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Fusion protein 210 and applications thereof in virus replication optimization

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, few reports in the field of virology, cell apoptosis, etc., and achieve high expression efficiency and time-consuming detection Shortened, easy-to-purify effect

Inactive Publication Date: 2017-08-15
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 applications thereof in virus replication optimization
  • Fusion protein 210 and applications thereof in virus replication optimization
  • Fusion protein 210 and applications thereof in virus replication optimization

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0036] Using E. coli engineered 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: cut the pET-28a vector with restriction enzymes NdeI / EcoRI, cut the target gene obtained in step 1) and ligate it with the vector to obtain the recombinant vector pET-210 containing the target gene;

[0039] 3. Cell transformation and expression: Transform BL21(DE3) with recombinant vector pET-210, and culture to OD at 37°C on a shaker 590 0.8-1.0, add inducer IPTG to a final concentration of 1mM, and continue to incubate at 37°C for 4h;

[0040] 4. Protein purification: the induce...

Embodiment 2

[0047] Example 2 Study on the effect of fusion protein 210 on virus virulence by immunoblotting, cytopathic and qRT-PCR

[0048] Western-blot: Wash the virus-infected cells with pre-cooled pH 7.5 PBS for 3 times, scrape the cells, lyse the cells with ultrasound and extract the total protein for 3-5 seconds, and compare them to 6×SDS-PAGE The sample buffer was mixed, boiled for 10 minutes, and SDS-PAGE electrophoresis was performed. After electrophoresis, the protein on the gel was transferred to the PVDF membrane by an electro-membrane transfer machine. Block with PBST buffer containing 5% skimmed milk powder for 1 hour at room temperature, and add primary antibody to incubate for 2 hours with shaking at 4°C. Wash the membrane 4 times with PBST, 5 min each time, and incubate the corresponding HRP-labeled secondary antibody with 1:8000 dilution at room temperature for 1 h, wash the membrane 4 times, 5 min each time, and finally develop the color with SuperSignal West Pico Chemilum...

Embodiment 3

[0065] Example 3 Cytotoxicity test of fusion protein 210

[0066] Lactate dehydrogenase (LDH) experiment is used to study whether the polypeptide or protein is toxic to cells, and it is completed with a toxicity detection kit purchased from Roche. Add the following final concentrations of fusion protein 210 to CEF cells cultured to a monolayer: 5μM, 25μM, 50μM, 100μM, 250μM, 500μM, 1.0mM, and gently mix them evenly. After 24 hours, determine the toxicity index according to the kit instructions . The experiment was repeated three times. The results showed that the fusion protein 210 had no toxic effect on cells at a concentration of 50 μM and 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 invention relates to the field of virus isolation and identification and 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 virological 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 viral antigens, the detection of antibodies produced by the virus, and the detection of viral genes. The detection of virus antigens and virus antibodies usually requires an ELISA test kit; 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 limitations, th...

Claims

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Application Information

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Patent Type & Authority Applications(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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