Feline calicivirus VP1-VP2 recombinant protein as well as preparation method and application thereof

A VP1-VP2, feline calicivirus technology, applied in biochemical equipment and methods, viruses, viral peptides, etc., has achieved great clinical significance, wide application prospects, and improved sensitivity

Pending Publication Date: 2021-02-05
杭州亿米诺生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] However, current detection methods and tools for the detectio

Method used

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  • Feline calicivirus VP1-VP2 recombinant protein as well as preparation method and application thereof
  • Feline calicivirus VP1-VP2 recombinant protein as well as preparation method and application thereof
  • Feline calicivirus VP1-VP2 recombinant protein as well as preparation method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Embodiment 1 Contains the construction of feline calicivirus VP1-VP2 fusion protein gene expression vector

[0062] Feline calicivirus VP1 gene was designed according to the protein sequence of NCBI Gene bank: P27406.1. The VP2 gene was designed according to the protein sequence of NCBI Gene bank: ALM55430.

[0063] The amino acid sequence of the VP1-VP2 recombinant protein is as follows (SEQ ID NO.1):

[0064] ADDGSITAPEQGTMVGGVIAEPSAQMSTAADMATGKSVDSEWEAFFSFHTSVNWSTSETQGKILFKQSLGPLLNPYLEHLAKLYVAWSGSIEVRFSISGSGVFGGKLAAIVVPPGVDPVQSTSMLQYPHVLFDARQVEPVIFCLPDLRSTLYHLMSDTDTTSLVIMVYNDLINPYANDANSSGCIVTVETKPGPDFKFHLLKPPGSMLTHGSIPSDLIPKTSSLWIGNRYWSDITDFVIRPFVFQANRHFDFNQETAGWSTPRFRPISVTITEQNGAKLGIGVATDYIVPGIPDGWPDTTIPGELIPAGDYAITNGTGNDITTATGYDTADIIKNNTNFRGMYICGSLQRAWGDKKISNTAFITTATLDGDNNNKINPCNTIDQSKIVVFQDNHVGKKAQTSDDTLALLGYTGIGEQAIGSDRDRVVRISTLPETGARGGNHPIFYKNSIKLGYVIRSIDVFNSQILHTSRQLSLNHYLLPPDSFAVYRIIDSNGSWFDIGIDSDGFSFVGVSGFGKLEFPLSASYMGIQLAKIRLASNIRSPMTKLMNSILGLIDTVTNTIGKAQQIE...

Embodiment 2

[0070] Embodiment 2 contains the expression of feline calicivirus VP1-VP2 fusion protein

[0071]Transform the synthetic feline calicivirus VP1-VP2 fusion gene plasmid into Escherichia coli BL21, smear it on an LB plate containing 50 μg / mL kanamycin (Shanghai Sangong, product number: K0408), and culture overnight at 37°C , picked a single clone colony, cultured in 300mL LB medium containing the same concentration of kanamycin at 37°C until the OD600 reached about 0.6, and induced expression with IPTG (Shanghai Sangong, product number: IB0168) with a final concentration of 1mM, The induction conditions are: 25°C, 200rpm rotation speed, 4h. After induction, the culture solution was centrifuged at 4° C. at 7000 rpm for 10 min to collect the bacteria.

Embodiment 3

[0072] Embodiment 3 Purification and renaturation containing feline calicivirus VP1-VP2 fusion protein

[0073] 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 500w, ultrasonic 2s, interval 5s, a total of 100 times; finally 12000rpm, 30min, 4℃ 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.

[0074] 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 tak...

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Abstract

The invention discloses a feline calicivirus VP1-VP2 recombinant protein, and belongs to the field of animal virus antibody detection. The recombinant protein comprises an amino acid sequence shown inSEQ ID NO.1 or an amino acid sequence shown in SEQ ID NO.1. The invention further discloses a gene of the recombinant protein, a vector containing the gene, a host cell containing the gene, a preparation method of the recombinant protein and application of the recombinant protein in detection of a feline calicivirus antibody. When the recombinant protein is used for detecting the feline calicivirus, the recombinant protein is convenient, quick and high in sensitivity, has no cross reaction with other pathogens, is high in specificity, and has huge clinical significance and wide application prospect.

Description

technical field [0001] The invention belongs to the field of animal virus antibody detection, and in particular relates to a feline calicivirus VP1-VP2 recombinant protein and a preparation method and application thereof. Background technique [0002] Feline calicivirus (Feline Calicivirus, Fcv) is a common pathogen of feline animals. It can be infected through the mouth, nose and other routes. Clinically, sneezing, eye and nose bleeding, conjunctivitis, stomatitis, gingivitis, oral ulcers, pneumonia, lameness, miscarriage, chronic gastroenteritis, skin edema and ulcers can also occur in severe cases . Feline calicivirus infection is a frequently-occurring disease in cats, with high morbidity and low mortality. Symptoms caused by FCV are very similar to feline herpes virus and other infections, and can also cause many complicated atypical symptoms. It is difficult to diagnose the pathogen from clinical symptoms alone, and it needs to be confirmed in combination with labora...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/70C12N1/21G01N33/569C12R1/19
CPCC07K14/005C12N15/70G01N33/56983C12N2800/22C12N2770/16022C07K2319/00G01N2333/08
Inventor 李晓光
Owner 杭州亿米诺生物科技有限公司
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