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Fusion protein for preparing hepatitis B vaccine and its carrier

A fusion protein and hepatitis B vaccine technology, applied in the field of bioengineering, can solve the problem of inability to clear viruses or bacteria

Inactive Publication Date: 2007-09-19
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because for viruses and intracellular bacteria, neutralizing antibodies can block their entry into the cell, but cannot clear the virus or bacteria that have entered the cell

Method used

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  • Fusion protein for preparing hepatitis B vaccine and its carrier
  • Fusion protein for preparing hepatitis B vaccine and its carrier
  • Fusion protein for preparing hepatitis B vaccine and its carrier

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1: Design, Construction and Identification of Antibody Hepatitis B Vaccine

[0062] 1. Amplification of PreS2-S and murine IgG1 Fc-encoding genes

[0063] The antibodyized hepatitis B vaccine designed in the present invention is composed of hepatitis B surface antigen and mouse IgG1 Fc segment, and no linker is added in the middle. The N-terminus of the fusion molecule and the Fc segment are at the C-terminus of the fusion molecule, similar to a complete antibody molecule.

[0064] Hepatitis B surface antigen PreS2-S encoding gene was amplified by PCR with plasmid PEHH as template, and the primers adopted were shown in Table 1 (SEQ ID NO: 5 and SEQ ID NO: 6), and the enzyme cleavage site Hind was introduced while designing primers III and EcoR V.

[0065] The gene encoding the Fc segment of mouse IgG1 was amplified from mouse-derived hybridoma cells 520C9, and the cells were cultured in RPMI1640 (containing 10% calf serum, 1000u / ml penicillin, 100mg / ml strepto...

Embodiment 2

[0074] Example 2: In vitro transfection of PCMFS

[0075] According to Lipofectamine 2000 Instructions for eukaryotic transfection reagents: C2C12 cells were cultured in complete 1640 medium, and the cells were cultured at 5 × 10 a day before transfection. 5 C2C12 cells were added to a 35mm six-well plate and transfected when the cell density was 80%. Take 10 μl Lipofectamine 2000 Add to 90 μl of incomplete 1640 medium, which is solution A; take 2 μl (about 3-5 μg) of the plasmid purified by QIAGEN kit, which is solution B; add solution A to solution B, mix gently, and leave at room temperature. Act for 20-25 minutes to promote complex formation. During incubation at room temperature, the culture supernatant was aspirated, and the cells were washed once with PBS. The A and B complexes were added dropwise to the six-well plate, and then 800 μl of incomplete 1640 medium was added and mixed gently. 37℃ 5%CO 2 After 5 hours of culture, complete 1640 medium containing 20% ​​N...

Embodiment 3

[0079] Example 3: Antibody Hepatitis B Vaccine Injected Through Muscle Gene

[0080] In this example, the plasmid PCMFS was injected.

[0081] The mice were intraperitoneally anesthetized with 0.75% pentobarbital sodium (about 120-150ul / mouse), took the abdominal position, and adjusted the posture of the legs to expose the tibialis anterior muscle. After disinfection, use a 1ml syringe with a plastic cannula to insert the needle vertically in the middle of the muscle. The insertion depth of the needle tip is determined by the cannula, which is about 2-3mm. Slowly advance, and the injection volume is 100ul (containing 100ug PCMFS plasmid). After the injection muscle expands, turn the needle 90°, stay for 5 seconds, and pull it out slowly.

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Abstract

The invention discloses a fusion protein of antibody Hepatitis B vaccine belonging to the bio-engineering field. It has two functional domains which composed of preS2 of Hepatitis B surface antigen in N-terminal and S protein, and IgG1 Fc in rats. The invention also discloses the vector containing the gene coding the said fusion protein. The invention also discloses a antibody Hepatitis B vaccine. The constructed antibody Hepatitis B vaccine may induce a strong humoral-mediated immune response and a cell-mediated immune response which has a better effect than the traditional vaccine.

Description

technical field [0001] The invention belongs to the field of bioengineering, and relates to a structural fusion molecule, in particular to a fusion protein for preparing an antibodyized hepatitis B vaccine and a carrier containing the gene encoding the fusion protein. Background technique [0002] Hepatitis B is a major disease that endangers human health, especially in my country. Due to the poor safety and low yield of traditional blood-derived vaccines, they have been replaced by genetically engineered vaccines. Since most hepatitis B patients were infected with the virus in childhood and are in a state of immune tolerance to HBSAg, although the existing genetic engineering vaccines can induce antibodies in most people, they have little effect on patients who have been infected with hepatitis B virus. Because for viruses and intracellular bacteria, neutralizing antibodies can block their entry into the cell, but cannot clear the virus or bacteria that have entered the ce...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/09C12N15/63A61K38/16C12P21/02A61K39/29
Inventor 熊思东王缨蒋正刚储以微
Owner FUDAN UNIV
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