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Encephalitis b virus non-structural protein NS1 truncated mutant as well as coding gene and application thereof

A Japanese encephalitis virus, non-structural protein technology, applied in the field of viruses, can solve the problems that affect the full-length NS1 protein for the treatment of Japanese encephalitis, and the low expression level of the full-length NS1 protein in Escherichia coli.

Active Publication Date: 2018-10-26
JIANGXI AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the existing technology discloses the problem that the expression level of the full-length NS1 protein in E. coli is not high, which directly affects the application of the full-length NS1 protein in the research and development of clinical vaccines and the treatment of Japanese encephalitis

Method used

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  • Encephalitis b virus non-structural protein NS1 truncated mutant as well as coding gene and application thereof
  • Encephalitis b virus non-structural protein NS1 truncated mutant as well as coding gene and application thereof
  • Encephalitis b virus non-structural protein NS1 truncated mutant as well as coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Material source description

[0055] (1) Viruses, plasmids, strains and laboratory animals

[0056] JEV SA14-14-2 strain, JEV P3 virulent strain, BHK-21 cells, E.coli DH5α, E.coli BL21(DE3), plasmid pET-28a(+) are stored in our laboratory. Female BALB / c mice aged 3–5 weeks were purchased from mice and purchased from Hunan Slack Jingda Experimental Animal Co., Ltd.

[0057] (2) Reagent source

[0058] His-tag antibody and HRP-labeled goat anti-mouse IgG secondary antibody were purchased from Wuhan Sanying Biotechnology Co., Ltd. Various restriction endonucleases, Taq DNA polymerase, Pyrobest DNA polymerase and T4 DNA ligase are all products of Bao Bioengineering (Dalian) Co., Ltd. DMEM and fetal bovine serum for cultured cells were purchased from GIBCO. Trizol RNA extraction kit was purchased from invitrogen company. Ni column was purchased from QIAGEN company. Mouse Japanese encephalitis (JE) antibody (IgG) enzyme-linked immunoassay kit was purchased from Wuhan Huamei Bioe...

Embodiment 2

[0064] (1) Primer design and synthesis

[0065] The primers were designed according to the NS1 gene sequence of JEV SA14-14-2 strain (AF315119) and synthesized by Shenggong. The primer sequence is as follows: NS1 forward primer: 5′-CGC GGATCC GACACTGGATGTGCC-3′; full-length NS1 reverse primer: 5′-CCG CTCGAG AGCGGCGACTAGCA-3′; NS1 △63 Reverse primer: 5’-CCG CTCGAG AGCATCAACCCGTGATC-3’. The forward and reverse primers were introduced into the BamHI and XhoI restriction sites (underlined).

[0066] (2) Cell and virus culture

[0067] BHK-21 cells are cultured in DMEM medium containing 10% fetal bovine serum. When the confluence density is about 80%, they are inoculated with the Japanese encephalitis virus SA14-14-2 strain. When about 80% of the cells are diseased, harvest the cells and extract the total protein or Total RNA.

[0068] (3) Amplification of full-length and truncated NS1 gene

[0069] The Trizol RNA extraction kit was used to extract JEV RNA, and reverse transcription ...

Embodiment 3

[0073] Construction of recombinant expression plasmid

[0074] The PCR-amplified full-length and truncated NS1 gene fragments obtained in Example 2 were digested with XhoI and BamH Ⅰ, and then respectively ligated with plasmid pET-28a(+) digested with the same enzymes to transform competent E. coli DH5α, screen positive clones, extract plasmids, identify them by PCR and double enzyme digestion, and send them for sequencing. The sequenced plasmids were named pET28a-NS1 and pET28a-NS1 △63 , Transform E.coli BL21(DE3).

[0075] NS1 and NS1 △63 Prokaryotic expression of gene recombinant plasmid pET28a-NS1 and pET28a-NS1 △63 After BamHI and XhoI digestion, the expected size of NS1 ( image 3 -A) and NS1 △63 Fragment( image 3 -B), sent to Biotech for sequencing, the BLAST alignment sequence was correct, indicating that the recombinant plasmid was constructed successfully.

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Abstract

The invention relates to an encephalitis b virus non-structural protein NS1 truncated mutant as well as a coding gene and application thereof, which belongs to the technical field of viruses. According to the encephalitis b virus non-structural protein NS1 truncated mutant, on the basis of original NS1, two strong hydrophobic zones on an end C are deleted, a truncated mutant NS1 delta63 gene with63 amino acids on the end C being deleted, then the truncated mutant NS1 delta63 gene is cloned to a prokaryotic expression vector pET-28a(+), a recombinant expression vector pET28a-NS1delta63 is obtained, escherichia coli is converted, and by virtue of the induction of IPTG, the NS1 delta63 can be efficiently and stably expressed in the escherichia coli. The NS1 delta63 protein expressed in the Escherichia coli purifies and immunizes a mouse, and the NS1 anti-serum titer of the immunized mouse reaches 1:12150 by virtue of ELISA detection. The protection capability test of the immunized mouseproves that the NS1 delta63 has the protection activity. The NS1 delta63 can lay a foundation for later research of the virus vaccine and diagnosis kit.

Description

Technical field [0001] The invention belongs to the field of virus technology, and specifically relates to a truncated mutant of Japanese encephalitis virus non-structural protein NS1 and its coding gene and application. Background technique [0002] The disease caused by Japanese Encephalitis Virus (JEV) is called Japanese encephalitis (Japanese encephalitis for short), which is a mosquito-borne zoonotic disease that seriously threatens the health of humans and animals. Pigs are the most important storage and multiplication host of JEV in nature. JEV infection can cause piglet encephalitis and breeding problems, causing huge economic losses to the pig industry. my country is an area with a high prevalence of Japanese encephalitis. This is related to the vast area of ​​my country, the common pig raising in various places, the close relationship between humans and pigs, and the large rice planting area, and the widespread distribution of Japanese encephalitis transmission vectors ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/18C12N15/40C12N15/11C12N15/10C12N15/70A61K39/12A61P31/14G01N33/569
CPCA61K39/12A61P31/14C07K14/005C12N2770/24122C12N2770/24134G01N33/56983G01N2333/185Y02A50/30
Inventor 孔令保徐晶郭韫丽
Owner JIANGXI AGRICULTURAL UNIVERSITY
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