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A synthetic polypeptide epitope based vaccine composition

A vaccine composition and vaccine antigen technology, which is applied in the field of vaccine compositions based on synthetic polypeptide epitopes, and can solve problems such as co-infection, destructive effects on medical systems, etc.

Active Publication Date: 2020-04-21
BHARAT BIOTECH INTERNATIONAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Often the two viruses co-transmit and cause co-infection, with devastating effects on healthcare systems in many Asian countries

Method used

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  • A synthetic polypeptide epitope based vaccine composition
  • A synthetic polypeptide epitope based vaccine composition
  • A synthetic polypeptide epitope based vaccine composition

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Embodiment 1: express recombinant protein of the present invention by MEV1 and MEV2 gene construct

[0071] Plasmids pET11b and pET11b-MEV1 were transformed into E. coli BL21(DE3) chemically competent cells. Incubate transformed BL21(DE3) cells at 37°C with shaking until OD 600 0.4 to 0.6 were reached, and then induced with different concentrations of isopropyl β-D-1-thiogalactoside (IPTG) for 4 hours at 37°C. IPTG is a molecular mimic of lactose metabolites that triggers the transcription of the lac operon, and thus induces the expression of recombinant proteins whose genes are controlled by the lac operator. The optimal concentration of IPTG for expression was determined, and then induction of the multi-epitopic gene (MEV1) for expression was also determined in various temperature environments ( image 3 ). Optimal conditions for expression were evaluated for subsequent batches of experiments. pET11b-MEV2 was also transformed into BL21(DE3) cells, followed by incu...

Embodiment 2

[0072] Example 2: Cell Lysis with Lysis Buffer-I and Lysis Buffer-II

[0073] Cell pellets of MEV1 transformed BL21(DE3) after IPTG induction were collected by centrifuging the cells at 10,000 rpm for 5 min. Cells were suspended in Suspension / Lysis Buffer-I containing 50 mM Na 2 HPO 4 , 0.3M NaCl, 1% TritonX-114, 0.5mg / ml lysozyme, 1mM AEBSF, pH 7.4 solution to prepare. Cell lysis was performed first by performing three freeze-thaw cycles followed by three sonications of 20 seconds duration with 30 second intervals in between. The position of the expected protein was checked again by SDS-PAGE. Most of the expected protein was found in cellular debris, indicating the formation of insoluble / inclusion-like bodies. Then, the cells were lysed at 4°C for 3-16 hours in lysis buffer-II with different concentrations of urea (3M-6M) in the presence of 5mM-10mM imidazole, 50mM Na 2 HPO 4 , 0.3M NaCl and 1 mM AEBSF.

[0074] The results showed that the recombinant enterovirus vacci...

Embodiment 3

[0076] Example 3: Immobilized Metal Affinity Chromatography (IMAC) and Washing of MEV1 with Wash Buffer-I, Wash Buffer-II and Wash Buffer-III

[0077] The urea-denatured protein solution in the presence of 5 mM-10 mM imidazole was added to the Ni-NTA IMAC column after equilibration with Lysis Buffer-II and incubated at 4°C for 3-16 hours for proper binding. Then, the column was washed with Wash Buffer-I prepared by preparing a solution containing: 50 mM Na 2 HPO 4 , 0.3M NaCl, 1 mMAEBSF, 3M urea, pH 7.4, and 0.1% Triton X-114 and 5mM-10mM imidazole.

[0078] Perform the next wash in Wash Buffer-II prepared by making a solution containing: 50 mM Na 2 HPO 4 , 0.3M NaCl, 1 mM AEBSF, 3M-6M urea, pH 7.4, and 0.1% TritonX-114 and 20 mM imidazole. Subsequently, depending on the initial concentration of urea used in Example 2, at least two or more additional washes were performed with washing buffer-III in the presence of decreasing concentrations of urea through Prepared contain...

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Abstract

Conserved epitopes selected from EV71 and CVA16, the two major causative agents of Hand Foot and Mouth Disease has been used to develop sub-unit bivalent vaccine antigen construct. The said vaccine described in this invention is capable to provide cross-protection to diverse EV71 and CVA16 infection causing strains. Further disclosed are the expression of the multi- epitope vaccine antigen codinggene and the purification process involved thereof. This present invention also discloses vaccine formulations against Hand Foot and Mouth Disease and other enterovirus infections comprising the recombinant vaccine antigen construct of the present invention.

Description

[0001] field of invention [0002] The present invention relates to vaccine compositions based on synthetic polypeptide epitopes. More specifically, a conserved epitope selected from EV71 and CVA16 is used to develop a subunit (sub-unit) bivalent vaccine antigen construct, and a vaccine composition is provided using the developed subunit bivalent vaccine antigen construct, Among them, the vaccine can provide cross-protection against different strains causing EV71 and CVA16 infection. The expression of the polyepitope vaccine antigen coding gene and the purification method involved are also disclosed. The invention also discloses a vaccine preparation against Hand Foot and Mouth Disease and other enterovirus infections, the vaccine preparation comprises the recombinant vaccine antigen construct of the invention. [0003] Background of the invention [0004] Hand-foot-mouth disease (HFMD) is a common pediatric disease mainly caused by enterovirus-71 (EV71) and coxsackievirus A1...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/12
CPCA61K39/12A61K2039/55505A61K2039/55561A61K2039/55566A61K2039/55572A61K2039/70C12N2770/32134C12N2770/32334A61P31/14A61K39/135C07K1/22C07K1/36C07K14/09
Inventor 阿米特·拉伊乔度里
Owner BHARAT BIOTECH INTERNATIONAL
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