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FLIP protein and novel use of encoding gene thereof

A technology for encoding genes and proteins, which is applied in the field of FLIP protein and its encoding genes, can solve the problems of lack of enzyme activity, etc., and achieve the effects of reducing follicular atresia, improving reproductive performance of female animals, and promoting cell proliferation

Inactive Publication Date: 2009-08-12
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

cFLIPS has only 2 DEDs, and cFLIPL also contains a caspase homology domain besides 2 DEDs, that is, the caspase proteolytic enzyme region, but because the two catalytic amino acid residues Cys and His are replaced by Tyr and Arg respectively, No enzymatic activity

Method used

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  • FLIP protein and novel use of encoding gene thereof
  • FLIP protein and novel use of encoding gene thereof
  • FLIP protein and novel use of encoding gene thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1, the cloning of bovine FLIP gene

[0042] 1. Bovine ovary total RNA extraction and cDNA synthesis

[0043] The ovaries of healthy cows were aseptically removed, quickly placed in liquid nitrogen, the total RNA was extracted by the Trizol method, and the OD value was measured by an ultraviolet spectrophotometer. 260 / OD 280 RNA >1.8 should be stored at -70°C for later use. cDNA was synthesized by reverse transcription using the reverse transcription kit from TaKaRa Company.

[0044] 2. PCR amplification of the target gene

[0045] According to the full-length cDNA sequence of the bovine FLIP gene (GenBank Accession Number: NM_001012281), the specific primer pair A was designed as follows:

[0046] Upstream primer: 5'-TTCCTTGGAATGACACTGTA-3';

[0047] Downstream primer: 5'-CTTTTTATTTGTGAGAGAGG-3'.

[0048] Primers were synthesized by Beijing Saibaisheng Bioengineering Company.

[0049] Using the pair of specific primers, the cDNA synthesized in step 1 ...

Embodiment 2

[0061] Example 2, construction and identification of recombinant plasmid (pAcGFP-bFLIP)

[0062] 1. Design of site-specific primers with enzyme cleavage

[0063] According to the restriction map of bovine FLIP gene and the multiple cloning site of pAcGFP-N1, BglII and EcoRI were selected as cloning sites. Design primers from both ends of the complete reading frame sequence of the FLIP gene:

[0064] Upstream primer: 5'-ACT AGATCT GCCACCATG TCTGCTGAAGTCAT-3';

[0065] Downstream primer: 5'-ACT GAATTC CTTTGTGAGAGAGGAAGA-3'.

[0066] The upstream primer adds a BglII restriction site and four protective bases before ATG, and at the same time adds a Kozak sequence to improve the efficiency of initial transcription and expression of the inserted gene in eukaryotic cells. When designing the downstream primers, delete the stop codon TAA of the bovine FLIP gene, and then add a C base and an EcoRI restriction site to make the FLIP reading frame consistent with the downstream Ac...

Embodiment 3

[0080] Embodiment 3, expression of recombinant plasmid (pAcGFP-bFLIP) in bovine ovary granulosa cells

[0081] 1. Extraction of pAcGFP-bFLIP plasmid

[0082] The pAcGFP-bFLIP plasmid was amplified in DH5α, and the endotoxin-free pAcGFP-bFLIP was prepared with an endotoxin-free ultrapure plasmid extraction kit (Beijing Tiangen Biochemical Company, catalog number: DP117), and stored at -20°C.

[0083] 2. Isolation, purification and culture of bovine follicular granulosa cells

[0084] ①Wash the bovine ovary obtained from the slaughterhouse with PBS repeatedly, then use a 2ml syringe to draw the cells in the follicles into a petri dish, and remove the oocytes.

[0085] ②Use a Pasteur tube to collect the granular cell mass, and use fresh DMEM / F 12 Wash three times to remove red blood cells (200g / min, centrifugation for 5min).

[0086] ③ Granulosa cells were resuspended in DMEM / F 12 Medium, 37°C, 5% CO2 Incubate in the incubator for 15 minutes.

[0087] ④ Then use DMEM / F 12 C...

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Abstract

The invention discloses an FLIP protein and new applications of encoded genes thereof. The new application of the invention refers to the application of the FLIP protein and the encoded genes thereof in blocking apoptosis induced by FADD and the application in inhibiting apoptosis of ovary granular cells and the application in promoting reproductive performance of animals. The FLIP protein and the encoded genes thereof can obviously inhibit apoptosis of the ovary granular cells induced by FADD, promote growth of oocyte, reduces follicular atresia and serve as potential medicines for improving reproductive performance of cows. The protein and the new applications of the invention provide relatively reliable and concrete experimental basis for increasing ovulation rate of dams and improving reproductive performance thereof, thus enjoying a wide application prospect in improving the reproductive performance of female animals.

Description

technical field [0001] The invention relates to a new application of FLIP protein and its coding gene. Background technique [0002] FLIP (FLICE-inhibitory-protein, also known as CASH, Casper, I-FLIP, CLARP, FLAMME-1, MRTT) is a type of apoptosis protein containing death effect domains (death effect domains, DED) discovered in recent years. Inhibitory proteins are widely found in viruses, eukaryotes, mammals and many other species. FLIP has many similarities with Caspase-8 in structure and sequence, and its overexpression can inhibit the activation of caspase-8 (caspase-8), prevent Caspase-8 from binding to the death-inducing signaling complex DISC, thereby preventing Block Fas-mediated apoptosis signal transduction, effectively reduce the occurrence of transiently enhanced apoptosis in various diseases of the body under pathological conditions. [0003] As an important apoptosis inhibitory protein, FLIP was first discovered in viruses. In the early 1990s, researchers dis...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00A61P15/08
Inventor 许尚忠杨润军李俊雅
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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