Vector for recombinant expression of panda follicle-stimulating hormone, expression system and preparation method thereof

A follicle-stimulating hormone and expression system technology, applied in the field of expression system and preparation, carrier for recombinant expression of giant panda follicle-stimulating hormone, can solve problems such as decline in estrus effect, adverse reactions in animals, and affecting the reproductive rate of giant pandas, and achieve The preparation method is simple, the risk of adverse reactions is reduced, and the cost is low

Active Publication Date: 2020-03-31
CHINA CONSERVATION & RES CENT FOR THE GIANT PANDA SICHUAN
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  • Application Information

AI Technical Summary

Problems solved by technology

[0002] Improving the reproductive rate of giant pandas usually uses heterogeneous exogenous gonadotropins. Studies have shown that long-term use of heterogeneous exogenous gonadotropins to induce estrus and ovulation in animals will cause adverse reactions to animals, and the estrus effect will decline, seriously affecting The reproductive rate of giant pandas
At present, there is no effective method to solve the above technical problems

Method used

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  • Vector for recombinant expression of panda follicle-stimulating hormone, expression system and preparation method thereof
  • Vector for recombinant expression of panda follicle-stimulating hormone, expression system and preparation method thereof
  • Vector for recombinant expression of panda follicle-stimulating hormone, expression system and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] This example provides the construction method of pCDH-FSHβ-T2A-FSHα expression vector.

[0045] The build materials used are as follows:

[0046] Strains: CHO-K1 cells and HEK293T cells, purchased from ATCC, USA; vector pcDNA3.1(+), purchased from Invitrogen, USA; vector pCDH-CMV-MCS-EF1-Puro, purchased from SBI, USA; lentivirus system packaging plasmid psPAX2+pMD2.G was purchased from Invitrogen, USA.

[0047] Reagents: The first gene, the second gene and T2A sequence were synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd., and the primers were synthesized by Beijing Liuhe Huada Gene Technology Co., Ltd.; restriction enzymes and ligases were selected from Baoriyi Biotechnology Co., Ltd. (Beijing) Co., Ltd.

[0048] The construction method includes the following steps:

[0049] 1. Enzyme digestion and ligation reaction: Use restriction enzymes EcoR I and BamH I to carry out double enzyme digestion reaction on the synthetic insert fragment and expression vector p...

Embodiment 2

[0054] This example provides a method for constructing the CHO-K1 cell expression system.

[0055] 1. Culture HEK293T cells (ATCC, USA) in a 6-well plate, and transfect when the cells are 80% full.

[0056] 2. Add pCDH-FSHβ-T2A-FSHα plasmid (1.4μg) and packaging plasmids psPAX2 (1.4μg) and pMD2.G (0.7μg) to 200μl buffer, vortex for 10 seconds and centrifuge briefly.

[0057] 3. Add 3 μl The transfection reagent (Dakota, USA) was vortexed for 10 seconds, centrifuged briefly, and allowed to stand at room temperature (22-26° C.) for 10 minutes.

[0058] 4. Add 200 μl transfection mixture dropwise to the 6-well plate, and shake to distribute the reagent evenly.

[0059] After 5.4 hours, the medium was changed, and the growth medium containing antibiotics and 10% serum was used, and the cells were grown in an incubator at 37°C and 5% CO2.

[0060] 6. At 48 o'clock after transfection, collect the culture medium into a 15-ml sterile conical centrifuge tube with a cover, filter ...

Embodiment 3

[0064] This example provides the pGL3-CRE-luciferase luciferase reporter system for detecting the recombinantly expressed FSH active protein in Example 2.

[0065] So far, the giant panda genome (Panda release 92:ailMel1) annotation shows that many genes, including the giant panda follicle-stimulating hormone receptor gene (FSHR), need to be further annotated. Given that giant panda follicle-stimulating hormone (FSH) relies on binding to its specific receptor

[0066] (FSHR) exerts a biological effect, so this embodiment clones the mouse FSHR gene, based on the fact that both the giant panda FSHR and the mouse FSHR encode a receptor protein with 692 amino acids, and have the characteristics of 85% amino acid sequence homology , the present invention uses mouse FSHR for expression to construct a eukaryotic expression vector (pcDNA3.1-FSHR) to detect the activity of recombinant FSH protein.

[0067] The detailed operation is as follows: the ovarian tissue of C57BL / 6 mouse (Chen...

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Abstract

The invention discloses a vector for recombinant expression of panda follicle-stimulating hormone, an expression system and a preparation method thereof, which relate to the technical field of gene engineering. The vector comprises a first gene for encoding a panda FSH alpha subunit and a second gene for encoding a panda FSH beta subunit, the sequence of the first gene is shown as SEQ ID NO.1, andthe sequence of the second gene is shown as SEQ ID NO.2. By constructing the vector and the expression system for recombinant expression of the panda follicle-stimulating hormone, stable and continuous active expression of the panda FSH alpha subunit, the FSH beta subunit and subunit compounds thereof can be realized. By using the panda-derived follicle-stimulating hormone, the risk of adverse reaction of organisms is reduced, and the breeding and propagation of varieties of rare endangered felines in China, such as giant pandas, small pandas and the like, are facilitated. In addition, the recombinant preparation method is simple and low in cost.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a vector, an expression system and a preparation method for recombinantly expressing giant panda follicle-stimulating hormone. Background technique [0002] Improving the reproductive rate of giant pandas usually uses heterogeneous exogenous gonadotropins. Studies have shown that long-term use of heterogeneous exogenous gonadotropins to induce estrus and ovulation in animals will cause adverse reactions to animals, and the estrus effect will decline, seriously affecting The reproductive rate of giant pandas. At present, there is no effective method to solve the above technical problems. [0003] In view of this, the present invention is proposed. Contents of the invention [0004] The object of the present invention is to provide a vector, an expression system and a preparation method for recombinantly expressing giant panda follicle-stimulating hormone to solve t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C07K14/59C12P21/02
CPCC12N15/86C07K14/59C12N2800/107C12N2740/15043
Inventor 黄炎张志忠屈元元凌珊珊
Owner CHINA CONSERVATION & RES CENT FOR THE GIANT PANDA SICHUAN
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