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Genetic engineering heat stable vaccine and preparation method thereof

A technology for thermal stability and vaccines, applied in pharmaceutical formulations, medical preparations with non-active ingredients, drug delivery, etc., can solve problems such as toxicity of organisms or vaccines, complex processing processes, and inability to produce good protective effects, and achieve breakthroughs Technical barriers, widening scope, effect of good biocompatibility

Inactive Publication Date: 2014-10-15
ZHEJIANG UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The above strategies have some limitations in the application of heat-stable vaccines. For example, methods (1)(2)(3) generally require complex treatment processes, method (3) cannot produce good protective effects, and method (4) directly mineralizes The strategy requires a large number of groups with high adsorption capacity for calcium ions on the surface of the vaccine, such as phospholipid molecules in the lipid bilayer of enveloped viruses, so this method can only be applied to some specific enveloped virus vaccines, and the mineralization requires High concentrations of calcium ions, which may be toxic to organisms or vaccines

Method used

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  • Genetic engineering heat stable vaccine and preparation method thereof
  • Genetic engineering heat stable vaccine and preparation method thereof
  • Genetic engineering heat stable vaccine and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0102] Embodiment 1, preparation has the genetic engineering virus vaccine of self-mineralization ability

[0103] In this example, the EV71 attenuated strain A12 was used as the model skeleton. First, the full-length genome clone of the EV71 attenuated strain A12 was constructed using the pEV plasmid (reformed from the pBR32 plasmid) as a vector, and then the EV71 structural protein VP1 β-( The coding sequence of the target polypeptide is inserted between the 100-101 amino acids of the BC)-loop region, that is, the corresponding nucleotide sequence is introduced between the 2737-2738 nucleotides of the full-length genome clone. In view of the similar three-dimensional structure and genome composition of polio vaccine and enterovirus EV71, the following examples use the attenuated strain A12 of EV71 as a model.

[0104] Step 1. Construction of full-length clone of EV71 attenuated strain A12

[0105] (1) Extraction of EV71 virus RNA

[0106] The EV71 AH / 08 / 06 strain was inocu...

Embodiment 2

[0178] Embodiment 2, preparation EV71, EV71-N6, EV71-NW and EV71-W6 vaccine solution and its biological characteristics analysis

[0179] Step 1, preparation of EV71, EV71-N6, EV71-NW and EV71-W6 virus liquid

[0180] (1) Enterovirus EV71, genetically engineered virus vaccine EV71-N6, EV71-NW, and EV71-W6 were used to infect human RD cells (using low-sugar DMEM medium), incubated at 37°C for 36-72 hours, and collected after the cells were completely damaged Virus vaccine.

[0181] (2) Centrifuge the culture system obtained in step (1) (16000g, 10 min), collect the supernatant and filter (using a filter membrane with a pore size of 0.22 microns), and collect the filtrate, which is the virus vaccine liquid (after testing, the pH 7.2), it was named as EV71, EV71-N6, EV71-NW and EV71-W6 vaccine liquid.

[0182] Step 2. Characterization of plaque characteristics

[0183] The EV71, genetically engineered virus vaccines EV71-N6, EV71-NW and EV71-W6 were subjected to the following ...

Embodiment 3

[0190] Calcium phosphate self-mineralization of EV71, EV71-N6, EV71-NW and EV71-W6 virus vaccine liquid prepared by embodiment 3, embodiment 2:

[0191] Step 1, Preparation of EV71-CaP, EV71-N6-CaP, EV71-NW-CaP, EV71-W6-CaP Vaccine Liquid-A

[0192] In EV71, genetic engineering virus vaccine EV71-N6, EV71-NW or EV71-W6 solution (virus vaccine titer is 1×10 7 PFU / mL, that is, 1×10 10 PFU / L) by adding CaCl 2 , so that Ca 2+ The ion concentration reaches 5.5 mM (including the newly added Ca 2+ ions and the Ca originally present in the vaccine solution 2+ Ion; Ca in vaccine solution 2+ The ion is theoretically equal to the Ca in the low-sugar DMEM medium 2+ ions, that is, the concentration is 1.8 mM), which is the initial system; incubate the initial system at 37°C for 2 hours, which is the termination system; add polyacrylic acid to the termination system to make the concentration reach 10 μg / mL, which is EV71-CaP, EV71-N6-CaP, EV71-NW-CaP or EV71-W6-CaP vaccine solution...

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Abstract

The invention discloses a method for preparing a heat stable vaccine, particularly relates to a method for preparing the heat stable vaccine by the combined use of genetic engineering and biomimetic mineralization technology, and provides a method for introduction of polypeptides with inorganic mineralization induction ability on vaccine surface by use of gene engineering technology. The method is as follows: (1) constructing a vaccine gene clone; (2) inserting a polypeptide nucleic acid sequence into the vaccine gene clone to obtain a recombinant clone; and (3) expressing the recombinant clone in cells or in Escherichia coli to obtain a vaccine with the self-mineralization ability. The invention also provides a method for preparing and calcium mineralization of the vaccine with the self-mineralization ability. The method for preparing and calcium mineralization of the vaccine with the self-mineralization ability is as follows: (1) adding Ca<2 +> ions into a vaccine liquid with acid radicals, and (2) incubating the system to obtain a calcium phosphate coated vaccine. Through vaccine mineralization, thermal inactivation phenomena in vaccine transportation and storage can be effectively solved, the vaccine fiscal expenditure can be significantly reduced, and the method can be well used in production of a new heat stable vaccine.

Description

technical field [0001] The invention relates to biotechnology, in particular to a genetically engineered heat-stable vaccine and a preparation method thereof, in particular to a vaccine with self-mineralization ability, a preparation method thereof, and a method for mineralizing the vaccine. Background technique [0002] Infectious diseases pose a serious threat to public health and cause millions of deaths every year. Vaccination, as the most effective medical treatment against infectious diseases, has saved countless lives. In particular, live attenuated vaccines (ie, attenuated strains of viruses) have emerged as the most important form of vaccine, playing a major role in the eradication of smallpox and polio. Live attenuated vaccines still in clinical use include polio vaccine, Japanese encephalitis vaccine, chickenpox vaccine, influenza vaccine, yellow fever vaccine, rubella vaccine, rotavirus vaccine, hepatitis A vaccine, and adenovirus vaccine. [0003] Since vaccin...

Claims

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Application Information

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IPC IPC(8): A61K47/48A61K9/00A61K39/12A61P31/12
CPCY02A50/30
Inventor 王广川秦成峰唐睿康李晓峰徐旭荣秦鄂德
Owner ZHEJIANG UNIV
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