Converted attenuated listeria introducing EB virus LMP2A nucleotide sequence and vaccine of converted attenuated listeria

A technology of nucleotide sequence and Listeria, which is applied in the field of transforming attenuated Listeria and its vaccine, can solve the problems that are not suitable for treatment and prevention of nasopharyngeal carcinoma, and achieve good prevention and treatment of tumors, high safety and reliability Effect

Inactive Publication Date: 2014-10-01
NANJING MEDICAL UNIV
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  • Description
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Problems solved by technology

[0005] The Chinese patent with the application number "201310598574.9" discloses a transformed attenuated Listeria vaccine using the PKSV7 shuttle plasmid to introduce the human CD24 nucleotide sequence to express the human CD24 protein, but the Listeria Express and secrete Epstein-Barr v

Method used

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  • Converted attenuated listeria introducing EB virus LMP2A nucleotide sequence and vaccine of converted attenuated listeria
  • Converted attenuated listeria introducing EB virus LMP2A nucleotide sequence and vaccine of converted attenuated listeria
  • Converted attenuated listeria introducing EB virus LMP2A nucleotide sequence and vaccine of converted attenuated listeria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1 Preparation method of attenuated Listeria vaccine

[0023] The strain in this example was provided by Professor Franckle of the University of Pennsylvania. This Listeria species is a defective strain, which lacks the D-alanine synthetase that synthesizes the bacterial cell wall and cannot survive independently. The P1565 vector we use contains subtilis D-alanine synthase gene of Bacillus. When the P1565 plasmid vector is introduced, the bacteria can synthesize the D-alanine of Bacillus subtilis, so that the bacteria can survive independently to a certain extent and self-replicate to a limited extent without causing obvious damage to tissues and organs.

[0024] The medium / reagent that embodiment relates to:

[0025] Improved BHI (heart-brain perfusion fluid) medium: 100ml deionized water + 3.7g BHI dry powder + 20mg D-alanine;

[0026] BHIS (sucrose heart and brain perfusion solution): 100ml deionized water + 3.7g BHI dry powder + 17.115g sucrose + 20mg D-...

Embodiment 2

[0049] Example 2 The Second Homologous Recombination Experiment of Attenuated Listeria Secreting Epstein-Barr Virus LMP2A Protein

[0050] After the attenuated Listeria is transferred into the P1565-LMP2A shuttle plasmid by electroporation, if it is to become a qualified vaccine, it must be able to continuously and stably secrete LMP2A protein to effectively activate the body's immune response.

[0051] The present invention continues to culture the second homologous recombination colony obtained in Example 1 in the BHI medium, utilizes the TCA / acetone precipitation method to extract the secreted protein in the BHI medium, and uses the western blot method to identify the protein in the protein expression. Identification, experimental results show that the attenuated Listeria vaccine can stably secrete LMP2A protein, figure 1 Immunoblot of Epstein-Barr virus LMP2A protein secreted for the transformed attenuated Listeria vaccine, wherein A is the protein extracted from CNE-1...

Embodiment 3

[0052] Example 3 To detect the distribution in vivo of the recombined attenuated Listeria vaccine of Example 1, the method is:

[0053] Experimental group: the recombinant attenuated Listeria vaccine provided in Example 1;

[0054] Select 9 C57BL / 6 mice with a body weight of 10 g, and use the vaccine of the experimental group to immunize the mice three times through tail vein injection, 10 5 CFU / mouse, immunized once a week, for three consecutive weeks; the liver, spleen, lung, and brain of the mice were extracted from the liver, spleen, lung, and brain of the mice on the 1st and 3rd days after the third immunization, and the results showed that the recombinant Listeria Bacteria were mainly distributed in the liver and spleen tissue, a small amount of Listeria was found in the kidney, almost no Listeria was distributed in the lung and brain tissue, and there was no significant statistical difference in the distribution of liver and spleen. content in figure 2 shown. Simult...

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Abstract

The invention discloses a converted attenuated listeria introducing the sequence of EB virus LMP2A nucleotide and a vaccine of the converted attenuated listeria, wherein the EB virus LMP2A nucleotide is positioned on a p1565 shuttle plasmid, and replicated and expressed on the attenuated listeria or the offspring of the attenuated listeria; the vaccine provided by the invention is of relatively high security and reliability, so that the vaccine has no obvious damage effects on various tissues and organs; meanwhile, the attenuated listeria without chloramphenicol resistance can avoid that chloramphenicol drug resistance occurs during a curing process of a nasopharynx cancer patient.

Description

technical field [0001] The invention relates to the field of microbes, in particular to a transformed attenuated listeria introduced with the nucleotide sequence of Epstein-Barr virus LMP2A and a vaccine thereof. Background technique [0002] Tumors and their related diseases have become more and more threats to people's health. At present, according to statistics, 80% of the world's nasopharyngeal carcinoma occurs in my country. It is one of the most frequent malignant tumors in southern my country, and its mortality rate ranks fifth among malignant tumors. Nasopharyngeal carcinoma tends to occur in the pharyngeal recesses with complex anatomical structure. The tumor grows insidiously and is characterized by early lymph node metastasis. Its strong invasiveness and high recurrence rate bring great difficulties to clinical treatment. [0003] Studies have shown that Epstein-barr virus (EBV) infection is closely related to the occurrence of nasopharyngeal carcinoma, and EB...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/74A61K39/02A61P35/00C12R1/01
Inventor 陈云杨雨林喆孙倍成姜润秋万昕姚堃赵玮唐俊伟卓晗
Owner NANJING MEDICAL UNIV
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