Human epididymal expression sperm binding protein HEL-28 as well as encoding gene and application thereof

Abstract

The invention belongs to the biotechnology and the medical field and discloses a new human epididymal specific expression sperm binding protein HEL-28 as well as preparation and application thereof. The invention discloses an amino acid sequence of the HEL-28 protein and provides a recombinant expression vector which carries a DNA sequence used for encoding the protein. The protein is located in the back region of the head of a mature sperm, and an RT-PCR result indicates that the protein has low expression level in an epididymis and various tissues of a humna body. A band of 187 KD is detected in the body part of the epididymis by using Western blot. The HEL-28 protein can be used as a target protein for developing an immunological contraceptive and clinically diagnosing and treating male infertility. A method for detecting a corresponding gene or protein based on development of the HEL-28 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-expressed sperm-binding protein hereinafter referred to as HEL-28 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 d...

Examples

Embodiment 1

[0039] Example 1. Preparation of Human Epididymis Expressed Protein HEL-28 Using Genetic Engineering Technology

[0040] Gene cloning and sequence analysis

[0041] 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-28. 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-28 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-sib...

Embodiment 2

[0045] Example 2. Preparation of anti-HEL-28 polyclonal antiserum

[0046] Polyclonal antibodies were prepared by immunizing BALB / C mice with recombinant HEL-28 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-28 extracted from human epididymis fluid.

Embodiment 3

[0047] Example 3. Human HEL-28mRNA tissue expression profile research

[0048] In order to determine the expression pattern of HEL-28, semi-quantitative RT-PCR was used to analyze the expression difference of HEL-28 gene in human epididymis head, body, tail, testis, heart, liver, spleen, lung, kidney, stomach and other tissues. Use TRIzol (Tiangen, Beijing, China) to extract total RNA, 1ug ​​total RNA uses 20UAMV reverse transcriptase (Promega) and 0.3ugoligod T 18 (Promega) was reverse transcribed into cDNA. Then, 2ul of the synthesized cDNA was amplified by PCR using gene-specific primers. 20ul reaction system contains: 2ul10×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-28) / 49°C (β-actin) for 30s; 72°C, 1min (35 cycles); 72°C extension for 7min. The expression of β-actin was used as an internal ...

technical field

[0001] The invention belongs to the field of biotechnology and medicine. Specifically, the present invention relates to a new human epididymis-expressed sperm-binding protein hereinafter referred to as HEL-28 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 d...

Examples

Embodiment 1

[0039] Example 1. Preparation of Human Epididymis Expressed Protein HEL-28 Using Genetic Engineering Technology

[0040] Gene cloning and sequence analysis

[0041] 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-28. 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-28 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-sib...

Embodiment 2

[0045] Example 2. Preparation of anti-HEL-28 polyclonal antiserum

[0046] Polyclonal antibodies were prepared by immunizing BALB / C mice with recombinant HEL-28 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-28 extracted from human epididymis fluid.

Embodiment 3

[0047] Example 3. Human HEL-28mRNA tissue expression profile research

[0048] In order to determine the expression pattern of HEL-28, semi-quantitative RT-PCR was used to analyze the expression difference of HEL-28 gene in human epididymis head, body, tail, testis, heart, liver, spleen, lung, kidney, stomach and other tissues. Use TRIzol (Tiangen, Beijing, China) to extract total RNA, 1ug ​​total RNA uses 20UAMV reverse transcriptase (Promega) and 0.3ugoligod T 18 (Promega) was reverse transcribed into cDNA. Then, 2ul of the synthesized cDNA was amplified by PCR using gene-specific primers. 20ul reaction system contains: 2ul10×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-28) / 49°C (β-actin) for 30s; 72°C, 1min (35 cycles); 72°C extension for 7min. The expression of β-actin was used as an internal ...

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