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Contraception birth control DNA vaccines and preparation method thereof

A DNA vaccine and plasmid technology, applied in the biological field, can solve problems such as failure, irreversibility, and physical harm.

Inactive Publication Date: 2016-05-11
PEKING UNIV SHENZHEN HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are few contraceptive measures available to men, and these measures have serious disadvantages such as harmful to the body, possible failure and irreversibility

Method used

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  • Contraception birth control DNA vaccines and preparation method thereof
  • Contraception birth control DNA vaccines and preparation method thereof
  • Contraception birth control DNA vaccines and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1: Preparation of the DNA vaccine of the present invention

[0051] Use pYA3149-Bin1b as a template to carry out PCR amplification reaction to obtain a target fragment with a size of 337bp, which is consistent with the expected size by agarose gel electrophoresis ( Image 6 ). Among them, the PCR amplification program is:

[0052] Pre-denaturation at 94°C for 5 minutes;

[0053] Denaturation at 94°C for 30s, renaturation at 55°C for 30s, extension at 72°C for 60s, 30 cycles;

[0054] Extend at 72°C for 10 minutes and store at 4°C.

[0055] The PCR amplification system is:

[0056]

[0057] Add ddH 2 O to 50ul, mix well.

[0058] The PCR product and pSG.SS.C3d3.YL and pSG.SS.YL were digested with BglⅡ, and recovered with PCR product purification kit. The recovered products were digested with BamHI respectively, and digested with 0.8% agarose gel electrophoresis. After the product is produced, the gel containing the target fragment and the carrier is cut separately under ul...

Embodiment 2

[0060] Example 2: Verification of recombinant plasmid

[0061] The extracted plasmids pSG.SS.C3d3.Bin1b and pSG.SS.YL.Bin1b were identified by double enzyme digestion with BglII and BamHI, and the PCR results were used as controls. The digestion result of 0.8% agarose gel electrophoresis showed that the band near 337bp was the target fragment after digestion, and the sequencing result also showed that the fragment was the target gene, indicating pSG.SS.C3d3.YL.Bin1b and pSG. SS.YL.Bin1b was successfully constructed.

Embodiment 3

[0062] Example 3: Indirect immunofluorescence detection of recombinant plasmid Bin1b expression

[0063] pSG.SS.C3d3.YL.Bin1b and pSG.SS.YL.Bin1b were transfected into human embryonic kidney HEK293 cells and transfected with Lipofectamine transfection kit.

[0064] After 48 hours of transfection, remove the cover glass, fix it with ice acetone for 5 minutes, perform enzyme repair and antigen blocking according to the SP9000 kit, add Bin1b antibody (1:400), incubate for 40 minutes, wash off the antibody with PBS, and add FITC-labeled antibody Goat IgG (diluted 1:500) was incubated at room temperature for 30 minutes, and the results were observed under a fluorescence microscope.

[0065] Compared with the negative immunofluorescence result of pSG.SS.C3d3.YL empty vector, the expression results of pSG.SS.YL.Bin1b and pSG.SS.C3d3.YL.Bin1b showed that a large number of target proteins were expressed in the cytoplasm and nucleus , It shows that the DNA vaccine of the present invention suc...

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Abstract

The invention relates to the field of biotechnology and discloses contraception birth control DNA vaccines. The contraception birth control DNA vaccines comprise plasmids for coding and expressing a nucleotide sequence shown in the formula of SEQ ID NO: 1. A nucleic acid sequence comprising 35th-371th bases of a Bin1b cDNA is used as a target antigen gene and pSG. SS. YL and pSG. SS. C3d. YL are used as appropriate plasmid carriers. Through the target antigen gene and the appropriate plasmid carriers, pSG. SS. YL. Bin1b and pSG. SS. C3d. YL. Bin1b recombinant eukaryotic expression plasmids are constructed and are used as DNA vaccines. The DNA vaccines can realize effective contraception birth control.

Description

Technical field [0001] The invention relates to the field of biotechnology, in particular to a contraceptive and birth control DNA vaccine and a preparation method thereof. Background technique [0002] Family planning is a basic national policy of our country. Effectively controlling the population and significantly improving the quality of the population are related to the prosperity of the country and the prosperity of the country and nation. The study of safe, effective, controllable, reversible, and simple contraceptive measures not only has important scientific significance, but also has important practical and long-term historical significance for the country and the nation. [0003] With the development of genetic recombination technology and people's deepening understanding of the molecular basis and detailed mechanism of the fertilization process, the development of effective immune contraception and birth control vaccines may become an ideal measure to control the excess...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00A61K39/00A61P15/16
Inventor 孙中义蒋滢周波曾凡春周青松
Owner PEKING UNIV SHENZHEN HOSPITAL
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