Vector, expression system and preparation method for recombinantly expressing giant panda follicle-stimulating hormone
A follicle-stimulating hormone and expression system technology, applied in the field of recombinant expression of giant panda follicle-stimulating hormone, expression system and preparation field, can solve problems such as adverse reactions of animals, decrease in estrus effect, influence on reproduction rate of giant pandas, etc. The effect of reducing the risk of adverse reactions, simple preparation method and low cost
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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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