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Placenta-like chondroitin sulfate A immunization composition and application

An immune composition and placenta technology, applied in the directions of drug combinations, vaccines, contraceptive vaccine components, etc., can solve the problems of inability to produce antibody titers, affect the normal function of the body, affect the contraceptive effect, etc., and achieve increased contraceptive effect, rich content, The effect of structural stabilization

Active Publication Date: 2018-09-25
SHENZHEN INST OF ADVANCED TECH CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The problems of the above-mentioned very promising target antigen vaccines include: first, targeting hormones and their receptors may cause endocrine disorders in the body and affect the normal function of the body; second, targeting germ cells may cause tissue damage and cause irreversible infertility Infertility; the third target vaccine molecule cannot produce sufficient antibody titers, which affects the contraceptive effect and causes contraceptive failure

Method used

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  • Placenta-like chondroitin sulfate A immunization composition and application
  • Placenta-like chondroitin sulfate A immunization composition and application
  • Placenta-like chondroitin sulfate A immunization composition and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Expression and localization of embodiment 1pl-CSA and its core protein CSPG4

[0068] This part was performed by immunofluorescence. First, collect human delivery placenta, mouse placenta of 15 days of pregnancy and pigs' placenta of 70 days of pregnancy, fully fixed with 4% paraformaldehyde; 2 o 2 (10min) to inactivate endogenous peroxidase; pH6.0 citrate buffer in 95°C water bath for 10min for antigen retrieval; rabbit anti-CSPG4 antibody (1:200) and self-made rVAR2-His-tag (1 :100) reagent and incubated overnight at 4°C; PBST (containing 0.1% Triton X-100) was soaked 3 times, 5min each time; FITC-labeled goat anti-rabbit secondary antibody (1:1000) and Dylight650-labeled anti-6×His monoclonal antibody Cloned antibody (1:1000), incubate at room temperature for 40min; soak in PBST 3 times, 5min each; The slides were sealed with mounting medium, observed and photographed under a fluorescent confocal microscope. For experimental results, see image 3 . From image...

Embodiment 2

[0069] The preparation of embodiment 2 immune agent

[0070] Preparation and purification of placenta-like chondroitin sulfate A:

[0071] (1) Broken pig placenta or semi-mechanical separation of continuously cultured trophoblast cell line to obtain tissue cell slurry;

[0072] (2) The tissue cell slurry obtained in step (1) is mixed with the lysate (V cell slurry: V lysate=1:10), the lysate contains RNase, DNase I, trypsin and protease K, and the lysed tissue is obtained cell plasma;

[0073] (3) inactivating the enzyme in the lysate of the lysed tissue cell serum obtained in step (2); adding 1 / 4 volume of Sevag solution to remove protein to obtain crude pl-CSA;

[0074] (4) Add a BamHI restriction site to the 5' end of the erythrocyte surface antigen VAR2CSA gene sequence SEQ ID No.1 of Plasmodium infection, and add a Sal I restriction site to the 3' end; combine the gene fragment SEQ ID No.1 with pET28a (+) The prokaryotic expression vector was connected, transformed int...

Embodiment 3

[0082] Immunogenicity and contraceptive effect evaluation of embodiment 3pl-CSA

[0083] Table 2. pl-CSA vaccine immunization groups

[0084]

[0085]

[0086] ① Immune grouping: 6-week-old female BALB / c mice were randomly divided into 4 groups, with PBS as negative control, 20 mice in each group (Table 2), and 40 10-week-old BALB / c male mice were fed.

[0087] ②Immunization procedure: Female mice in each group had free access to food and water, 2 mice per cage, and the time for inoculation of pl-CSA and pl-CSA+CBZ-1 vaccines was 0, 1, and 2 weeks, respectively (set to 0 at the time of purchase). Weeks), a total of 3 times of immunization, the first time using Freund's complete adjuvant to mix with the immunogen, the second and third times using Freund's incomplete adjuvant to mix with the immunogen. From the 4th week, the female mice of each group were caged with the male mice, and B-ultrasound was used to monitor whether they were pregnant on the 9th day after the vag...

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Abstract

The invention relates to a placenta-like chondroitin sulfate A immunization composition and application, and particularly discloses a contraceptive immunization composition. The placenta-like chondroitin sulfate A immunization composition comprises at least one type of placenta trophoblast cell specific expression polysaccharides and / or protein antigens and B cell dominant antigen epitopes of placenta-like chondroitin sulfate A core proteins CSPG4. The embryo or placenta trophoblast cell specific expression polysaccharides are selected from placenta-like chondroitin sulfate A. The placenta-like chondroitin sulfate A immunization composition and the application have the advantages that the opinion that implantation execution critical cytotrophoblast cells are targeted by contraceptive vaccine for early implantation which is a placentation and embryogenesis critical and sensitive period is proposed for the first time, and accordingly the contraception sensitivity and effectiveness can beimproved.

Description

technical field [0001] The invention relates to the field of biopharmaceuticals, in particular to a placenta-like chondroitin sulfate A immune composition and its application. Background technique [0002] At present, there is no contraceptive vaccine used in clinical practice. The targets of contraceptive vaccines are mainly targeting hormones or certain components of germ cells, which usually play a key role in the reproductive process, such as by inhibiting the production of germ cells (sperm or eggs) or inhibiting the function of germ cells , and hinder normal pregnancy progression and maintenance [1] . Among these components, sperm became the first contraceptive vaccine target. In 1899, Nobel Prize winners Landsteiner and Metnikoff respectively carried out research on anti-sperm antibodies, and injection of anti-sperm antibodies can achieve contraceptive effects; The first U.S. patent for an anti-sperm contraceptive vaccine was filed in 2000. Although this vaccine ca...

Claims

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Application Information

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IPC IPC(8): A61K39/00A61K45/06A61P15/18
CPCA61K39/0006A61K45/06A61K2039/53A61K2039/575A61K2300/00
Inventor 张居作范秀军张键陈指龙汪宝蓓黄晨陈杰李梦霞
Owner SHENZHEN INST OF ADVANCED TECH CHINESE ACAD OF SCI
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