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Equine infectious anemia virus p26-gp90 recombinant protein as well as preparation method and application thereof

A p26-gp90, equine infectious anemia technology, applied in the direction of botany equipment and methods, biochemical equipment and methods, applications, etc., can solve the problems of difficult grass-roots promotion, achieve pollution-free training, short color development time, specificity strong effect

Pending Publication Date: 2021-03-19
杭州亿米诺生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the above methods such as immunofluorescent staining technology and enzyme-linked immunosorbent assay (ELISA) require the use of designated equipment, corresponding test conditions and skills, and are difficult to promote at the grassroots level

Method used

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  • Equine infectious anemia virus p26-gp90 recombinant protein as well as preparation method and application thereof
  • Equine infectious anemia virus p26-gp90 recombinant protein as well as preparation method and application thereof
  • Equine infectious anemia virus p26-gp90 recombinant protein as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1 Construction of equine infectious anemia virus p26-gp90 fusion protein gene expression vector

[0062] The p26 gene of equine infectious anemia virus was designed according to the protein sequence of NCBI Gene bank: ABE03841. The surface protein (Envelope polyprotein) gp90 gene was designed according to the protein sequence of NCBI Gene bank: AAC24024.

[0063] The amino acid sequence of the p26-gp90 recombinant protein fusion protein is as follows (SEQ ID NO.1):

[0064] EFIDGAGNRNFRPLTPRGYTTWVNTIQQHNLLNEASVNLFGILSVDCTSEEMNAFLDVVPGQAGQKQVLLDALDKIAEDWDNRHPLPNAPLVAPPQGPIPMTARFIRGLGVPRERQMEPAFDQFRQTYRQWIIEAMTEGIKVMTGKPKAQNIRQGPKEPYPEFVDRLLSQIESEGHSTEITRFLTDTLTIQNANEECRNAMRHLRPEDSLEEKMYACRDFGSTKLSKNSMAESKEARDQEMNLKEESKEEKRRNDWWKIGMFLLCLAGTTGGILWWYEGLPQQHYIGLVAIGGRLNGSGQSNAIECWGSFPGCRPFQNYFSYETNRSMHMNNNTATLLEAYHREITFIYKSSCTDSDHCQEYQCKKVDLINSSSNSVRVVENETTTEYWGFKWLECNQTENLKTILVPENEMVNINDSDTWIPKGCNETWARVKRCPIDILYGIHPIRLCVQPPFFLVQEKGIANNSRISNCGPTIFLGVLEDNKGVIRGNSTICKV...

Embodiment 2

[0069] Example 2 Expression of Equine Infectious Anemia Virus p26-gp90 Fusion Protein

[0070] The equine infectious anemia virus p26-gp90 fusion gene plasmid was transformed into Escherichia coli BL21, spread on LB plates containing 50 μg / mL kanamycin (Shanghai Sangong, product number: K0408), cultured overnight at 37°C, and picked Take a single clone colony, culture it with 300mL LB medium containing the same concentration of kanamycin at 37°C until the OD600 reaches about 0.6, and induce expression with IPTG (Shanghai Shenggong, product number: IB0168) with a final concentration of 1mM. For: 25 ℃, rotating speed 200rpm, 4h. After induction, the culture solution was centrifuged at 4° C. at 7000 rpm for 10 min to collect the bacteria.

Embodiment 3

[0071] Example 3 Purification and renaturation of equine infectious anemia virus p26-gp90 fusion protein

[0072] Use 50mL of loading buffer Binding Buffer (50mM Tris, 0.2M Nacl, pH8.0) 50mL to break up the bacteria; then ultrasonically break, the condition is 550w, ultrasonic 2s, interval 5s, a total of 100 times; finally 12000rpm, 30min, 4°C Collect the supernatant by centrifugation, and the target protein is in the supernatant. Then, it was purified by Ni column, and the target protein was eluted with Elution Buffer (50mM Tris, 0.2M Nacl, 0.5M Imidazole, pH8.0). The target protein was detected by PAGE gel electrophoresis, and the results were as follows figure 1 shown.

[0073] Depend on figure 1 It can be seen that the purified fusion protein has a high purity. The purified recombinant protein was dialyzed with a dialysis buffer (50mM Tris, 0.2M Nacl, pH8.0), and the dialysis solution was changed every 12 hours for a total of 3 times. The dialyzed protein solution was ...

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Abstract

The invention discloses an equine infectious anemia virus p26-gp90 recombinant protein, and belongs to the field of animal virus antibody detection. The recombinant protein comprises an amino acid sequence as shown in SEQ ID NO.1 or consists of the amino acid sequence as shown in SEQ ID NO.1. The invention also discloses a preparation method of the recombinant protein. The invention further discloses a gene of the recombinant protein, a vector containing the gene, a host cell, a preparation method of the recombinant protein and application of the recombinant protein in detection of an equine infectious anemia virus antibody. Detection of the equine infectious anemia virus antibody using the recombinant protein is convenient, quick and high in sensitivity, no cross reaction with other pathogens exists, the specificity is high, and the recombinant protein has a huge clinical significance and wide application prospects.

Description

technical field [0001] The invention belongs to the field of animal virus antibody detection, and in particular relates to a p26-gp90 recombinant protein of equine infectious anemia virus and its preparation method and application. Background technique [0002] Equine infectious anemia (EIA, Equine Infectious Anemia) is an infectious disease of horses, mules, and donkeys caused by the equine infectious anemia virus (Equine infectious anemia virus) in the lentiviridae subfamily of the Retroviridae family. Equine infectious anemia virus belongs to the retroviridae lentivirus genus, and is the pathogen of equine infectious anemia, an infectious disease that seriously harms equine animals. Equine infectious anemia is characterized by intermittent fever, emaciation, progressive weakness, anemia, hemorrhage, and edema; symptoms gradually decrease or disappear temporarily during the absence of fever. [0003] The agar gel immunodiffusion test was successfully developed by American...

Claims

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Application Information

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IPC IPC(8): C07K14/155C12N15/49C12N15/70C12N1/21C12P21/02G01N33/68G01N33/569G01N33/558C12R1/19
CPCG01N33/6854G01N33/56983G01N33/558G01N2333/155G01N2469/20
Inventor 李晓光何坚锋王哲侃
Owner 杭州亿米诺生物科技有限公司
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