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Ankara vaccinia virus genetic engineering vaccine for pig replication and respiration complex

A genetically engineered vaccine and respiratory syndrome technology, applied in the field of porcine reproductive and respiratory syndrome vaccine, genetically engineered vaccine, can solve the problems of immune failure, high probability of virulence reversion, unsatisfactory immune effect, etc.

Inactive Publication Date: 2008-07-02
WUCHANG SHIPBUILDING IND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it has been confirmed that attenuated vaccines have potential safety hazards such as loose virus and a high probability of virulence returning to strength. Many foreign countries have banned attenuated vaccines
Although inactivated vaccines are safe, they not only require repeated vaccinations, but also have unsatisfactory immune effects, and often lead to immune failure

Method used

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  • Ankara vaccinia virus genetic engineering vaccine for pig replication and respiration complex
  • Ankara vaccinia virus genetic engineering vaccine for pig replication and respiration complex
  • Ankara vaccinia virus genetic engineering vaccine for pig replication and respiration complex

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1. Synthesis of GP5 sequence

[0039] 1) Design a pair of primers based on the GP5 sequence.

[0040] GP1: 5′-CGGGATCCGCCACCATGGGCATGTTGGGGAAATGCTTGACC-3′

[0041] GP2: 5′-GGAATTCCTAGAGACGACCCCATTGTTCCGC-3′

[0042] 2) Using GP1 and GP2 as primers, and using porcine reproductive and respiratory syndrome virus as a template to carry out PCR, the reaction system and reaction conditions for amplifying the GP5 sequence are:

[0043] The reaction system is: ddH 2 O 18.5 μL, 10× buffer 2.5 μL, dNTP (2.5 mmol / L) 2 μL, GP1 (10 μmol / L) 0.5 μL, GP2 (10 μmol / L) 0.5 μL, pfu DNA polymerase (5u / μL) 0.5 μL, PRRSV ( 50-fold dilution) 0.5 μL.

[0044]The reaction parameters are: 94°C for 2min, 94°C for 15s, 60°C for 15s, 72°C for 30s, 30 cycles, and 72°C for 3min. The amplified GP5 fragment is 630bp in length (see Figure 4).

Embodiment 2

[0045] Example 2. Optimizing the synthesis of GP5A sequences.

[0046] First, under the premise that the amino acid sequence of porcine reproductive and respiratory syndrome virus GP5 protein remains unchanged, the porcine reproductive and respiratory syndrome virus GP5 sequence is optimized by using porcine preferred codons, and the optimized sequence is synthesized by a gene synthesizer. The optimized sequence is called GP5A. Sequence optimization and synthesis of optimized sequences were entrusted to GenScript Company, and the synthesized sequences were cloned in pUC57 ( Figure three ), GP5A sequence see Figure II .

Embodiment 3

[0047] Example 3. Modified synthesis of GP5-DB sequence.

[0048] 1) Based on the optimized GP5A, design two pairs of primers respectively:

[0049] GPA1: 5' CTCGAGGTTTAAAC CCACCATGCTG-3' (the underlined parts are the restriction sites of XhoI and PmeI, respectively);

[0050] GPA2: 5′-GTTGCTGGCGTTATAAATCAACTGAATATGAGACAGGTAGAAGGGCAC-3′;

[0051] GPB1: 5'-CAGTTGATTTATAACGCCAGCAACAACAACAG-3';

[0052] GPB2: 5′- GTCGACGGCGCGCC TCACAGGCGGCCCCACG-3' (the underlined parts are the restriction sites of SalI and AscI, respectively).

[0053] Wherein the primer GPA2 replaces the epitope A in the optimized GP5 with the epitope B of the natural porcine reproductive and respiratory syndrome virus GP5 gene.

[0054] 2) PCR was performed with two pairs of primers as primers and the optimized GP5A sequence as a template. The reaction system and reaction parameters of GPA and GPB were the same, respectively:

[0055] The reaction system is: ddH 2 O 18.5μL, 10×buffer 2.5μL, dNTP (2.5m...

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Abstract

The invention discloses a porcine reproductive and respiratory syndrome-Ankara vaccinia virus genetic engineering vaccine, the preparation method is that: 1. the PCR amplification of GP5 gene is carried out from the natural porcine reproductive and respiratory syndrome virus; 2. the porcine preferred codon is used for replacing the codon in the natural GP5 gene, so as to carry out GP5 gene optimization and the synthesis of the optimized sequence GP5A; 3. the epitope A in the optimized GP5A gene is replaced by the epitope B of the GP5 gene, so as to obtain the GP5-DB gene; 4. the GP5 and GP5-DB genes are respectively cloned to the expression carrier JN-2; 5. the constructed JN-2-GP5 and the JN-2-GP5-DB plasmid are recombined with Ankara vaccinia virus, so as to obtain the porcine reproductive and respiratory syndrome GP5 and GP5-DB gene Ankara vaccinia virus recombinant vaccine. Experiments confirm that the invention has excellent immune protection in BALB / c mice and pigs.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to genetic engineering vaccines, in particular to porcine reproductive and respiratory syndrome vaccines. Background technique [0002] Porcine reproductive and respiratory syndrome virus (porcine reproductive and respiratory syndrome virus, PRRSV) is the pathogen that causes porcine reproductive and respiratory syndrome, which mainly manifests as severe reproductive impairment in pregnant sows, respiratory symptoms and high mortality in piglets. Since PRRS was first reported and discovered in the United States in 1987, it has spread to all major pig-raising countries and regions in the world, and has become one of the most serious infectious diseases that endanger the pig industry. The prevention and control of PRRS mainly rely on vaccination. At present, the commercial vaccines used for the prevention and control of PRRS mainly include attenuated vaccines and inactivated vaccines. H...

Claims

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Application Information

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IPC IPC(8): A61K48/00A61K39/285A61P31/20
Inventor 王忠德封江南孙永林胡勤勤吴娟王迁迁魏佳李超严俊
Owner WUCHANG SHIPBUILDING IND
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