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A Japanese encephalitis virus non-structural protein ns1 truncated mutant and its coding gene and application

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: 2020-08-25
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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  • A Japanese encephalitis virus non-structural protein ns1 truncated mutant and its coding gene and application
  • A Japanese encephalitis virus non-structural protein ns1 truncated mutant and its coding gene and application
  • A Japanese encephalitis virus non-structural protein ns1 truncated mutant and its coding gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Description of source of material

[0054] (1) Viruses, plasmids, strains and experimental animals

[0055] JEV SA14-14-2 strain, JEV P3 virulent strain, BHK-21 cells, E.coli DH5α, E.coli BL21(DE3), plasmid pET-28a(+) are preserved by our laboratory. 3–5-week-old female BALB / c mice were purchased from Hunan Slack Jingda Experimental Animal Co., Ltd.

[0056] (2) Reagent source

[0057] 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 Treasure Bioengineering (Dalian) Co., Ltd. DMEM and fetal bovine serum used to culture cells were purchased from GIBCO. Trizol RNA extraction kit was purchased from Invitrogen. Ni column was purchased from QIAGEN company. Mouse Japanese encephalitis (JE) antibody (IgG) ELISA kit was purchased from Wuhan Huamei Bioengineering Co., Ltd.

[0...

Embodiment 2

[0063] (1) Primer design and synthesis

[0064] Primers were designed according to the NS1 gene sequence of JEV SA14-14-2 strain (AF315119) and synthesized by Sangong. The primer sequences are 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'. Forward and reverse primers respectively introduced BamHI and XhoI restriction sites (underlined part).

[0065] (2) Cell and virus culture

[0066] BHK-21 cells were cultured with DMEM medium containing 10% fetal bovine serum. When the confluence density was about 80%, the SA14-14-2 strain of Japanese encephalitis virus was inoculated. When about 80% of the cells showed pathological changes, the cells were harvested and the total protein or total RNA.

[0067] (3) Amplification of full-length and truncated NS1 genes

[0068] Trizol RNA extraction kit was used to extract JEV RNA, and the r...

Embodiment 3

[0072] Construction of recombinant expression plasmids

[0073] The PCR-amplified full-length and truncated NS1 gene fragments obtained in Example 2 were double-digested with XhoI and BamHI, and then respectively ligated with the plasmid pET-28a(+) digested with the same enzymes, and transformed into competent Escherichia coli DH5α , screening positive clones, extracting plasmids, identified by PCR and double enzyme digestion, and sent to Shenggong for sequencing. The plasmids with correct sequencing were named pET28a-NS1 and pET28a-NS1 respectively △63 , transforming E.coli BL21(DE3).

[0074] NS1 and NS1 △63 Gene expression in recombinant plasmids pET28a-NS1 and pET28a-NS1 △63 NS1 of the expected size can be obtained by digestion with BamHI and XhoI △63 ( image 3 -A) and NS1 fragments ( image 3 -B), sent to Sangon for sequencing, and the BLAST alignment sequence was correct, indicating that the recombinant plasmid was successfully constructed.

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Abstract

The invention relates to a Japanese encephalitis virus non-structural protein NS1 truncated mutant, its coding gene and application, and belongs to the technical field of viruses. The non-structural protein NS1 truncation mutant of Japanese encephalitis virus deletes two strong hydrophobic regions at the C-terminus on the basis of the original NS1, and obtains a truncation mutant NS1 with 63 amino acids at the C-terminus deleted △63 Gene, cloned into the prokaryotic expression vector pET‑28a(+), and obtained the recombinant expression vector pET28a‑NS1 △63 , transformed into E. coli, induced by IPTG, NS1 △63 It can be expressed efficiently and stably in Escherichia coli. NS1 expressed in E. coli △63 After protein purification and immunization of mice, the titer of NS1 antiserum of immunized mice reached 1:12150 by ELISA. Protection tests in immunized mice showed that NS1 △63 Has protective activity. The NS1 △63 The protein can lay the foundation for the development of later virus vaccines and diagnostic kits.

Description

technical field [0001] The invention belongs to the technical field of viruses, and in particular relates to a truncated mutant of non-structural protein NS1 of Japanese encephalitis virus, its coding gene and application. Background technique [0002] The disease caused by Japanese Encephalitis Virus (JEV) is called Japanese Encephalitis (abbreviated as Japanese Encephalitis). Japanese Encephalitis is a mosquito-borne zoonotic disease that seriously threatens the health of humans and animals. Pigs are the most important storage and multiplication hosts of JEV in nature. JEV infection can cause encephalitis in piglets and reproductive disorders in breeding pigs, causing huge economic losses to the pig industry. my country is a high-incidence area of ​​JE epidemic, which is related to the vast territory of our country, the widespread pig breeding in various places, the close relationship between humans and pigs, the large area of ​​rice planting, and the widespread distributi...

Claims

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Application Information

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Patent Type & Authority Patents(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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