicine and animal reproduction, in particular to a recombinant porcine FSH-CTP fusion protein and a preparation method and application thereof. Background technique [0002] Porcine Follicle-stimulating hormone (pFSH) is a glycoprotein gonadotropin secreted by the anterior lobe of the pig pituitary gland. pFSH can promote the growth of sow endometrium, ovary and follicles; promote the synthesis and secretion of estrogen ; Induce the development of seminiferous tubules and maintain spermatogenesis in boars. In the field of animal reproduction, pFSH is commonly used in estrus synchronization, superovulation, embryo transfer, and the treatment of ovarian diseases in female animals. [0003] pFSH contains two subunits, α and β, the α subunit is responsible for signal transcription, and the β subunit participates in receptor binding to exert biological functions. The α-subunit of pFSH is identical to t...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62A61K38/09A61P15/08A61P15/00
CPCA61K38/09A61P15/00A61P15/08C07K14/59C07K2319/00
Inventor 罗昊澍师磊韩国
Owner BEIJING VJT BIO CO LTD
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