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Fusion protein 210 and its use in optimizing viral replication

A fusion protein and virus replication technology, which is applied in the field of virus isolation and identification and whole virus vaccine preparation, can solve the problems of obvious cytotoxicity, cell apoptosis, few reports in the field of virology, etc., and achieves high expression efficiency, easy purification, Detect the effect of shortening time-consuming

Inactive Publication Date: 2020-01-24
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 there are few reports in the field of virology
[0006] Previous studies have found that P60 protein can promote the replication of several viruses (Rhadoviridae VSV, Flaviviridae HCV, Parvoviridae EV71), but cytotoxicity experiments show that P60 whole protein can induce significant cell apoptosis, and cytotoxic

Method used

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  • Fusion protein 210 and its use in optimizing viral replication
  • Fusion protein 210 and its use in optimizing viral replication
  • Fusion protein 210 and its use in optimizing viral replication

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Prokaryotic expression and purification of embodiment 1 fusion protein 210 gene

[0036] Using Escherichia coli engineering bacteria to express fusion protein 210, the specific method includes the following steps:

[0037]1. Gene cloning: Using glioma tumor suppressor candidate gene 2 (GenBank: KJ898763.1) as a template, design primers for subcloning of the target gene; among them, the upstream primer: 5'-CGAGGTCTGTCCCACGCCCG-3', the downstream primer: 5 '-CAGCTCCGAGCTCAGCTGCA-3';

[0038] 2. Vector construction: the pET-28a vector was digested with restriction enzyme NdeI / EcoRI, and the target gene obtained in step 1) was also double-digested and connected to the vector to obtain the recombinant vector pET-210 containing the target gene;

[0039] 3. Cell transformation and expression: transform BL21(DE3) with the recombinant vector pET-210, and culture on a shaker at 37°C until OD 590 0.8-1.0, add the inducer IPTG to a final concentration of 1mM, and continue to cult...

Embodiment 2

[0047] Example 2 Western blotting, cytopathic method and qRT-PCR method to study the influence of fusion protein 210 on virus virulence

[0048] Western-blot: Wash the virus-infected and treated cells 3 times with pre-cooled pH 7.5 PBS, scrape off the cells, extract the total protein by ultrasonic lysis for 3-5 seconds, and print on 6×SDS-PAGE The sample buffer was mixed, boiled for 10 min, and subjected to SDS-PAGE electrophoresis. After electrophoresis, the protein on the gel was transferred to a PVDF membrane by an electrotransfer apparatus. Block with PBST buffer containing 5% skimmed milk powder at room temperature for 1 h, add primary antibody and incubate with shaking at 4 °C for 2 h. Wash the membrane with PBST 4 times, 5min each time, dilute the corresponding secondary antibody labeled with HRP at 1:8000, incubate at room temperature for 1h, wash the membrane 4 times, 5min each time, and develop color with SuperSignalWest Pico Chemilumineseent HRP Substrate ECL, and u...

Embodiment 3

[0065] Example 3 Cytotoxicity experiment of fusion protein 210

[0066] Lactate dehydrogenase (LDH) assay was used to study whether the polypeptide or protein is toxic to cells, and the toxicity detection kit purchased from Roche Company was used to complete. Add the following final concentrations of fusion protein 210 to the CEF cells cultured to a monolayer: 5 μM, 25 μM, 50 μM, 100 μM, 250 μM, 500 μM, 1.0 mM, mix gently, and measure the toxicity index according to the kit instructions after 24 hours . Experiments were repeated three times. The results showed that fusion protein 210 had no toxic effect on cells at a concentration of 50 μM or below.

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Abstract

The invention provides fusion protein 210 and applications thereof in virus replication optimization. The fusion protein 210 is a truncated part of P60 protein, preserves a function for promoting replication of a plurality of viruses of the P60, and does not generate toxicity on cells in an effective concentration range. The fusion protein 210 can increase the virus titer by two orders of magnitudes in a wide-spectrum range (0.0001-0.1 MOI), and therefore virus identification and detection can be facilitated, rhabdovirus VSV, flaviviridae HCV, parvoviridae EV71, and the like can be detected, and time for detection is shortened by 50% or less. The fusion protein 210 obtained by a prokaryotic expression system is high in expression efficiency and easy to purify, the one-step purification efficiency can be 85% or above, and the protein final concentration can be 3 mg / mL. Accordingly, through directly adding the fusion protein 210 into a virus culture medium, a plurality of virus isolate strains can be detected under low titer conditions, and operation is simple, convenient and rapid. The fusion protein 210 has important influences and significance in the fields of production, learning, researching, and the like of the virology.

Description

technical field [0001] The present invention relates to the field of isolation and identification of viruses and the preparation of whole virus vaccines, in particular to a fusion protein 210 and its application in optimizing virus replication. Background technique [0002] The identification and detection of viruses is a prerequisite for cultivating a large number of viruses, conducting virology experiments, and preparing vaccines and specific diagnostic reagents. The establishment of a new technology for virus detection should basically be considered from three aspects, namely, the detection of virus antigens, the detection of antibodies to viruses, and the detection of virus genes. The detection of virus antigens and virus antibodies usually requires ELISA detection kits; the detection of virus genes requires the use of laboratory molecular means, such as PCR technology and gene chip technology for identification. At present, these two detection methods have certain limi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/70C12N7/00
CPCC07K14/4747C07K2319/21C12N7/00C12N2710/16051C12N2720/12251C12N2750/14351C12N2760/16051C12N2760/18051C12N2760/20051C12N2770/10051C12N2770/20051
Inventor 王晓佳李翠翠
Owner CHINA AGRI UNIV
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