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Application of HBcAg (hepatitis B core antigen) virus-like particle serving as cancer therapeutic vaccine carrier

A technology of hepatitis B core antigen and therapeutic vaccine, applied in the fields of molecular biology and immunology, which can solve problems such as undetermined

Inactive Publication Date: 2016-04-20
INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, whether HBcAg virus-like particles have the potential to serve as therapeutic vaccine vectors and promote antigen-specific cellular immune responses remains undetermined

Method used

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  • Application of HBcAg (hepatitis B core antigen) virus-like particle serving as cancer therapeutic vaccine carrier
  • Application of HBcAg (hepatitis B core antigen) virus-like particle serving as cancer therapeutic vaccine carrier
  • Application of HBcAg (hepatitis B core antigen) virus-like particle serving as cancer therapeutic vaccine carrier

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Embodiment 1: recombinant expression plasmid p HBcAg-E7 49-57 build

[0026] Code E7 49-57 The oligonucleotide sequence (positive strand: 5'-gatctCGTGCTCACTACAACATCGTTACCTTCggatccggtg-3'; negative strand: 5'-aattcaccggatccGAAGGTAACGATGTTGTAGTGAGCACGa-3') was synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. In a 10 μl system, the positive and negative oligonucleotide fragments were annealed and annealed at a concentration of 0.1 mM 1:1. The specific renaturation system was: 2 μl of positive and negative oligonucleotides, 1 μl of 10×PCRBuffer, and 5 μl of water. Denature at 95°C for 30 seconds, slowly cool down to room temperature and anneal and renature to form double-stranded DNA fragments encoding antigenic peptides. The formed double-stranded DNA fragment was constructed between the restriction sites of BamHI and EcoRI of the pThioHisA-HBcAg vector. The vector pThioHisA-HBcAg, constructed by our laboratory, is to clone the modified HBcAg (1-149 amino a...

Embodiment 2

[0027] Embodiment 2: recombinant expression plasmid p HBcAg-E7 49-57 induced expression of

[0028] The recombinant plasmid was transformed into Escherichia coli DH5α competent cells, and a single clone was picked for induction and expression. The specific method was to inoculate the positive clone into LB medium that had been added with ampicillin (100 mg / mL), culture it for 16 hours and then inoculate it with 1 :5 ratio transfer bacteria solution into the new LB medium with ampicillin, when the strain grows to the logarithmic phase, add IPTG (1mmol / L) to induce expression, control the induction expression temperature at 37°C, induce expression 4 hours ( figure 2 ).

Embodiment 3

[0029] Embodiment 3: recombinant protein p HBcAg-E7 49-57 preparation and purification of

[0030]Centrifuge the sample after overnight induction at 12,000g at room temperature for 10 minutes to collect the bacteria, resuspend the bacteria in PBS, and then ultrasonically disrupt the cells. The crushing condition is 15% maximum power, work for 5 seconds, stop for 5 seconds, and last for 10 minutes. The crushed sample was centrifuged at 12000g for 10 minutes, and the supernatant was collected and subjected to 40% ammonium sulfate precipitation at room temperature for 30 minutes. Centrifuge at 12,000rpm (~13000g) and 20°C for 10min. Discard the supernatant, wash the pellet with 20% ammonium sulfate solution, centrifuge at 12,000rpm (~13000g) at 20°C for 10min, repeat the washing three times, resuspend the pellet with 500μl PB, centrifuge at 12,000rpm (~13000g) at 20°C for 15min, and collect the supernatant. clear.

[0031] After SDS-PAGE analysis, the recombinant protein w...

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Abstract

The invention relates to the field of molecular biology and immunology, in particular to an application of an HBcAg (hepatitis B core antigen) virus-like particle serving as a cervical cancer therapeutic vaccine carrier. A preparation method comprises steps as follows: an HPV16 E749-57CTLs epitope peptide fragment is selected, a DNA (deoxyribose nucleic acid ) fragment of the HPV16 E749-57CTLs epitope peptide fragment is inserted between 78 and 79 amino acids of the HBcAg through genetic recombination, an obtained recombinant plasmid pHBcAg-E749-57 is converted into Escherichia coli DH5alpha, and an HBcAg virus-like particle vaccine presenting E749-57 is obtained after induction expression and purification. After a tumor-bearing mouse is immunized with the virus-like particle vaccine, the body of the mouse can be induced to generate a higher HPV16E7 specific cellular immunologic response, and growth of tumors is remarkably inhibited.

Description

technical field [0001] The invention relates to the fields of molecular biology and immunology, in particular to a construction method and application of a hepatitis B core antigen virus-like particle (HBcAg) as a tumor therapeutic vaccine carrier. Background technique [0002] Cervical cancer is one of the most serious diseases that threaten women's health, and it ranks second in the incidence of female tumors. Worldwide, there are approximately 493,000 new cases and 274,000 deaths each year, and HPV can be detected in almost all cervical cancer cases. HPV infection also causes anal cancer, vulvar cancer, vaginal cancer and penile cancer. In addition, HPV infection is also an important cause of head and neck cancer and other non-genital tract cancers. It is now clear that the persistent infection of high-risk HPV is a necessary factor for the occurrence and existence of cervical cancer. Among high-risk HPV types, types 16 and 18 are associated with 70% of cervical can...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/12A61P35/00C12N15/66
CPCA61K39/12A61K2039/5258A61K2039/6075C12N15/66C12N2710/20034
Inventor 马雁冰褚晓杰李杨龙琼夏烨黄惟巍
Owner INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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