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Transformed and attenuated Listeria monocytogenes introducing human CD24 (cluster of differentiation 24) nucleotide sequence, and vaccine thereof

A nucleotide sequence, Listeria technology, applied in the field of attenuated Listeria, to achieve high safety and reliability, prevent drug resistance, and avoid the effect of chloramphenicol resistance

Inactive Publication Date: 2014-05-28
NANJING XINSAIERSI BIOLOGICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, using Listeria as a vaccine carrier, through molecular cloning technology, it can overexpress and secrete human-derived CD24 protein, activate the body's immune response, and produce corresponding antibodies, which can be used to treat liver cancer patients resistant to chemotherapy and radiotherapy and prevent postoperative liver cancer patients. Metastasis and recurrence have not been reported

Method used

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  • Transformed and attenuated Listeria monocytogenes introducing human CD24 (cluster of differentiation 24) nucleotide sequence, and vaccine thereof
  • Transformed and attenuated Listeria monocytogenes introducing human CD24 (cluster of differentiation 24) nucleotide sequence, and vaccine thereof
  • Transformed and attenuated Listeria monocytogenes introducing human CD24 (cluster of differentiation 24) nucleotide sequence, and vaccine thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1 Preparation method of attenuated Listeria vaccine

[0021] The strains in this example were provided by Professor Franckle of the University of Pennsylvania.

[0022] The medium / reagent that embodiment relates to:

[0023] Improved BHI (heart-brain perfusion fluid) medium: 100ml deionized water + 3.7g BHI dry powder + 20mg D-alanine;

[0024] BHIS (sucrose heart and brain perfusion solution): 100ml deionized water + 3.7g BHI dry powder + 17.115g sucrose + 20mg D-alanine;

[0025] SGWB (glycerol-sucrose buffer): 1000ml deionized water + 171.15g sucrose + 100ml glycerol + 0.2383g 4-hydroxyethylpiperazineethanesulfonic acid;

[0026] BHI medium: 100ml deionized water + 3.7g BHI dry powder;

[0027] BHI plate: 100ml+deionized water+3.7gBHI dry powder+100g agarose;

[0028] BHI chloramphenicol resistance medium: 100ml deionized water + 3.7g BHI dry powder + 1mg chloramphenicol;

[0029] BHI chloramphenicol resistance plate: 100ml deionized water + 3.7g BHI dr...

Embodiment 2

[0048] Example 2 The Second Homologous Recombination Experiment of Attenuated Listeria Secreting Human CD24 Protein

[0049] After the attenuated Listeria is transferred into the PKSV-CD24 shuttle plasmid by electroporation, it must be able to continuously and stably secrete CD24 protein to effectively activate the body's immune response in order to become a qualified vaccine.

[0050] In the present invention, the second homologous recombination colony obtained in Example 1 is further cultured in BHI medium for 40 generations, and the 5th, 10th, 15th, 20th, 30th and 40th generation attenuated Listeria progenies are extracted respectively Bacteria culture medium, use TCA / acetone precipitation method to extract secreted protein in BHI medium, use western blot method to identify the protein in the expression of the protein for identification, the experimental results show that with the increase of the number of passages, the attenuated Listeria monocytogenes Can continuously and...

Embodiment 3

[0051] Example 3 Experiment of Infection Ability of Recombined Attenuated Listeria Live Vaccine to Cells and Tissues

[0052] Attenuated Listeria is a monocytogenes Gram-positive bacterium whose ability to infect host cells plays a key role in activating cellular and humoral immune responses.

[0053] The infectivity of the recombined attenuated Listeria vaccine in Example 1 was tested in vivo to observe whether exogenous gene recombination would affect the survival and reproduction of the vaccine strain. the way is:

[0054] Experimental group: the recombinant attenuated Listeria vaccine provided in Example 1;

[0055] Control group: attenuated Listeria vaccine provided by Professor Franckle of the University of Pennsylvania;

[0056] a) Select HepG2, Snu423, SMMC-7721, Huh-7 liver cancer cell lines and mouse macrophage cell line Raw264.7, and infect them in vitro with the vaccines of the experimental group and the control group. There was no significant difference in the ...

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Abstract

The invention relates to a transformed and attenuated Listeria monocytogenes introducing a human CD24 (cluster of differentiation 24) nucleotide sequence, and a vaccine thereof. The transformed and attenuated Listeria monocytogenes is characterized in that human CD24 nucleotide is located on a shuttle plasmid PKSV7, and is duplicated and expressed in the attenuated Listeria monocytogenes or a progeny of the attenuated Listeria monocytogenes. The vaccine provided by the invention overcomes various defects in liver cancer treatment in the prior art, is used for clinic treatment and prevention of drug resistance of liver cancer to chemo-radiotherapeutic drugs and postoperative metastasis and relapse, and is relatively high in safety and reliability.

Description

technical field [0001] The invention relates to the field of microorganisms, in particular to a transformed attenuated listeria introduced with a human source CD24 nucleotide sequence. Background technique [0002] Tumors and their related diseases have become more and more threats to people's health. At present, worldwide, hepatocellular carcinoma accounts for 80-90%, ranking fifth among the most common tumors and third among tumor-related deaths. Worldwide, 50% of HCC cases are caused by HBV infection. In China, due to the high incidence of HBV infection, the incidence of HCC is as high as 1 / 10000. Due to the high degree of malignancy and poor prognosis of liver cancer, the current treatment mainly focuses on antiviral therapy, radiotherapy, drug chemotherapy and surgical treatment. However, liver cancer often produces chemoradiotherapy resistance and postoperative recurrence and metastasis. Liver cancer stem cells have been proposed as a new concept to explain the...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/74A61K39/00A61P31/04C12R1/01
Inventor 侯嘉杰杨雨孙倍成陈云
Owner NANJING XINSAIERSI BIOLOGICAL TECH
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