Feline coronavirus recombinant antigen, its genetic engineering subunit vaccine and application

A feline coronavirus and recombinant antigen technology, applied in genetic engineering, virus antigen components, viruses, etc., can solve the problems of poor prevention and treatment of FcoV infection, exacerbation of disease, and enhancement of FIPV infection, etc., to achieve vaccine production cost reduction, immunogen strong, immunogenicity-enhancing effects

Active Publication Date: 2022-03-08
苏州沃美生物有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Some conventional vaccines are not effective in the prevention and treatment of FCoV infection, mainly because of the ADE phenomenon of FIPV, and the FCoV antibodies previously present in the host body will enhance the infection of FCoV when fighting against surface proteins, resulting in aggravation of the disease

Method used

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  • Feline coronavirus recombinant antigen, its genetic engineering subunit vaccine and application
  • Feline coronavirus recombinant antigen, its genetic engineering subunit vaccine and application
  • Feline coronavirus recombinant antigen, its genetic engineering subunit vaccine and application

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preparation example Construction

[0053] For example, in a specific embodiment of the embodiments of the present invention, a method for preparing a feline coronavirus genetically engineered subunit vaccine may specifically include:

[0054] (1) Preparation for encoding the aforementioned recombinant protein (S RBD -dimer protein) nucleic acid molecules;

[0055] (2) respectively cloning the nucleic acid molecule encoding the recombinant protein prepared in step (1) into a shuttle vector to obtain a recombinant shuttle vector containing the gene of interest;

[0056] (3) transform the recombinant shuttle vector obtained in step (2) into DH10Bac bacteria, select the recombinant bacteria, extract the genome and transfect Sf9 cells (or other aforementioned insect cells), to obtain recombinant baculovirus;

[0057] (4) cultivating the Sf9 cells (or other aforementioned insect cells) and then recombinantly expressing and producing recombinant proteins;

[0058] (5) adding the obtained recombinant protein into an ...

Embodiment 1

[0063] Example 1 transfer vector pF-S RBD - dimer construction and identification

[0064] 1. S RBD -dimer gene amplification and purification

[0065] Codon-optimized S was synthesized at Nanjing GenScript Biotechnology Co., Ltd. RBD - dimer gene (SEQ IDNO: 1) and cloned on the pUC17 vector, obtain pUC-S RBD -dimer plasmid vector. pUC-S RBD -dimer plasmid as template, S RBD -dimer-F, S RBD -dimer-R was used as upstream and downstream primers for PCR amplification (S RBD -dimer-F, S RBD The gene sequence of -dimer-R is shown in SEQ ID NO: 3, 4), and the amplification system is shown in Table 1.

[0066] Table 1 S RBD -dimer gene amplification system

[0067]

[0068] The reaction conditions were: 95°C pre-denaturation for 5 minutes; 94°C denaturation for 45 seconds, 54°C annealing for 45 seconds, 72°C extension for 1 minute, 35 cycles; 72°C extension for 10 minutes.

[0069] Perform gel electrophoresis on the PCR product to verify the size of the target gene, su...

Embodiment 2

[0085] Embodiment 2 recombinant baculovirus genome Bac-S RBD -dimer build

[0086] 1. Transformation of DH10Bac bacteria

[0087] Take 1 μl of pF-S obtained in Example 1 RBD Add -dimer plasmid to 100 μl of DH10Bac competent cells and mix well, ice bath for 30 minutes, heat shock in 42°C water bath for 90 seconds, ice bath for 2 minutes, add 900 μl LB liquid medium without Amp, and incubate at 37°C for 5 hours. Afterwards, 100 μl of the bacterial solution was diluted 81 times, and then 100 μl of the diluted bacterial solution was applied to LB solid medium containing gentamicin, kanamycin, tetracycline, X-gal and IPTG, and incubated at 37°C for 48 hours.

[0088] 2. Picking Single Clones

[0089] Use an inoculation needle to pick large white colonies, then streak on LB solid medium containing gentamicin, kanamycin, tetracycline, X-gal, and IPTG, culture at 37°C for 48 hours, and then pick single colonies Inoculate the LB liquid medium containing gentamicin, kanamycin, and tet...

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Abstract

The invention discloses a feline coronavirus recombinant antigen, its genetic engineering subunit vaccine and application thereof. The feline coronavirus recombinant antigen includes a recombinant protein having the sequence shown in SEQ ID NO: 2 or its added or truncated sequence. The vaccine comprises the recombinant protein and a pharmaceutically acceptable carrier. The feline coronavirus recombinant antigen provided by the invention has high immunogenicity, can produce high-concentration neutralizing antibodies, and will not produce ADE effect. At the same time, the present invention expresses the feline coronavirus recombinant antigen by adopting the baculovirus insect cell expression system and using the method of suspension culture Sf9 cells, not only the expression level is high, the protein immunogenicity is good, but also the vaccine prepared thereby It also has the advantages of easy quality control, batch-to-batch stability, and low production cost.

Description

technical field [0001] The invention relates to a genetic engineering vaccine, in particular to a feline coronavirus recombinant antigen, its genetic engineering subunit vaccine and its application, and belongs to the technical field of animal immune medicines. Background technique [0002] Feline Coronavirus (FCoV) is a common pathogen in felines, mainly including feline enteric coronavirus (FECV) and feline infectious peritonitis virus (Feline Infectious Peritonitis Virus, FIPV). Among them, the infection of FECV is limited to the intestinal tract and is highly contagious. It can cause symptoms such as body temperature rise, loss of appetite and even dehydration in cats, and the fatality rate is low. FIPV is a highly virulent mutant strain of FECV, which has acquired the ability to replicate in macrophages, which can escape the restriction of the intestine, thereby causing more organs to be infected. The pathogenic mechanism of FIPV mainly depends on the antibody-dependen...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/165C12N15/50C12N15/866C12N5/10A61K39/215A61P31/14G01N33/569
CPCC07K14/005C12N15/86A61K39/12A61P31/14G01N33/56983C12N2770/20022C12N2770/20034C12N2710/14043G01N2333/165
Inventor 方鹏飞孔迪曹文龙滕小锘张大鹤
Owner 苏州沃美生物有限公司
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