Method for rapid preparation of epidemic and infectious bronchitis vaccine

A bronchitis and infectious technology, applied in the field of rapid preparation of epidemic infectious bronchitis vaccine, can solve the problems of time-consuming and labor-intensive separation and purification of viruses, contamination of exogenous viruses, long production cycle, etc., and achieve high protection effect and satisfy Effect of large-scale application and small damage to cilia

Active Publication Date: 2020-01-21
SOUTH CHINA AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The invention overcomes the time-consuming and labor-intensive separation and purification of viruses during the development of traditional attenuated vaccines, the uncertainty of continuous passage of attenuated viruses, and the return of virulence in field applications, etc., and avoids high costs, long production cycles, and possible pollution. In view of the disadvantages of exogenous viruses, etc., a method for the rapid preparation of recombinant virus vaccines with strong timeliness and high rescue efficiency for epidemic infectious bronchitis is provided. The method is based on the infectious clone of infectious bronchitis virus H120 vaccine strain It is a skeleton carrier, and then the antigen gene in the skeleton carrier is replaced with the target antigen gene of the epidemic strain of infectious bronchitis, so as to obtain the method of recombinant bronchitis virus, the target antigen gene is S gene or S1 gene, and the S The gene is chimerized with the S gene fragments of infectious bronchitis epidemic strains of different serotypes / genotypes, and the signal peptide region of the original S1 gene in the backbone vector needs to be retained during the replacement

Method used

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  • Method for rapid preparation of epidemic and infectious bronchitis vaccine
  • Method for rapid preparation of epidemic and infectious bronchitis vaccine
  • Method for rapid preparation of epidemic and infectious bronchitis vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] In this embodiment, a test is carried out in the substitution mode S1+N.

[0047] 1) PCR amplification of the screening marker gene with homology arms to the target replacement gene (SI and N genes on GL15)

[0048] Using the plasmid pUC57-amp-ccdB as a template, the primers ΔS1z-ccdB-F / R, ΔNz-ccdB-F / R were used to PCR amplify the selection marker genes ΔS1z / ccdB-amp, ΔNz / ccdB-amp containing homology arms . .

[0049] Primer ΔS1z-ccdB-F:

[0050]

[0051] Primer ΔS1z-ccdB-F-R:

[0052]

[0053] Primer ΔNz-ccdB-F:

[0054]

[0055] Primer ΔNz-ccdB-R:

[0056]

[0057] Refer to Table 1 for the PCR amplification system. Take 5uL of the PCR product and observe the amplification result by 1% agarose gel electrophoresis. The remaining PCR product is recovered and purified with a PCR product recovery kit. The size of the recovered product is about 1.5Kb by 1% agarose gel electrophoresis, which is consistent with the expectation , and use the gel recovery kit...

Embodiment 2

[0106] This example is the replacement of the chimeric GL15-GZ14 S gene

[0107] 1) Preparation of target genes GL15-S1 and GZ14-S2: using plasmids pRK5-GL15 S and pRK5-GZ14 S as templates respectively, using primers GL15-S1-F / R and GZ14-S2-F / R to amplify the homologous Arm GL15-S1 and GZ14-S2. The high-fidelity DNA polymerase PrimeSTAR Max DNA Polymerase (Takara) was used for PCR amplification. Refer to Table 1 to prepare the system. The PCR amplification parameters were 98°C for 2min; 98°C for 10s; 55°C for 5s; 72°C for 20s; amplification for 30 cycles . The PCR product was subjected to 1% agarose gel electrophoresis, recovered and purified with a gel extraction kit, and the nucleic acid concentration was determined, and a small amount was sent to BGI for sequencing.

[0108] Primer GL15-S1-F:

[0109]

[0110] Primer GL15-S1-R:

[0111]

[0112] Primer GZ14-S2-F:

[0113]

[0114] Primer GZ14-S2-R:

[0115]

[0116] 2) Preparation of screening marker genes...

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Abstract

The invention provides a method for rapid preparation of epidemic and infectious bronchitis vaccine. The method takes infectious clone of an infectious bronchitis virus H120 vaccine strain as a skeleton carrier, then replaces an antigen gene in the skeleton carrier with a targeted antigen gene of an infectious bronchitis epidemic virus strain, so as to obtain the method of recombining bronchitis virus, and the targeted antigen gene is prepared from S gene fragments of infectious bronchitis epidemic strains with different serotypes / genotypes in an gomphosis mode; replacement can also be the simultaneous replacement of the targeted antigen gene and a N gene, and a signal peptide region of an original S1 gene in the skeleton carrier needs to be preserved during the replacement. The method forthe rapid preparation of the epidemic and infectious bronchitis vaccine has the beneficial effects that an operation method is simple and easy to conduct, therepeatability is high, the generation stability is good, the frequent variation of IBV epidemic can be quickly and efficiently tackled, and the method provides a new idea for the construction of a carrier vaccine.

Description

technical field [0001] The invention belongs to the field of biotechnology, and more specifically relates to a method for rapidly preparing an epidemic infectious bronchitis vaccine. Background technique [0002] Avian infectious bronchitis (Infectious bronchitis, IB) is an acute, highly contagious disease of chickens caused by avian infectious bronchitis virus (IBV). It is characterized by respiratory symptoms, nephritis, and decreased production performance. Especially, broiler chickens have a high mortality rate after nephropathy characterized by nephropathy, and the death rate of white feather broilers can reach More than 30%, high-quality broilers can cause about 15% mortality, the weight gain and feed remuneration of diseased broilers are reduced, and mixed infections with Escherichia coli, mycoplasma, etc. cause air sacculitis and reduce the quality of broilers. Infection of IB in laying chicks will not only cause death, but also cause permanent irreversible damage t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/63
CPCC12N7/00C07K14/005C12N2770/20021C12N2770/20022C12N2770/20034A61K39/12A61K2039/5254A61K2039/575A61K2039/51A61P31/14
Inventor 谢青梅封柯宇符军邵冠明张新珩
Owner SOUTH CHINA AGRI UNIV
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