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Feline herpes virus type I gB-gD recombinant protein and preparation method and application thereof

A feline herpes virus and recombinant protein technology, applied in biochemical equipment and methods, viruses, viral peptides, etc., can solve problems such as difficult promotion and time-consuming, and achieve the effects of improving sensitivity, reducing cross-reaction, and pollution-free training

Pending Publication Date: 2021-03-09
杭州爱谨生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although virus isolation is the most reliable detection method, it is time-consuming and generally not used for routine diagnosis of FHV-1 infection
Immunofluorescence, enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR) and other methods 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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  • Feline herpes virus type I gB-gD recombinant protein and preparation method and application thereof
  • Feline herpes virus type I gB-gD recombinant protein and preparation method and application thereof
  • Feline herpes virus type I gB-gD recombinant protein and preparation method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] The construction of embodiment 1 feline herpesvirus type I gB-gD fusion protein gene expression vector

[0064] Feline herpesvirus type I gB gene is designed according to the protein sequence of NCBI Gene bank: YP_003331552.2. According to the hydrophilicity and hydrophobicity of the protein ( https: / / web.expasy.org / protscale / ) analysis, the gB (1-100aa) sequence was selected for fusion in the area predicted to have a higher hydrophilic content. The gD gene was designed according to the protein sequence of NCBI Gene bank: YP_003331589.1. According to the hydrophilicity and hydrophobicity of the protein ( https: / / web.expasy.org / protscale / ) analysis, the gD (275-374aa) sequence was selected for fusion in the predicted higher hydrophilic content region.

[0065] The amino acid sequence of the gB-gD recombinant protein fusion protein is as follows (SEQ ID NO.1):

[0066] MSTRGDLGKRRRGSRWQGHSGYFRQRCFFPSLLGIAATGSRHGNGSSGLTRLARYVSFIWIVLFLVGPRPVEGQSGSTSEQPRRTVATPEVGG...

Embodiment 2

[0071] Embodiment 2 Expression of feline herpesvirus type I gB-gD fusion protein

[0072] Feline herpesvirus type I gB-gD 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

[0073] Embodiment 3 Purification and renaturation of feline herpesvirus type I gB-gD fusion protein

[0074] Use 50mL loading buffer Binding Buffer (50mM Tris, 0.2M Nacl, pH8.0) 50mL to crush the bacteria; then ultrasonically break, the condition is 600w, 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.

[0075] 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 taken out, f...

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Abstract

The invention discloses a feline herpes virus type I gB-gD recombinant protein, and belongs to the field of animal virus antibody detection. The recombinant protein comprises an amino acid sequence asshown in SEQ ID NO.1 or consists of an amino acid sequence as shown in SEQ ID NO.1. The invention further discloses a gene of the recombinant protein. The gene comprises a vector containing the gene,a host cell. The invention also discloses a preparation method of the recombinant protein and application of the recombinant protein in detection of feline herpes virus type I antibody. The recombinant protein is used for detecting the feline herpes virus type I antibody, is convenient and rapid, has high sensitivity, does not have cross reaction with other pathogens, has high 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 herpesvirus type I gB-gD recombinant protein and a preparation method and application thereof. Background technique [0002] Feline herpesvirus type 1 (Feline herpesvirus 1, FHV-1), also known as feline rhinotracheitis virus, belongs to the α-herpesviridae family. Respiratory diseases. The virus mainly affects young cats and spreads through direct contact, with a morbidity rate of up to 100% and a mortality rate of up to 50%. The disease was first discovered in the United States, and then found and spread in Canada, the United Kingdom and other places. At present, the case has been reported many times in my country, and the virus has been isolated. [0003] Cats with recessive FHV-1 infection and recovery after infection can carry and shed the virus for a long time and become the source of infection. Like other herpes viruses, FHV-1 can be do...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/70C12N1/21G01N33/569G01N33/558G01N33/543C12R1/19
CPCG01N33/56994G01N33/558G01N2333/03G01N2469/20G01N33/54387G01N33/532G01N33/587Y02A50/30
Inventor 李晓光何坚锋王哲侃
Owner 杭州爱谨生物科技有限公司
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