ESC42 protein, preparation method and use thereof

A protein and amino acid technology used in bioinformatics, molecular immunology and medicine, reproductive biology, molecular biology

Inactive Publication Date: 2006-10-04
李建远
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Fully figuring out the relationship between this programmed gene expression and the sperm maturation process will require a lot of hard work

Method used

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  • ESC42 protein, preparation method and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1: Construction of a healthy young epididymis tissue cDNA library

[0025] 1. Extraction of total RNA

[0026] Human epididymis tissues donated by accidental victims were collected, total RNA was extracted, purified and identified. Take a complete epididymis tissue, weigh it, quickly add Trizol, chop it up, homogenize it electrically, place it at room temperature, extract the total RNA according to the instructions of the Trizol reagent, and use a UV spectrophotometer to identify its concentration and purity by colorimetry at 260nm and 280nm wavelengths. Its integrity was determined by electrophoresis on a denaturing agarose gel with 1.1% formaldehyde. After total RNA extraction, A 260 / A 280 =1.85, 1.1% formaldehyde denatured agarose gel electrophoresis, three bright bands of 28S, 18S and 5S can be seen, and the brightness ratio of 28S:18S is about 2:1, clearly without degradation (Figure 1). 2. Synthesis of the first strand of cDNA: Add RNA lug from human ...

Embodiment 2

[0031] Example 2: Fishing the human epididymis-specific expression gene ESC42 from the library

[0032] Two pairs of primers were designed according to the mRNA sequence of the gene reported in GenBank, ESC42 upstream primer: 5'-CGGAATTCTACAGCGGTGAAAAAAAATGC-3', downstream primer: 5'-TCCCCCCCGGGAAGTCATGAGCTATGGTGAAC-3', 1ul of human epididymis cDNA library was used as a template, and ESC42 primers participated Downstream PCR amplification successfully amplified the target fragment ( FIG. 5 ), and the length of the amplified product was identified by 2.0% agarose electrophoresis gel to be 333bp.

Embodiment 3

[0033] Example 3: Recombinant expression of hESC42 protein with prokaryotic expression vector

[0034] 1. Construction of ESC42 prokaryotic expression plasmid: design primers 5- GGAATTCCATATG TACAGCCGGTGAAAAAAAATGC-3;5 CGGAATTC AAGTCATGAGCTATGGTGAAC-3. They contain EcoRI and NdeI restriction sites (indicated by horizontal lines), respectively.

[0035] 2. Recombination and screening of the pET-ESC42 plasmid: 1 μl of the library was used for PCR, and β-actin was used as a positive control. The amplification conditions were denaturation at 94°C for 2 min, 30 cycles at 94°C for 30 s, 1 min at 60°C, 2 min at 68°C, and extension at 68°C for 7 min. The amplified product was first digested with NdeI overnight, then digested with EcoRI at 37°C for 1 hour, and the large fragment was recovered by electrophoresis; the vector pET-25b(+) was double digested with NdeI and EcoRI, and the large fragment was recovered. : 1 molar ratio and mix well, transform Escherichia coli BL21 after li...

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Abstract

The provided human epididymis specific express protein ESC42 with coded sequence can be prepared by chemical method or genetic engineering technique, and can be used to prepare antibacterial drug or new contraceptive.

Description

technical field [0001] The invention relates to the fields of molecular biology, bioinformatics, reproductive biology, molecular immunology and medicine. Specifically, the present invention relates to the biological function and application of a protein related to male fertility produced by a novel human epididymis-specific expression gene ESC42. The biotechnology involved includes: the construction of human epididymis cDNA library, the screening, cloning, expression and preparation of monoclonal antibody of human epididymis-specific expression gene ESC42, and also involves the expression protein in male reproduction, contraception, anti-infection and anti-tumor and other uses. Background technique [0002] Biotechnology is the most eye-catching high-tech in the field of life science at the end of the 20th century. It has brought hope for the prevention and treatment of human diseases, and to solve problems such as population expansion, energy shortage, and environmental p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435C07K14/47C07K16/18C12N15/12C12N15/70C12N15/79A61K38/17A61P31/00A61P35/00A61P15/16G01N33/53G01N33/68
Inventor 李建远王海燕沈肖方
Owner 李建远
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