Immunological contraception vaccine and preparation method thereof

A technology for nucleic acid vaccines and immune contraception, which can be used in pharmaceutical formulations, medical preparations containing active ingredients, gene therapy, etc., and can solve problems such as human life threats

Inactive Publication Date: 2014-08-06
INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In addition, rats and other harmful mammals can spread hemorrhagic fever, plague an...

Method used

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  • Immunological contraception vaccine and preparation method thereof
  • Immunological contraception vaccine and preparation method thereof
  • Immunological contraception vaccine and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Bioinformatics analysis of sperm-egg fusion key membrane protein Izumo specifically expressed in sperm

[0029] Sperm protein Izumo is a type I membrane protein specifically expressed in mouse testis tissue and sperm, and belongs to the immunoglobulin superfamily. Its coding gene is located on mouse chromosome 7, and its nucleotide sequence number is XM_133424. According to the full-length mRNA sequence analysis of mouse Izumo, its coding gene is 1194bp in length, the precursor protein is composed of 397 amino acids, the amino terminal (1-21aa) has a signal peptide sequence, and the carboxy terminal (320-340aa) is hydrophobic the sequence of the transmembrane region. The likely functional domain of mouse Izumo is the extracellular immunoglobulin-like domain (161-251 aa). In this domain, Asn 204 is a potential N-linked glycosylation site, and Cys182 and Cys233 form an intramolecular disulfide bond. Homology analysis showed that the amino acid sequence of mous...

Embodiment 2

[0030] The PCR amplification of the mouse sperm Izumo gene of embodiment 2

[0031] Reagents and instruments:

[0032] Trizol Reagent (Invitrogen, USA)

[0033] EcoRI and XhoI restriction endonucleases, T4DNA ligase, MLV reverse transcriptase, and RNase Inhibitor were purchased from TaKaRabao Bioengineering (Dalian) Co., Ltd.

[0034] Cloning vector pBluescript II KS+ (Stratagene, USA)

[0035]High-fidelity pfu DNA polymerase (NEB company, USA)

[0036] Ordinary DNA Gel Recovery Kit (Bomad Technology Co., Ltd., China)

[0037] UV spectrophotometer (Bio-Rad, USA)

[0038] DNA Engine Peltier Thermal Cycler (Bio-Rad, USA)

[0039] experimental method:

[0040] 1. By analyzing the mouse sperm Izumo nucleotide sequence (sequence number XM_133424) published in GenBank, a pair of specific primers for amplifying the mouse sperm Izumo coding region sequence 1194bp was designed online using Primer Premier5.0 software. For cloning and expression, restriction sites EcoRI and XhoI s...

Embodiment 3

[0045] Example 3 Construction and identification of eukaryotic expression vector pCAGGS-Izumo and large-scale preparation of plasmids Reagents and instruments:

[0046] EcoRI and XhoI restriction enzymes, T4DNA ligase (TaKaRabao Bioengineering (Dalian) Co., Ltd.)

[0047] Eukaryotic expression vector pCAGGS (Invitrogen, USA)

[0048] Transfection reagent Lipofectamine2000 (Invitrogen, USA)

[0049] HEK293T cells were donated by Professor Zhang Xuemin of the National Center for Biomedical Analysis

[0050] DMEM high glucose medium, special grade fetal bovine serum and trypsin (HyClone company, USA)

[0051] Mouse monoclonal antibody against Flag tag (Sigma, USA)

[0052] Horseradish peroxidase (HRP)-labeled goat anti-mouse IgG (Cell Signaling, USA)

[0053] Western Luminescence Detection Kit (Viglass Biotechnology Co., Ltd., China)

[0054] A large number of high-purity endotoxin-free plasmid extraction kits (Bomaide Technology Co., Ltd., China)

[0055] UV spectrophotome...

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Abstract

The invention provides an immunological contraception vaccine, including a DNA sequence coding a mouse sperm-specific membrane protein Izumo. The DNA sequence coding the mouse sperm-specific membrane protein Izumo is inserted into an eukaryotic expression vector which may be pCAGGS. The invention further provides a preparation method for the immunological contraception vaccine. After the nucleic acid vaccine is used to immunize a mouse, generation of a high-titer specific antibody against mouse Izumo in the mouse is induced, and reproductive capacity of the mouse is reduced. Moreover, the specific antibody generated by the mouse immunized by the nucleic acid vaccine can undergo a specific reaction with mouse sperm proteins and effectively inhibit egg-sperm binding of the mouse.

Description

technical field [0001] The invention relates to an immunocontraceptive nucleic acid vaccine and a preparation method thereof, wherein the immunocontraceptive nucleic acid vaccine comprises the nucleic acid of Izumo, a key membrane protein of sperm-egg fusion specifically expressed by mouse sperm. The immunocontraceptive nucleic acid vaccine provided by the invention can be used for birth control of mice. Background technique [0002] Rats and other harmful mammals cause serious losses to global agriculture, forestry and animal husbandry every year. At present, according to the statistics of the World Health Organization (WHO), there are more than 17 billion rats in the world, and more than 4 billion rats in my country. The world's food loss due to rodent damage can reach up to 15%-20% of the harvest, which is equivalent to The gross national income of 25 poor countries is enough to feed 150 million people for a whole year. In my country, nearly ten million mu of forest and ...

Claims

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Application Information

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IPC IPC(8): A61K48/00A61K39/00A61P15/18C12N15/85C12N15/66
Inventor 汪莉王玉民侯小强田利源郭振东张斌胡亚欧
Owner INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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