Preparation and application of sperm binding protein HEL-127 specifically expressed in human epididymis

A technology of HEL-127 and protein, which is applied in the fields of biotechnology and medicine, and can solve problems such as no reports and no homology

Inactive Publication Date: 2009-09-09
李建远
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Nucleic acid sequence and amino acid sequence analysis show that the gene encodes a 9kD secreted pr...

Method used

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  • Preparation and application of sperm binding protein HEL-127 specifically expressed in human epididymis
  • Preparation and application of sperm binding protein HEL-127 specifically expressed in human epididymis
  • Preparation and application of sperm binding protein HEL-127 specifically expressed in human epididymis

Examples

Experimental program
Comparison scheme
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Embodiment 1

[0039] Example 1 Tissue-specific RT-PCR analysis

[0040] Trizol reagent was used to extract the total RNA of human heart, lung, liver, spleen, kidney, stomach, testis, head of epididymis, body of epididymis and tail of epididymis, the quality of total RNA was identified by agarose gel electrophoresis, and RNA was quantified by UV spectrophotometer. Then reverse transcription (RT) was performed. cDNA synthesis system: primers (oligo d T 18, 25pmol / mL) 3ul, total RNA (300ng / ul) 3ul, ddH 2O (DEPC treatment) 4ul, total volume 10ul; denature at 65°C for 5min, quickly place on ice, then add the following components: 5×AMV buffer 4ul, 10mmol / L dNTP mix 4uL, AMV 1ul, ddH 2 O (DEPC treatment) has a total volume of 20ul; extended at 42°C for 1h, incubated at 70°C for 10min to terminate the reaction, and stored at -20°C for later use.

[0041] The reversed cDNA template was amplified by PCR to detect tissue-specific expression of the gene. The primer sequence used: upstream: 5'-TTGG...

Embodiment 2

[0042] Example 2HEL-127 expression vector construction, protein purification and molecular weight identification

[0043] According to the gene sequence of HEL-127, synthesize a pair of specific primers for amplifying the mature coding region: upstream SC112-F: 5'-TT GGTACC GACGACGACGACAAGCAGTGCATAACATGCCACC-3', downstream SC112-R: 5'-GGC GAATTC TCAGAGTTTAAAGTTACAGTAATTGTAG-3, the upstream and downstream endonuclease sites KpnI and EcoRI were introduced respectively. Using the human epididymis cDNA library as a template, the target gene was directly amplified by PCR, and then cloned into the cloning vector pGM-T vector for sequencing identification. The SC112 gene identified by sequencing was cloned into the expression vector pET32b(+) through KpnI and EcoRI sites, making it consistent with the reading frame of the fusion tag. The recombinant expression vector pET32b(+)-sc112 was transferred into E.coliBL21(DE3) competent cells, and the engineered strain obtained was induce...

Embodiment 3

[0045] Embodiment 3 HEL-127 polyantiserum preparation

[0046] The purified recombinant protein was used to immunize Kunming white mice, and SC112 protein was fully mixed with Freund's complete adjuvant at a dose of 26ug for each mouse, and injected intramuscularly at multiple points on the back; Immunization was boosted three times with incomplete adjuvant; 7 days later, blood was collected from the orbit, and ELLSA was used to determine the titer, and then the blood was collected from the tibia, and the antiserum was separated and stored at -70°C for future use.

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Abstract

The invention belongs to the field of biotechnology and medicine science, and provides sperm binding protein HEL-127 specifically expressed in human epididymis and a nucleic acid sequence for coding the protein. The invention also discloses a preparation method and application of the protein HEL-127. The protein is associated with the mature and defense function of sperms. The HEL-127 protein can be used for preparing medicaments of resisting infections and tumors, provides a novel molecular target for developing novel contraceptive medicine, carrying out aid diagnosis and treating infertility, and is favorable for researches on male reproductive function.

Description

Technical field: [0001] The invention relates to the fields of biotechnology and medicine. The invention provides a novel human epididymis-specifically expressed sperm-binding protein HEL-127, and discloses the amino acid sequence encoding the HEL-127 protein and the DNA sequence encoding the protein. The invention also discloses the preparation and detection method of the protein, as well as the biological function. This protein plays an important role in sperm maturation, normal fertilization and early embryonic development. The HEL-127 protein can be used as a target protein for the research and development of immune contraceptives, as well as the clinical diagnosis and treatment of male infertility. The research and development of this protein is of great significance to male reproductive medicine. Background of the invention: [0002] In recent years, the incidence of infertility among couples of childbearing age has been increasing. According to the research result...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/63C12Q1/68C07K14/47C07K16/18G01N33/577A61K38/17A61P15/00A61P15/16A61P31/00A61P35/00
Inventor 李建远王海燕李宁
Owner 李建远
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