Vector for recombinant expression of luteinizing hormone of panda, expression system and preparation method thereof

The technology of a luteinizing hormone and an expression system is applied to the vector, expression system and preparation field of recombinant expression of giant panda luteinizing hormone, which can solve the problems of adverse reactions of the body, affecting long-term effects, etc., achieves wide application prospects, is beneficial to The effect of high breeding quality and safety

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

AI Technical Summary

Problems solved by technology

[0003] In order to improve the quality of giant panda conservation, my country has used heterogeneous exogenous gonadotropins (for example: follicle-stimulating hormone (FSH) from horses, luteinizing hormone (LH) from horses) to induce estrus and ovulation in giant pandas for many years. However, repeated The use of heterogeneous exogenous hormones may induce adverse reactions in the body, thereby affecting its long-term effect
At present, there is no suitable method to solve the above problems

Method used

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  • Vector for recombinant expression of luteinizing hormone of panda, expression system and preparation method thereof
  • Vector for recombinant expression of luteinizing hormone of panda, expression system and preparation method thereof
  • Vector for recombinant expression of luteinizing hormone of panda, expression system and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] This example provides the construction method of pCDH+LHβ-T2A-LHα expression vector. The build materials used are as follows:

[0051] 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; lentiviral system plasmid psPAX2 +pMD2.G was purchased from Invitrogen, USA.

[0052] 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., and the transfection reagent was purchased from Daktronics.

[0053] Build method:

[0054] 1. Enzyme digestion reaction of vector and insert fragment.

[0055] The α and β subunit genes of giant panda LH and the T2A sequence connecting them were directly synthesized by Jinwei...

Embodiment 2

[0089] This example provides a method for constructing the CHO-K1 cell expression system.

[0090] Recombinant virus packaging and target cell infection, screening experiments.

[0091] (1) Culture HEK293T cells in a 6-well plate, and transfect when the cells are 80% full, and the transfection steps are performed according to the instructions of the kit.

[0092] (2) Add pCDH-LH plasmid (1.4μg) and packaging plasmid psPAX2 (1.4μg) and pMD2.G (0.7μg) to 200μl buffer, vortex for 10 seconds and centrifuge briefly.

[0093] (3) Add 3μl For the transfection reagent, vortex for 10s, centrifuge briefly, and let stand at room temperature (22-26°C) for 10min.

[0094] (4) Add 200 μl transfection mixture dropwise to the 6-well plate, and shake to distribute the reagent evenly.

[0095] (5) Change the medium after 4 hours, use growth medium containing antibiotics and incubate at 37°C, 5% CO 2 incubator culture growth.

[0096] (6) At 48 o'clock after transfection, collect the cul...

Embodiment 3

[0100] This example provides the pGL3-CRE-luciferase luciferase reporter system for detecting the recombinantly expressed LH active protein in Example 2.

[0101] So far, the sequence BLAST in the giant panda genome (Panda release 92:ailMel1) has suggested that there is a NNNN deletion in the 5-terminal sequence of the giant panda LHR gene, that is, there is a deletion in the genome sequence containing the start codon of the giant panda LHR gene, indicating that the giant panda genome Sequences await further annotation. In view of the fact that LH relies on binding to its specific receptor (LHR) to exert its biological effects, this embodiment clones the mouse LHR gene, and combines the giant panda LHR and the mouse LHR to encode a receptor protein with 690 amino acids, and It has a high amino acid sequence identity (87%), suggesting that the mouse LHR can be used to express and construct a eukaryotic expression vector (pcDNA3.1-LHR) to detect the activity of the recombinant L...

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Abstract

The invention discloses a vector for recombinant expression of luteinizing hormone of panda, 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 LH beta subunit and a second gene for encoding a panda LH alpha subunit, the sequence of the first gene is shown as SEQ ID NO.1, and the sequence of the second gene is shown as SEQ ID NO.3. By constructing the vector and the expression system for recombinant expression of the luteinizing hormone of the panda, stable and continuous activeexpression of the LH beta subunit, the LH alpha subunit and subunit compounds of the LH beta subunit and the LH alpha subunit of the panda can be realized. The expression product can be used for inducing oestrus and ovulation of pandas, and luteinizing hormone derived from pandas cannot induce adverse reactions of organisms, so that the breeding quality of pandas can be improved. In addition, therecombinant preparation method is simple, convenient and feasible, and has a wide application prospect.

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 luteinizing hormone. Background technique [0002] The giant panda is a rare and endangered animal in my country. The low reproductive rate restricts the number of giant panda populations and the quality of conservation. Studies have shown that the low reproductive rate of giant pandas may be related to insufficient secretion of gonadotropins such as follicle stimulating hormone (FSH) and luteinizing hormone (LH). [0003] In order to improve the quality of giant panda conservation, my country has used heterogeneous exogenous gonadotropins (for example: follicle-stimulating hormone (FSH) from horses, luteinizing hormone (LH) from horses) to induce estrus and ovulation in giant pandas for many years. However, repeated The use of heterogeneous exogenous hormones may induce a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C07K14/59C12P21/02
CPCC07K14/59C12N15/86C12N2740/15043C12N2800/107
Inventor 黄炎王亚军张志忠张剑南屈元元凌珊珊李娟
Owner CHINA CONSERVATION & RES CENT FOR THE GIANT PANDA SICHUAN
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