Human epididymis expression sperm binding protein HEL-172 as well as encoding gene and application thereof

A technology of HEL-172 and binding protein, applied in the direction of anti-animal/human immunoglobulin, application, gene therapy, etc., can solve the problem of little known protein function and achieve the effect of less side effects

Inactive Publication Date: 2010-06-09
李建远
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are about 200 secreted proteins in the epididymis that interact with sperm and participate in sperm maturation, but little is known about the functions of these proteins

Method used

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  • Human epididymis expression sperm binding protein HEL-172 as well as encoding gene and application thereof
  • Human epididymis expression sperm binding protein HEL-172 as well as encoding gene and application thereof
  • Human epididymis expression sperm binding protein HEL-172 as well as encoding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1. Preparation of Human Epididymal Expression Protein HEL-172 by Genetic Engineering Technology

[0039] Gene cloning and sequence analysis

[0040] Large-scale sequencing and screening of the human epididymis cDNA library constructed in our laboratory was performed to obtain expressed sequence tags (EST), and electronic cloning was performed using the Unigene database to obtain the full-length cDNA of human HEL-172. Using Expasy Translate tool (http: / / us.expasy.orghttp: / / us.expasy.org), InterProScan (http: / / www.ebi.ac.uk / Tools / http: / / www.ebi.ac. uk / Tools / ) and Protein roteinblast (http: / / www.ncbi.nlm.nih.gov / BLAST / http: / / www.ncbi.nlm.nih.gov / BLAST / ) etc. to predict the peptide sequence of HEL-172 and assumed functions. SignalP (http: / / www.cbs.dtu.dkhttp: / / www.cbs.dtu.dk), PSORTII and WoLFPSORT (http: / / psort.nibb.ac.jp) and other software analyzes and predicts the signal peptide of the protein and intracellular localization. ProfileScan (http: / / myhits.isb-si...

Embodiment 2

[0044] Example 2. Preparation of anti-HEL-172 polyclonal antiserum

[0045] Polyclonal antibodies were prepared by immunizing BALB / C mice with recombinant HEL-172 protein. Briefly, each mouse was injected with 50 μg of recombinant protein and the same amount of complete Freund's adjuvant (CFA) on day 1. Then on the 15th, 30th and 45th days, 25 μg of recombinant protein and the same amount of incomplete Freund's adjuvant (IFA) were injected to boost the immunization. On the 60th day, blood was collected from the eyeball, and the serum was separated to analyze the titer and specificity of the antibody by ELASA and western blot. ELISA analysis showed that the antibody titer reached 1:10000. Western blot showed good specificity for both recombinant protein and native HEL-172 extracted from human epididymis fluid.

Embodiment 3

[0046] Example 3. Study on Human HEL-172 mRNA Tissue Expression Profile

[0047] In order to determine the expression pattern of HEL-172, semi-quantitative RT-PCR was used to analyze the expression difference of HEL-172 gene in human epididymis head, body, tail, testis, heart, liver, spleen, lung, kidney, stomach and other tissues. Total RNA was extracted with TRIzol (Tiangen, Beijing, China), and 1 ug of total RNA was reverse-transcribed into cDNA with 20UAMV reverse transcriptase (Promega) and 0.3ugoligodT18 (Promega). Then, 2ul of the synthesized cDNA was amplified by PCR using gene-specific primers. 20ul reaction system contains: 2ul 10×PCR buffer (with MgCl 2 ), 2ul dNTP Mix (10mmol / L), 1ul each primer ul (25umol / L), 1ul Taq DNA polymerase (2.5U / ul), 2ulcDNA template and 11ulddH 2 O. The program of PCR reaction was: 94°C, 10min; 94°C, 1min; 54°C (for HEL-172) / 49°C (β-actin) for 30s; 72°C, 1min (35 cycles); 72°C extension for 7min. The expression of β-actin was used as...

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Abstract

The invention belongs to biotechnology and medical field and discloses a new human epididymis specific expression sperm binding protein HEL-172 and preparation and application thereof. The invention discloses an amino acid sequence of the HEL-172 protein and provides a recombination expression vector carrying a DNA sequence for encoding the protein. The invention further discloses a method for preparing the protein and detecting the biological function of the HEL-172 protein. The protein is positioned at the back region of the head of a mature sperm, has expression in epididymis and spermary, heart, liver, spleen, kidney and stomach. A band of about 34 KD is detected in the epididymis by using Western blot. The HEL-172 protein can be used as a target protein for the research and development of immunity acyeterion as well as clinical diagnosis and treatment of male infertility. The relevant gene or protein detecting method based on the protein HEL-172 can be widely applied to the reproductive medicine research field.

Description

technical field [0001] The invention belongs to the field of biotechnology and medicine. Specifically, the present invention relates to a new human epididymis expressing sperm-binding protein HEL-172 and its coding gene and application Background technique [0002] The development of human beings in this century is facing two serious problems: on the one hand, the global population is rapidly expanding and is expected to reach 9 billion in 2050; on the other hand, according to the World Health Organization survey, about 15% of couples are infertile The problem is that the incidence of infertility in developed countries has soared in the past 10 years, and it can be as high as 30% in developed countries in Europe, of which male factors account for about half. The World Health Organization predicts that with the increase of environmental pollution and sexually transmitted diseases and other pathogenic factors, infertility will become the third major disease after tumors and c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/47C12N15/12C12N15/63C12N5/10C07K16/18A61K38/17A61K31/7088A61K48/00G01N33/68C12Q1/68A61P15/16A61P31/00
Inventor 李建远
Owner 李建远
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