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Recombinant akabane virus capsid protein, its preparation method and uses

A technology of nucleocapsid protein and Akabane virus, which is applied in the field of genetic engineering, can solve the problems of high technical equipment requirements, cumbersome operation, maternal antibody interference, etc., and achieve the effect of easy purification and preparation, low cost and guaranteed specificity

Inactive Publication Date: 2006-05-10
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, the diagnostic methods reported in China include microneutralization test, agar diffusion test, and PCR and ELISA diagnostic methods. higher

Method used

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  • Recombinant akabane virus capsid protein, its preparation method and uses
  • Recombinant akabane virus capsid protein, its preparation method and uses
  • Recombinant akabane virus capsid protein, its preparation method and uses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] [Example 1] The construction of recombinant AKAV nucleocapsid protein gene prokaryotic expression vector

[0042] Use RNeasy  The Mini Kit RNA extraction kit was used to extract the total RNA of AKAV, and the primers were synthesized by Saibaisheng Company to amplify the S segment gene, the expected amplification length was 840bp, the primer sequence: p1: 5-TAA CTA CGC ATT GCA ATG GC-3 (SEQ ID NO: 3); p2: 5-AGT AGT GTGCTC CAC-3 (SEQ ID NO: 4). Amplification conditions: 42°C for 1h, 94°C for 5min and then cycle, the cycle parameters are 94°C for 60s, 55°C for 30s, 72°C for 60s, after 30 cycles, 72°C for 10min, 10g / L agarose gel electrophoresis identification PCR product.

[0043] The product purified by PCR was connected to pMD18-T Vector, transformed into E.coli JM109, coated with LB solid medium containing ampicillin (100ug / ml), X-gale and IPTG, picked white single colony after culturing overnight at 37°C, Inoculate into liquid LB containing ampicillin (100ug / ml), ...

Embodiment 2

[0045] [Example 2] Induction condition optimization test

[0046] Transform the recombinant plasmid PET-N into E.coli BL21(DE3) expression host bacteria, pick a single colony from the LB plate and insert it into 3ml LB medium (all medium contains kanamycin), culture overnight at 37°C, and then Introduce 100ml 2×YT medium at 150°C and culture at 37°C, do induction condition optimization test, use uninduced bacteria as control, do different induction time, different D 600nm The value of the initial induction, different IPTG amount induction test, and finally determine the best induction conditions.

[0047] Depend on Image 6 It can be seen that different induction times and different D 600nm The value has a significant effect on the expression rate of the fusion protein, but the induction by different IPTG amounts is not obvious. OK D 600nm When the value is 0.5-0.6, IPTG is used to induce the expression of the recombinant nucleocapsid protein, and the IPTG is 0.6-1.5 mM, a...

Embodiment 3

[0048] [Example 3] Identification, purification and expression measurement of recombinant AKAV nucleocapsid protein

[0049] After the engineered bacteria were induced, they were ultrasonically lysed and centrifuged at 12,000×g for 15 minutes. The expressed proteins were detected in the supernatant. Compared with the control, there was a bright band at 27ku, which was consistent with the expected size. ProBond TM After purification by Purification System, only this band remains, which shows that it is indeed an induced fusion protein. Bovine anti-AKAV positive serum was used as the primary antibody, and horseradish peroxidase-labeled rabbit anti-bovine IgG was used as the secondary antibody. After DAB color development, the results showed that there were obvious bands at the corresponding positions, while the control did not, which indicated that the expressed The fusion protein has immunological activity ( image 3 , Figure 4 ).

[0050] The recombinant fusion protein of...

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Abstract

The invention discloses a novel recombinant Akabane virus nucleotide capsid protein, gene encoding the recombinant protein and the process for preparing the recombinant protein. The recombinant protein has the amino acid sequence represented by SEQ ID No:1, the gene encoding the recombinant Akabane virus nucleotide capsid protein has the amino acid sequence represented by SEQ ID No:2. The preparing process of the recombinant protain comprises the following steps: constructing recombinant Akabane virus nucleotide capsid protein prokaryotic expression vectors, transforming bacillus coli with the recombinant Akabane virus nucleotide capsid protein prokaryotic expression vectors, inducing recombinant nucleotide capsid protein expression with IPTG, reclaiming and purifying the expressed recombinant nucleotide capsid protein.

Description

technical field [0001] The present invention relates to a recombinant protein, in particular to a recombinant Akabane virus nucleocapsid protein, a gene encoding the recombinant Akabane virus nucleocapsid protein, a preparation method of the recombinant Akabane virus nucleocapsid protein and its application in detecting or diagnosing Akabane virus The application of the antigen belongs to the field of genetic engineering. Background technique [0002] Akabane virus, also known as Akabane virus (AKAV), is an arbovirus belonging to the Simpovirus group of the genus Kobney virus, which can cause congenital malformations in cattle, sheep, and goats. Early, stillbirth and congenital arthrogryposis and hydrocephalus anencephaly (Arthrogryposis-Hydraencephaly, AH syndrome) is characterized. The AKAV virus was first isolated from the mosquitoes Cules Tritaeniorhynchis and AedesVexans collected in the cowshed of Akabane Village, Gunma County, Japan in 1959, so it was named Akabane v...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/08C12N15/40C12N15/63G01N33/569G01N33/68
Inventor 吴东来李少英孟庆文尹训南
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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