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Recombinant FCV antigen and feline calicirus genetic engineering subunit vaccine

An antigen and gene technology, applied in genetic engineering, antiviral agents, viruses, etc., can solve the problems of VLP and wild virus morphology, stability and immunogenicity differences, and achieve excellent immunogenicity, strong immunogenicity, and structure. stable effect

Pending Publication Date: 2022-01-07
苏州世诺生物技术有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The development and commercialization of new FCV vaccines in China are almost blank. CN108371710A proposes a method for preparing feline infectious rhinoconjunctivitis and feline panleukopenia dual vaccines using baculovirus expression system expression, but this method only expresses FCV The VP1 protein of the wild virus has obvious differences in the form, stability, and immunogenicity of the VLP formed by it.

Method used

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  • Recombinant FCV antigen and feline calicirus genetic engineering subunit vaccine
  • Recombinant FCV antigen and feline calicirus genetic engineering subunit vaccine
  • Recombinant FCV antigen and feline calicirus genetic engineering subunit vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1 Construction and Identification of Transfer Vector pVL-FCV-VLP

[0054] 1. FCV-VLP gene amplification and purification

[0055] The codon-optimized FCV-VLP gene (SEQ ID NO: 1) was synthesized in Nanjing GenScript Biotechnology Co., Ltd. and cloned into the pMD19-T vector to obtain the pMD-FCV-VLP plasmid vector. With pMD-FCV-VLP plasmid as template, FCV-VLP-F, FCV-VLP-R carry out PCR amplification (the gene sequence of FCV-VLP-F, FCV-VLP-R as SEQ ID NO: 4 , 5), the amplification system is shown in Table 1.

[0056] Table 1 FCV-VLP gene amplification system

[0057]

[0058] The reaction conditions were: 94°C pre-denaturation for 5 minutes; 95°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.

[0059] Perform gel electrophoresis on the PCR product to verify the size of the target gene, such as figure 1 As shown, the target band appeared at the position of 2.9kbp, the targe...

Embodiment 2

[0076] Embodiment 2 recombinant baculovirus rBac-FCV-VLP transfection and purification

[0077] 1. Transfection

[0078] Inoculate 2 × 10 in a tissue culture dish with a diameter of 6 cm 6 Sf9 cells, the cell confluence is 50-70%. Discard the medium after the cells adhere to the wall, and add 1.0ml of transfection buffer A. Mix 0.5 μg of BD BaculoGold LinearizedBaculovirus DNA and 2 μg of pVL-FCV-VLP plasmid in a sterile EP tube for 5 minutes, add 1.0ml of transfection reagent B, and mix well. Pipette the mixture dropwise into the tissue culture dish, incubate at 27°C for 4 hours, remove the transfection complex, wash the cell culture medium three times, add 3.0ml of fresh medium, seal the culture dish with parafilm, incubate at 27°C for 4 to 5 days and harvest Supernatant, that is, co-transfection virus supernatant.

[0079] 2. Plaque Purification

[0080] Inoculate 2.0 × 10 in a tissue culture dish with a diameter of 6 cm 6 ~2.5×10 6 Sf9 cells at room temperature for ...

Embodiment 3

[0086] Example 3 SDS-PAGE detection

[0087] The F0 generation recombinant baculovirus rBac-FCV-VLP cell culture harvested in Example 2 and the cell cultures of each control group were detected by SDS-PAGE, and Sf9 cells infected with empty baculovirus were used as a negative control. The specific operation is as follows: take 40 μl of harvested cell culture, add 10 μl of 5×loading buffer, bathe in boiling water for 5 minutes, centrifuge at 12000 r / min for 1 minute, take the supernatant and carry out SDS-PAGE gel (12% concentration gel) electrophoresis, After electrophoresis, the gel was stained and decolorized to observe the target band. Such as Figure 4 As shown, the rBac-FCV-VLP cell culture showed target bands at molecular weights of about 73kDa and 12kDa, and the protein expression levels of VP1 and VP2 showed a ratio of about 10:1, which was similar to wild virus; control group 1 had a molecular weight of about 73kDa The target band appeared at the target band; the co...

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Abstract

The invention discloses a recombinant FCV antigen and a Feline calicirus genetic engineering subunit vaccine. The recombinant FCV antigen comprises two proteins with the sequences shown as SEQ ID NO: 2 and SEQ ID NO: 3 respectively. The vaccine comprises the recombinant FCV antigen and a pharmaceutically acceptable carrier. The recombinant FCV antigen provided by the invention is easy for self-assembly to form a virus-like particle (VLP) with stable structure and excellent immunogenicity. The VLP has shape, stability and immunogenicity close to those of a wild virion, and can be prepared by large-scale serum-free suspension culture in a bioreactor through an insect cell expression system along with high expression level and good protein immunotherapy. The prepared vaccine is easy in quality control, stable in batches and low in production cost.

Description

technical field [0001] The invention relates to a recombinant protein, in particular to a recombinant FCV antigen and its application, such as the application in the preparation of feline calicivirus recombinant subunit vaccine, which belongs to the technical field of animal immune medicine. Background technique [0002] Feline Calicivirus (FCV) is an important pathogen that causes oral and respiratory diseases in cats. The clinical symptoms of FCV-infected animals mainly showed rhinitis, conjunctivitis, bronchitis, pneumonia, and blisters and ulcers of oral epithelial cells including tongue. The virus is distributed worldwide, and all cats are susceptible to FCV, and kittens under one year old are most susceptible. Animals infected by the virus have a high incidence rate and low mortality rate, but FCV is often mixed with bacteria, chlamydia, mycoplasma, parasites and other feline upper respiratory viruses, resulting in increased mortality. FCV is mainly transmitted in na...

Claims

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Application Information

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IPC IPC(8): C07K14/085C12N15/41C12N7/00C12N15/866A61K39/125A61P31/14G01N33/569
CPCC07K14/005C12N15/86A61K39/125A61P31/14G01N33/56983C12N2770/32021C12N2770/32022C12N2770/32023C12N2770/32034C12N2710/14043A61K2039/552G01N2333/085Y02A50/30
Inventor 孔迪徐蓉方鹏飞曹文龙滕小锘张大鹤
Owner 苏州世诺生物技术有限公司
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