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A fusion protein contraception vaccine with molecular adjuvant and its preparation

A fusion protein and adjuvant technology, which is applied in the direction of medical preparations containing active ingredients, drug combinations, and pharmaceutical formulas, can solve the problems of activating proto-oncogenes, destroying tumor suppressor genes, and causing cancer

Inactive Publication Date: 2005-01-26
THE OBSTETRICS & GYNECOLOGY HOSPITAL OF FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the restriction of the expression of gene vaccines in host cells and the integration of gene vaccines into the host cell genome, it is possible to activate proto-oncogenes or destroy tumor suppressor genes, theoretically there is a risk of carcinogenesis

Method used

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  • A fusion protein contraception vaccine with molecular adjuvant and its preparation
  • A fusion protein contraception vaccine with molecular adjuvant and its preparation
  • A fusion protein contraception vaccine with molecular adjuvant and its preparation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1. Construction of secretory eukaryotic expression plasmids phCMV1-6His-hCGβ-C3d3 and phCMV1-6His-hCGβ.

[0056] phCMV1-signal-6His-hCGβ construction strategy ( figure 2)

[0057] Using hCGβ with signal peptide as template, design the following 2 pairs of primers: 5'-CCAAGCTT ATG GAGATG TTC CAG GGG-3', 5'-CGGAATTC CCA ATG ATG ATG ATG ATG ATG TGC CCATGT CCC GCC CAT-3' and 5 '-CGGAATTC TCC AAG GAG CCGCTT CGG-3', 5'-CGGGATCC TTG TGG GAG GAT CGG-3'. Using pBS-hCGβ as a template, the Hind III-Signal-6His-EcoR I fragment was amplified with the first pair of primers. The EcoR I-hCGβ (no signal peptide and stop codon)-BamH I fragment was amplified with a second pair of primers. The Hind III-Signal-6His-EcoR I fragment was double-digested with Hind III and EcoR I, and the EcoR I-hCGβ (no signal peptide and stop codon)-BamH I fragment was double-digested with EcoR I and BamH I. Ligation obtained Hind III-Signal-6His-EcoR I-hCGβ (no signal peptide and stop codon)-BamH...

Embodiment 2

[0061] Example 2 Expression, identification and purification of hCGβ-C3d3 fusion protein and hCGβ protein.

[0062] Transfection of CHO cells with recombinant plasmids, selection of resistant clones and serum-free culture

[0063] When the CHO cells in the six-well plate were 95% confluent, use Lipofectamine2000 to mediate the transfection of CHO cells with phCMV1-6His-hCGβ-C3d3, phCMV1-6His-hCGβ, phCMV1, pcDNA3-hCGβ-C3d3 and pcDNA3-hCGβ respectively. And liposome dosage is 1μg:7μg. Positive clones were screened 24 hours after transfection with complete medium containing G418 (800ug / ml). The medium was changed every 3 days, and the selection was maintained for 8 days until the formation of drug-resistant cell clones. Select 10 positive clones of phCMV1-6His-hCGβ-C3d3 and phCMV1-6His-hCGβ respectively, maintain culture and amplification with culture medium containing G418 (300 μg / ml), and replace with CHO when 95% of the positive clones confluence into sheets cultured in ser...

Embodiment 3

[0072] Example 3. Animal Immunity and Immune Effects

[0073] Immunization method

[0074] 6-8 weeks old BALB / c mice (19-24g) and C57BL / 6 mice (20-25g) were divided into 3 groups, 5 mice in each group. They were immunized with hCGβ-C3d3 fusion protein, hCGβ alone and hCGβ plus Freund's adjuvant. The immunization was subcutaneously injected into the right back, and the immunization was done twice with an interval of 4 weeks. The immunization doses were: 50 pmol hCGβ-C3d3 fusion protein / 100 μl PBS, 50 pmol hCGβ / 100 μl PBS and 50 pmol hCGβ / 50 μl PBS+50 μl CFA formed complete emulsion. The hCGβ+CFA immunization group was immunized with hCGβ+IFA again.

[0075] Blood collection began 7 days after the first injection, once a week, a total of 4 times. Blood collection began 7 days after the second injection, once a week, a total of 3 times. Blood was collected from the retro-orbital venous plexus of mice, 100-150 μl each time, and 20-25 μl of serum was separated and stored at -2...

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Abstract

The invention belongs to biological pharmaceutical field. It relates to a contraception vaccine cross linked with the molecule adjuvant, in particular to a hCGª‰-C3d3 confluence protein contraception vaccine cross linked with the molecule adjuvant C3d3. The invention selects eucaryon to express carrier phCMV1, constructs separately of the hCGª‰-C3d3 confluence protein with six histidine purification signals and eucaryon expressed plasmid phCMV1-6His-hCGª‰-C3d3 and phCMV1-6His-hCGª‰ of the hCGª‰protein, in vitro expresses, identifies and purifies hCGª‰-C3d3 confluence protein and hCGª‰protein, applies freunds adjuvant to immune mouse in different species. It is proved that hCGª‰-C3d3 confluence protein vaccine has comparative strong anti generating potent function by detection of the antibody level, biological activity of antiserum and neutralization of the hCG and excretion level of the cell factor by the spleen cells.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals and relates to a contraceptive vaccine cross-linked with a molecular adjuvant, in particular to a hCGβ-C3d3 fusion protein contraceptive vaccine cross-linked with a molecular adjuvant C3d3. Background technique: [0002] Human chorionic gonadotropin (hCG) is a glycoprotein hormone secreted by placental syncytiotrophoblasts. Its main physiological function is to prolong the life of the corpus luteum, make it enlarge to become the corpus luteum of pregnancy, and increase the secretion of steroid hormones to maintain pregnancy. . hCG is a specific hormone that appears temporarily during pregnancy. Studies have confirmed that neutralizing the biological activity of hCG can terminate pregnancy without interfering with other reproductive biological functions. Therefore, hCG has become an ideal target antigen in the design of contraceptive vaccines . Based on the specificity of the β subunit in the hC...

Claims

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Application Information

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IPC IPC(8): A61K39/00A61K39/39A61P15/18C12N15/62
Inventor 李大金王秀丽
Owner THE OBSTETRICS & GYNECOLOGY HOSPITAL OF FUDAN UNIV
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