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Rescue system for paramyxoviruses and rescue method for paramyxoviruses

A paramyxovirus and rescue system technology, applied in the field of viral reverse genetics, can solve problems such as the inability to meet the needs of paramyxovirus vaccine development, achieve good application prospects, and avoid pollution

Pending Publication Date: 2020-06-02
CHENGDU INST OF BIOLOGICAL PROD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] In summary, the use of conventional virus rescue systems cannot meet the needs of paramyxovirus vaccine development

Method used

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  • Rescue system for paramyxoviruses and rescue method for paramyxoviruses
  • Rescue system for paramyxoviruses and rescue method for paramyxoviruses
  • Rescue system for paramyxoviruses and rescue method for paramyxoviruses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1 Preparation of experimental cells and electroporation plasmids

[0052] Passage the Vero cells required for electroporation into a T75 flask at a ratio of 1:4, at 37°C, 5% CO 2 Culture for 24-48h until the cells are more than 70% confluent or a thin monolayer. After the Vero cells were digested with trypsin, an appropriate amount of medium (6-8 ml) was taken to elute, and 10 μl was sucked with a pipette for counting on a hemocytometer. After counting the cells, calculate the required amount of cells, and add the calculated amount of cells into a 1.5ml EP tube after autoclaving. Add the required plasmid into the 1.5ml EP tube after autoclaving according to the amount calculated in advance, and mark it.

[0053] Put the cells in the EP tube into a centrifuge, centrifuge at 2000g×5min at room temperature, and remove the culture medium. For each experimental sample, use 110 μl electroporation buffer to fully and gently suspend the cells, transfer the cell suspe...

Embodiment 2

[0054] Example 2 Exploration of Electroporation Conditions

[0055] Buffer: This system selects two electrotransfer buffers for experiments, AT electrotransfer buffer and BT electrotransfer buffer.

[0056] The AT electrotransfer buffer is Beijing Entranster-H electrotransfer buffer, catalog number: 98668.

[0057] BT electrotransfer buffer is lonzanucleofector electrotransfer buffer, catalog number: V4LP-3520.

[0058] Plasmid: pCDNA3.1-RFP with CMV promoter (can express red fluorescent protein in eukaryotic cells, which is convenient for later evaluation of electroporation efficiency by observing the expression of fluorescent protein);

[0059] Cells: Vero (the amount of cells used in each experimental group was 5×10 5 indivual).

[0060] Electrode Cup: C Manufacturer: Haimen Aibide Experimental Equipment Co., Ltd. QIVON Electrode Cup Item No.: CUV502

[0061] D Manufacturer: Bole Electric Rotor Cup Item No.: 165-2086.

[0062] Cells and plasmids were mixed and transfer...

Embodiment 3

[0070] Example 3 Rescue of recombinant measles virus MV-GFP by electroporation of whole plasmid in Vero cells

[0071] Plasmid used: pT7-MV S191 N. pT7-MV S191 P, pT7-MV S191 L, pCDIBP-T7RNAP (see SEQ ID NO.1 in the appendix for the sequence), pT7-T7RNAP (see SEQ ID NO.2 in the appendix for the sequence); pT7-MV S191 - For the construction of GFP, see patent CN201710001203 - a system and method for rescuing measles virus and recombinant measles virus.

[0072] Electrotransfer buffer: AT electrotransfer buffer; BT electrotransfer buffer.

[0073] Electric cup: D manufacturer.

[0074] The experimental operation was carried out according to the optimal electroporation conditions explored in Example 1. The specific electroporation conditions and plasmid usage are shown in Table 3. If the test is successful, the resulting recombinant measles virus MV-GFP can express green fluorescent protein ( Figure 5 ) for later observation under a microscope.

[0075] Transfer the cells...

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Abstract

The invention provides a rescue method for paramyxoviruses. The method includes the following steps: (1) mixing a paramyxovirus genome plasmid, a N plasmid, a P plasmid, an L plasmid, a pT7-T7RNAP plasmid, a pCDIBP-T7RNAP plasmid and animal cells; (2) mediating multi-plasmid cotransfection by using a physical or chemical method; (3) cultivating cells; and (4) collecting the viruses, wherein the pT7-T7RNAP plasmid is a plasmid having a T7 promoter and a T7RNA polymerase DNA sequence, and the pCDIBP-T7RNAP plasmid is a plasmid having a CMV promoter and a T7RNA polymerase DNA sequence. The methodcan efficiently rescue the paramyxoviruses, and can be applied to production of a human live attenuated paramyxovirus vaccine.

Description

technical field [0001] The invention relates to the field of virus reverse genetics, in particular to a paramyxovirus rescue system and method. Background technique [0002] Paramyxovirus is a type of virus with special affinity with mucus protein. Measles, respiratory syncytial virus, Newcastle disease virus, etc. can infect humans or animals. People usually use attenuated live vaccines to prevent paramyxovirus infection. Live attenuated vaccines refer to strains in which the pathogen mutates and weakens its toxicity after undergoing various treatments, but still retains its immunogenicity. [0003] Paramyxovirus is a negative-strand RNA virus. The reverse genetics of RNA virus is to use the genetic material of the virus to rescue live virus or similar virus material in cultured cells or susceptible hosts. The reverse genetics system can be used to rescue and obtain the attenuated strain of the active ingredient virus in the live vaccine without going through the mutation...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/45C12N7/00
CPCC12N15/85C12N7/00C12N2760/18052
Inventor 刘兰军张勇侠高雅丽陈宗香
Owner CHENGDU INST OF BIOLOGICAL PROD
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