Yersinia pestis ghost, preparation method thereof, and application of Yersinia pestis ghost serving as vaccine

A technology of Yersinia and plague, applied in the fields of molecular biology and immunology, can solve the problems of high side reaction rate, poor production safety, and single ingredient, and achieve simple and easy preparation process, reduced possibility, strong The effect of immune memory

Inactive Publication Date: 2011-02-23
MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Its disadvantages include: poor production safety; low protection efficiency, short validity period, repeated immunization after 6 months; high side effect rate, serious side effects; high price, and poor protection against pneumonic plague
Although subunit vaccines (recombinant proteins or purified bacterial proteins) can effectively induce the body to produce a protective immune response, there are still some limitations, such as complex preparation process, need for adjuvants, high cost, and single ingredients cannot provide effective vaccines. Protection, etc.

Method used

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  • Yersinia pestis ghost, preparation method thereof, and application of Yersinia pestis ghost serving as vaccine
  • Yersinia pestis ghost, preparation method thereof, and application of Yersinia pestis ghost serving as vaccine
  • Yersinia pestis ghost, preparation method thereof, and application of Yersinia pestis ghost serving as vaccine

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Experimental program
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Embodiment 1

[0037] Preparation and identification of embodiment 1 plague EV slough

[0038] 1.1 Electrotransformation of Yersinia pestis with plasmid pMBE

[0039] The production method of EV competent cells is as follows: Inoculate a single plague EV colony into 5ml LB liquid medium, culture overnight at 37°C, then expand and transfer to 100ml LB medium, culture at 37°C until mid-logarithmic growth, and collect the bacteria by centrifugation Body, wash twice with PBS (PH7.4) and electrotransfer medium (0.5M sorbitol, 0.5M mannitol, 10% glucose, distilled water to volume), and finally add 1ml pre-cooled electroporation medium, 100ul aliquot , stored at -70°C.

[0040] The operation method of electroporation is as follows: After mixing the correctly identified plasmid pMBE with 100 μl of competent cells, inject it into a pre-cooled 0.1 cm electric shock cup (BIO-RAD), and set on the electric shock instrument: voltage 1500v, capacitance 25uF, resistance 200Ω , for electrical conversion. ...

experiment example 2

[0053] Experimental Example 2 Analysis of Immune Effect of Plague EV Sludge

[0054] BALB / c mice were immunized with plague EV slough by subcutaneous injection, and the immune response and immune protection induced by the slough were analyzed.

[0055] 2.1 Experimental animals and grouping

[0056] Healthy BALB / c female mice, 6-8 weeks old, weighing about 18 g, were purchased from the Experimental Animal Center of the Academy of Military Medical Sciences. Randomly divided into 3 groups, 5-6 in each group. In the experimental group (EV slough group), the freeze-dried slough was directly dissolved in PBS without adding any adjuvant, and each mouse was subcutaneously inoculated with 10 7 CFU / 100μL, a total of two immunizations (boosting immunization on the 21st day); at the same time, a positive control group (EV live vaccine group, Lanzhou Institute of Biological Products produced human plague EV live vaccine) was immunized once, and each mouse was vaccinated dose of 10 7 CF...

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Abstract

The invention provides a Yersinia pestis ghost, a preparation method thereof, and application of the Yersinia pestis ghost serving as a vaccine. The ghost has a complete outer membrane structure, can induce an organism to generate a protective antibody by inoculating a mouse without an adjuvant, generate strong immune memory and protect the mouse from being attacked by strong dose of plague live strains, and has higher safety compared with a live vaccine.

Description

technical field [0001] The invention relates to a Yersinia pestis slough, its preparation method and its application as a molecular delivery system, belonging to the technical fields of molecular biology and immunology. Background technique [0002] Bacterial Ghost (BG) is a complete bacterial empty shell that induces the expression of the E gene of phage PhiX174 in Gram-negative bacteria, and finally lyses the host cell. The PhiX174E gene encodes a hydrophobic protein consisting of 91 amino acids. This protein itself has no enzymatic activity, but can connect the inner and outer membranes through oligomerization. Electron micrographs show that the inner and outer membranes fuse at the channel and form a closed periplasmic space. The diameter of the cleavage channel ranges between 40nm and 200nm. The size of the lysis channel is affected by individual differences in cells, mainly related to the degree of cell autolysis, the size of the sieve holes on the peptidoglycan laye...

Claims

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Application Information

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IPC IPC(8): C12N15/63A61K9/19A61K39/02A61P31/04C12N1/21
CPCY02A50/30
Inventor 寇志华周育森温晶王效义于虹郭彦赵光宇杨瑞馥
Owner MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI
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