Human epididymis expression sperm binding protein HEL-81, encoding gene and application thereof

A HEL-81 and protein-binding technology, applied in the fields of biotechnology and medicine, can solve the problems of poorly understood protein functions 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-81, encoding gene and application thereof
  • Human epididymis expression sperm binding protein HEL-81, encoding gene and application thereof
  • Human epididymis expression sperm binding protein HEL-81, encoding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1. Preparation of human epididymal expression protein HEL-81 by genetic engineering technology

[0040] Gene cloning and sequence analysis

[0041] The human epididymal cDNA library constructed in our laboratory was screened by large-scale sequencing, and expressed sequence tags (EST) were obtained. The Unigene database was used for electronic cloning to obtain human HEL-81 full-length cDNA. Use 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 HEL-81 peptide sequence And assumed function. SignalP (http: / / www.cbs.dtu.dkhttp: / / www.cbs.dtu.dk), PSORTII and WoLFPSORT (http: / / psort.nibb.ac.jp) and other software analyze and predict the signal peptide of the protein And intracellular localization. ProfileScan (http: / / myhits.isb-sib.ch / cgi-bin / PFSCANhttp: / / myhits.isb-sib...

Embodiment 2

[0045] Example 2. Preparation of anti-HEL-81 polyclonal antiserum

[0046] Polyclonal antibodies were prepared by immunizing BALB / C mice with recombinant HEL-81 protein. Simply put, each mouse was injected with 50 μg of recombinant protein and an equivalent amount of complete Freund's adjuvant (CFA) on the first day. Then on the 15, 30 and 45 days, 25 μg of recombinant protein and an equal amount of Incomplete Freund's Adjuvant (IFA) were injected to boost the immunization. On the 60th day, blood was taken from the eyeball, and the serum was separated and analyzed for the titer and specificity of the antibody by ELASA and western blot. ELISA analysis showed that the antibody titer reached 1:10000. Westernblot showed good specificity for both recombinant protein and natural HEL-81 extracted from human epididymal fluid.

Embodiment 3

[0047] Example 3. Study on tissue expression profile of human HEL-81 mRNA

[0048] In order to determine the expression pattern of HEL-81, semi-quantitative RT-PCR was used to analyze the expression differences of HEL-81 gene in human epididymal 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 total RNA was reverse transcribed into cDNA with 20UAMV reverse transcriptase (Promega) and 0.3ugoligodT18 (Promega). Then, 2ul of 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 PCR reaction program is: 94°C, 10min; 94°C, 1min; 54°C (for HEL-81) / 49°C (β-actin) 30s; 72°C, 1min (35 cycles); 72°C extension for 7 minutes. The expression of β-actin serves as an internal contr...

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Abstract

The invention belongs to the field of biotechnology and medicine, and discloses a new human epididymis specific expression sperm binding protein HEL-81 as well as preparation and application thereof. The invention discloses an amino acid sequence of the HEL-81 protein and provides a recombination expression carrier carrying the DNA sequence for encoding the protein. The protein is located in the rear area of the head of a mature sperm. A RT-PCR result shows that the gene is weakly expressed in the epididymis and heart. A band of about 44 KD is detected in the corpus epididymidis part with Western blot. The HEL-81 protein can be used as a target protein for researching and developing immunity contraceptive as well as clinical diagnosis and treatment for male sterility. A method for detecting a corresponding gene or protein based on development of the HEL-81 protein can be widely used for the research field of reproductive medicine.

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-81 and its coding gene and application Background technique [0002] Human development 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 last 10 years, and it can reach as high as 30% in developed countries in Europe, and male factors account for about half. The World Health Organization predicts that with the increase of pathogenic factors such as environmental pollution and sexually transmitted diseases, infertility will become the third largest disease in this century after tumors and cardiov...

Claims

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Application Information

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Patent Type & AuthorityApplications(China)
IPC IPC(8): C07K14/47C12N15/12C12N15/63C12N5/10C07K16/18A61K38/17A61K31/7088A61K48/00G01N33/68C12Q1/68A61P15/16A61P31/00A61P35/00
Inventor李建远
Owner李建远