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Zika virus antigen and application thereof

A Zika virus and antigen technology, applied to Zika virus antigen and its application field, can solve the problem of inability to pass, and achieve the effects of strong specificity, good immunogenicity and high sensitivity

Active Publication Date: 2017-03-22
德诺杰亿(北京)生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

It causes a condition similar to a mild form of dengue fever, and there is currently no medicine or vaccine to prevent it

Method used

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  • Zika virus antigen and application thereof
  • Zika virus antigen and application thereof
  • Zika virus antigen and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Preparation and purification of Zika virus antigenic protein

[0029] Digest the nucleotide sequence shown in SEQ ID NO.2 with restriction endonucleases Nde I and Xho I respectively, collect the target fragment and connect it with the pET30a vector treated with restriction endonuclease to construct a recombinant expression plasmid pZE400; E protein expression, SDS-PAGE electrophoresis and image analysis system to determine the expression and yield.

[0030] 1. Transform the rebuilt plasmid pZE400 into BL21(de3) E. coli strain, smear the plate, pick a single clone in a medium test tube of 5ml LB medium (A+, 50mg / ml), and culture overnight at 37°C and 240rpm.

[0031] 2. Inoculate the overnight cultured strain into 300ml of LB medium (A+, 50mg / ml), and continue to cultivate at 37°C and 240rpm. When the OD600 of the bacterial solution is about 1, add IPTG with a final concentration of 1mm for induction for 3 hours.

[0032] 3 Centrifuge at 4000rpm, collect the b...

Embodiment 2

[0037] Example 2 The immune effect of Zika virus antigenic protein

[0038] The immune adjuvant uses aluminum hydroxide with a concentration of 500ug / ml, and the antigen concentration of Zika virus antigen protein is adjusted to 100ug / ml. After the two are mixed in equal proportions, rabbits are immunized and injected intramuscularly, each with 1ml. The immunization procedure is the first Three doses of immunization on day 0, day 10, and day 20 to detect Zika virus neutralizing antibody titer in rabbit serum, the experimental steps are as follows:

[0039]1. Culture Vero cells in a cell culture flask. When the number of cells in the culture flask is sufficient, digest all the cells with 0.25% trypsin, inoculate them on a 96-well plate and continue culturing. Use DMEM containing 10% fetal bovine serum and 1% antibiotics as Basic culture medium, keep the number of seeded cells in each well about 5-8×10 3 When inoculating cells, count them according to the following method:

[...

Embodiment 3

[0047] The IgM / IgG ELISA method and kit of embodiment 3 Zika virus antigenic protein

[0048] When detecting IgM, the method is to pre-coat the anti-human IgM antibody (anti-human μ chain) on the microtiter plate, then add the serum of the infected person to react and rinse, and then the Zika virus antigen protein labeled with horseradish peroxidase Add the microtiter plate to react and rinse, and then add horseradish peroxidase substrate for color development. The general detection process is as follows: Anti-human IgM antibody (anti-human μ chain) is diluted to 2 μg / ml with 0.05M carbonate buffer (pH9.6), coated with a microtiter plate (100 μl per well) and placed at 2°C-8 ℃ overnight. Discard the coating solution, add blocking solution (300 μl per well), and block for 1 hour at 37°C. Discard the blocking solution, add negative control substances N1, N2, and the sample to be tested (diluted to 20 times), respectively, 100 μl per well, and react at 37°C for 30 minutes. The...

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Abstract

The invention provides a Zika virus antigen and application thereof, and belongs to the technical field of protein engineering. DNA sequences corresponding to a protein sequence determined through DNA analysis and protein structure analysis are synthesized, Nde I and Xho I cleavage sites are introduced, a commercialized pET30a plasmid serves as a basic carrier, pZE400 is built, the protein sequence is as shown in SEQ ID NO.1, and the DNA sequences are as shown in SEQ ID NO.2. A protein C end is labeled with a protein band HIS expressed by the expression carrier and Zika virus protein is obtained through affinity chromatography purifying and protein renaturation. The protein is good in immunogenicity and high in specificity, can be used for preparing a Zika virus detection test strip or kit, can also be used for preparing Zika virus vaccines, and is remarkable in market value.

Description

technical field [0001] The present invention relates to the field of virus immunity, in particular to Zika virus antigen and its application. Background technique [0002] Zika virus (Zika Virus, ZIKV) belongs to the flavivirus genus of the Flaviviridae family. It is transmitted by the Aedes aegypti mosquito and can cause Zika virus infection (also known as Zika fever) in people bitten by mosquitoes. The virus was first isolated from macaques in Uganda's Zika forest in 1947, hence the name. According to the genotype, it is divided into two types: Asian type and African type, which have been found in Central Africa, Southeast Asia and India. Since 2015, the Zika virus epidemic has spread rapidly in Central and South America. Among them, more than 4,100 cases of microcephaly in Brazil were suspected to be related to Zika virus. Zika virus is closely related to dengue fever, yellow fever, Japanese encephalitis and West Nile virus. It causes a condition similar to a mild form...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/18C12N15/40C12N15/70C12N1/21G01N33/68G01N33/569A61K39/12A61P31/14
CPCA61K39/12A61K2039/55505C07K14/005C12N2770/24122C12N2770/24134G01N33/56983G01N33/6854G01N2469/20Y02A50/30
Inventor 曹健荣林小靖
Owner 德诺杰亿(北京)生物科技有限公司
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