Interference fragment of colipase-like 2 (Clpsl2) gene and application of interference fragment

A lipid-like, gene-based technology, applied in gene therapy, DNA/RNA fragments, diseases, etc.

Inactive Publication Date: 2013-12-18
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, although the functions of some genes and proteins specifically expressed in the epididymis have been analyzed, the diagnosis and treatment of infertility caused by male semen, or the development of corresponding drugs as targets for the design of male contraceptive drugs have not yet been completed. in design stage

Method used

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  • Interference fragment of colipase-like 2 (Clpsl2) gene and application of interference fragment
  • Interference fragment of colipase-like 2 (Clpsl2) gene and application of interference fragment
  • Interference fragment of colipase-like 2 (Clpsl2) gene and application of interference fragment

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Experimental program
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Effect test

Embodiment 1

[0020] Embodiment 1: In situ hybridization detects the expression localization of Clpsl2mRNA in epididymis tissue

[0021] Using ApaI to transfer pGEM-T-Clpsl2 vector containing Clpsl2 gene (Guan Yiqing, Yu Yanhong, Huang Dan, Pan Shanpei. Prokaryotic expression of mouse epididymis-specific colipase and preparation of its antibody[J]. Journal of Jinan University (Nature Science and Medicine Edition), 2010,31(2):105-110.) The plasmid was linearized, purified with phenol chloroform and labeled with Roche’s Digoxigenin Probe Kit, and the antisense probe was labeled with SP6 RNA polymerase to T7RNA Polymerase-labeled sense probe was used as a control, ethanol precipitated, and DEPC was dissolved in water.

[0022] The epididymis of 8-week-old sexually mature mice were taken, put into 4% paraformaldehyde by weight, fixed at 4°C for 2 hours, and washed 3 times with PBS, 5 minutes each time. Then add 30% sucrose solution by mass, and place at 4°C until the tissue pieces sink to the ...

Embodiment 2

[0023] Embodiment 2: Western blot detects the expression of Clpsl2 in each section of epididymis tissue

[0024]Eight-week-old sexually mature mice were killed by neck dislocation, and the epididymis were separated immediately. After removing excess fat and connective tissue, they were washed in pre-cooled PBS, and then the epididymis was separated into three parts: head, body and tail. Each sample taken was added to the Western and IP cell lysate (Mitsu) with a final concentration of 1mM phenylmethylsulfonyl fluoride (PMSF) and protease inhibitor mixture (Calbiochem, Merck Millipore, USA) at a ratio of mass volume ratio of 1:10. organisms), grind in an ice bath. After centrifuging at 12,000rpm at 4°C for 15 minutes, take the supernatant, use the BAC protein quantification kit to quantify the total protein content of each tissue, and then carry out Tricine SDS-PAGE electrophoresis by preparing the gel according to the concentration of the stacking gel at 4% and the concentrati...

Embodiment 3

[0025] Example 3: Immunohistochemical detection of Clpsl2 protein expression localization in epididymis

[0026] Frozen sections of epididymis tissue were taken and incubated with 3% hydrogen peroxide for 10 min. After blocking with 10% goat serum in a wet box at 37°C for 2-3 hours, incubate overnight in a wet box at 4°C with anti-meClpsl polyclonal antiserum (volume ratio 1:500) prepared with 2% BSA by mass. The next day, they were incubated with horseradish peroxidase-labeled goat anti-rabbit IgG (volume ratio 1:300) in a humid box at 37°C for 1 hour. DAB color development, hematoxylin counterstaining gradient alcohol dehydration, xylene transparent, neutral gum mounting, observation under an inverted microscope found that the expression of Clpsl2 protein can be detected in the wall cells of the entire head of the epididymis, and in the head proximal end ( image 3 A, F) The expression level was significantly higher than that in the distal end ( image 3 B), there is a sm...

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Abstract

The invention discloses an interference fragment of colipase-like 2 (Clpsl2) and an application of the interference fragment and belongs to the fields of biotechnology and medicines. Detection of expression positioning on mRNA (Messenger Ribonucleic Acid) and protein of Clpsl2 in epididymis shows that the Clpsl2 is synthesized in epithelial cells at the head of the epididymis, is secreted into a lumen and is specifically combined with perforatorium at the head of a sperm in an epididymis lumen and a main section at the tail of the sperm. The invention discloses an interference fragment of the Clpsl2, wherein the nucleotide sequence of the interference fragment is 5'-ACTCAGTGCTCCAGTGACTGT-3', after expression of the Clpsl2 is reduced by virtue of the interference fragment in the epididymis of a mouse, quantity of sperms at the tail of the epididymis of the mouse is reduced, motoricity of the sperms is weakened, and quantity of embryos of a pregnant mouse after the mouse is mated with a female mouse is reduced; meanwhile, development of multiple embryos is abnormal, so that the interference fragment of the Clpsl2 can be applied to preparation of male mouse contraceptive and preparation of mouse sperm maturity-related medicines.

Description

technical field [0001] The invention belongs to the field of biotechnology and medicine, and in particular relates to an interference fragment of a colipase-like2 (Clpsl2) gene and an application thereof. Background technique [0002] In recent years, human investigations have found that the quality and fertility of male semen are gradually declining. According to statistics, the incidence of infertility among couples of childbearing age in China is 10-15%, of which male factors account for about 40%. It is clinically found that many cases of infertility are associated with abnormalities in sperm count, density, and maturation (movement, capacitation, acrosome reaction, etc.). On the other hand, for a long time, contraception has been mainly aimed at women of childbearing age. With the signing of international human rights treaties, the society has put forward advocacy and requirements for male contraception. The development of male contraceptives with independent property ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113A61K48/00A61P15/16
Inventor 禹艳红陈雷曹佐武蔡冬青廉滋珍秦俊文齐绪峰武征
Owner JINAN UNIVERSITY
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