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Medium being able to optimize virus replication

A virus replication and virus technology, applied in antiviral agents, viral antigen components, viruses/phages, etc., to achieve the effects of high expression efficiency, increased titer, and easy purification

Inactive Publication Date: 2016-10-05
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The unique biochemical properties and complex functions of P60 make it quickly become a research hotspot, but in the field of virology, there are few reports in the literature

Method used

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  • Medium being able to optimize virus replication
  • Medium being able to optimize virus replication
  • Medium being able to optimize virus replication

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0037] 1. Preparation of cells

[0038] DMEM medium containing 5% fetal bovine serum and 100U penicillin and streptomycin was used for passage of Hep-2 and Vero cells, and DMEM medium containing 2% fetal bovine serum and 100U penicillin streptomycin was used as maintenance medium.

[0039] Primary chicken embryo fibroblast (CEF) cells: 9-10-day-old chicken embryos were purchased from Beijing Merial Company, the product catalog number is "SPF chicken embryos". Take out the 9-10-day-old chicken embryos and carefully remove the head and tail, bones and adipose tissue, wash the rest with PBS 3 times, cut into small pieces as much as possible, add 0.25% trypsin to digest for 30 minutes, wash 3 times with PBS, and then add 70ml of culture fluid (DMEM culture fluid containing 5% fetal bovine serum and 100U penicillin streptomycin, maintenance fluid with DMEM medium containing 2% fetal bovine serum and 100U penicillin streptomycin), vigorously wash out fibroblasts, containing Pass th...

Embodiment 1

[0053] Example 1 Cell half infection dose and chick embryo half infection dose of several viruses

[0054] HSV and NDV infected Hep-2 cells, and AIV infected Vero cells. CDV, MDV and IBV infect CEF cells. EV71 infection of RD cells.

[0055] Take 9-10 day-old SPF chicken embryos to prepare chicken embryo fibroblasts (CEF), inoculate CDV, MDV and IBV on CEF and culture for 48 hours, harvest cells and culture supernatant repeatedly freeze-thaw 3 times, and re-inoculate on CEF cells Continue to cultivate, so pass down to 3 generations. Dilute the virus solution 10 times, and inoculate cells in 96-well plates or chicken embryos. Virus measured cell half infectious dose (TCID 50 ), wherein MDV uses the formation of cytopathic acne spots to measure TCID 50 , In addition, IBV uses chicken embryo half infective dose EID 50 , to determine the virulence of the virus.

[0056] Calculated TCID for NDV 50 =2×10 9 / ml, TCID of HSV-1 50 =10 8.1 / ml, TCID of CDV 50 =TCID 50 =10 ...

Embodiment 2

[0057] Example 2 Overexpression of protein A, B, C, E

[0058] Cell transfection: When the cell density of CEF, Hep-2 or Vero cells reached 80%, according to the instructions of lipofectamine 2000, the eukaryotic expression plasmid pEGFP-E and the carrier pEGFP-N1 were respectively transfected into the above cells, each plasmid was 10 μg, Overexpression 40h.

[0059] The construction method of the pEGFP-E plasmid is as follows: using the glioma tumor suppressor candidate gene 2 as a template, design the upstream and downstream primers, which are respectively 5'-GAATTCTTTGAAGACCACCAG-3 (the nucleotide sequence shown in SeqID No.6)' and 5'-GGATCCTCACAACTGGATCTC-3' (the nucleotide sequence shown in Seq ID No.7), perform PCR amplification, and add the enzyme cutting sites EcoRI and BamHI to the PCR amplification products respectively; at the same time, select the enzyme that can stably express GFP For the vector pEGFP-N1, EcoRI and BamHI were selected upstream of EGFP. The prote...

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Abstract

The invention relates to a medium being able to optimize virus replication. The medium contains a fusion protein E with the concentration of 100-1000nM, and a cell line is a host cell line over-expressing the fusion protein E gene. The fusion protein E can improve the TCID50 tilter of various viruses by 1-3 lg units and improve the HA tilter by 2-4 lg2 units, the various viruses comprise coronavirus, paramyxovirus, orthomyxovirus, reovirus and herpes virus, the fusion protein E has no cytotoxicity in a prescribed dosage, breaks a boundary between DNA viruses and RNA viruses, and also breaks the boundary between viruses with envelopes and viruses without envelopes. The fusion protein E obtained through a prokaryotic expression system has a high expression efficiency, and can be easily purified, the one-step purification efficiency can reach 85% or above, and the final concentration of the protein can reach 3mg / ml. The fusion protein E can be directly added to the virus medium in order to improve the tilter of various virus vaccines, so the medium is simple and fast. The medium has great influences and meanings in the production, learning and research of the virus subject.

Description

technical field [0001] The invention relates to the field of whole virus vaccine preparation, in particular to a culture medium capable of optimizing virus replication. Background technique [0002] Whole virus vaccines (including attenuated vaccines and inactivated vaccines) account for a considerable proportion of modern vaccine applications. Whole virus vaccines are also known as conventional vaccines, including inactivated vaccines and live attenuated vaccines. Among them, human vaccines include mumps live attenuated vaccine, Japanese encephalitis attenuated vaccine, hepatitis A live attenuated vaccine, rabies attenuated vaccine, hand, foot and mouth disease inactivated vaccine, rotavirus inactivated vaccine, hepatitis A inactivated vaccine, B Brain inactivated vaccines, etc. The use of animal vaccines is more common. At the same time, considering the cost and convenience of administration, they are usually prepared as multiple vaccines. The most typical one is the can...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/66C12N15/70A61K39/215A61P31/14A61K39/155A61K39/145A61K39/15A61K39/245A61P31/22C12N7/00C12R1/93
Inventor 王晓佳
Owner CHINA AGRI UNIV
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