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Type ii grass carp reovirus vp4-ns38 fusion protein gene, expression vector, bacterial strain and application thereof

A technology of gcrvvp4-ns38-cotc and grass carp hemorrhagic disease, applied in the field of genetic engineering, can solve the problems of low immune protection rate, low immune protection rate and low protection rate of bacillus oral vaccine

Active Publication Date: 2022-03-15
PEARL RIVER FISHERY RES INST CHINESE ACAD OF FISHERY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] However, compared with traditional injection immunization, the immune protection rate of bacillus oral vaccine is low, which limits its application effect
Oral immunization of grass carp with recombinant Bacillus expressing GCRV VP4 protein using Bacillus subtilis as a carrier can effectively stimulate fish to produce immune protection, but compared with traditional attenuated vaccine injection immunization, the immune protection rate of recombinant Bacillus GCRV VP4 protein is lower Low, the immune protection effect is not ideal; GCRV NS38 protein orally immunizes grass carp, although it cannot stimulate grass carp to produce a specific immune response, it can significantly improve the level of non-specific immunity of grass carp
In addition, the results of the challenge protection experiment showed that the expression of GCRV VP4 protein and GCRV NS38 protein can stimulate the fish to produce a certain immune protection effect, but the two relative protection rates are relatively low.

Method used

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  • Type ii grass carp reovirus vp4-ns38 fusion protein gene, expression vector, bacterial strain and application thereof
  • Type ii grass carp reovirus vp4-ns38 fusion protein gene, expression vector, bacterial strain and application thereof
  • Type ii grass carp reovirus vp4-ns38 fusion protein gene, expression vector, bacterial strain and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Embodiment 1 Design the gene sequence of fusion protein

[0053] In this example, a fusion protein was prepared, which contained genotype II grass carp reovirus VP4 protein and NS38 protein. The nucleotide sequence encoding the gene type II grass carp reovirus VP4 protein corresponds to that shown in SEQ ID NO.1, specifically:

[0054] TTTGGCAATATTCGCAACATTACCGACTTCTCAATGTCTGCAATTTGGGAACCAGAGACAGTCAGCGCGGCAGGCAATTACTATCTATGGCCGACCGTAATCGGTGATGCATCAATGACATCAGATTGGGGGACAATTAGCACATCCCTAGCTAATGGCAGACTCCGTGTCGCACCTCTGGACCTGACTCATGCACTCCACAAAGGCAATGTCGTGGAGCCGATCGTACCATCTGATGTGCTAGGTAATGCCTCACCAGAGGAAATGCGTTCCGCGTTGCCAGCAGATGTGCTGACAGCTTTCAAAGCCAAGCTCACAACAGTGGCTTCCGTAGTCGGCCGTGCCTTAAATCCCAACGACAGTGCGCATGCACCATCATCCGGCACCGTCCTCGGCCCGCTTGCAATCGAAAACAAGGCCCAATCAAAACCTAAACCCGTATCAGATCTGTGGATAGCCGCTYGTCGTGGTGTGAATCTATTCGTAGCCTCTCCAAGCGCGAGTCTGCAAACAGGTGTGCCGGTCATGGGGGACAGCAGCGTGTTGACAAGCTTGACGGGTGGTGCAACTACCGCGTTGAATACTGGTGACATGGGTACCCCAAGCTTGGAGGCCACAGCGAAACGGGCAGCTAGAGTTGCTGGAGG...

Embodiment 2

[0063] Example 2 Construction of Expression Recombinant Vector PEB03-CotC-GCRV VP4-NS38

[0064] This example prepares an expression vector. Connect the GCRV VP4-NS38 gene fragment to the Bacillus subtilis plasmid vector PEB03 to obtain the recombinant vector PEB03-GCRV VP4-NS38, extract the Bacillus subtilis WB600 genomic DNA, and extract the CotC target fragment from the Bacillus subtilis genomic DNA with primers P5 and P6 After PCR amplification, restriction enzyme sites: Sal I and Bam H I were introduced at the same time, and the amplified fragment was connected to the recombinant vector PEB03-GCRV VP4-NS38 to construct PEB03-CotC that fused and expressed CotC protein and GCRV VP4-NS38 protein - GCRV VP4-NS38 recombinant vector.

[0065] The amino acid sequence of the GCRV VP4 protein is shown in SEQ ID NO.5:

[0066] FGNIRNITDFSMSAIWEPETVSAAGNYYLWPTVIGDASMTSDWGTISTSLANGRLRVAPLDLTHALHKGNVVEPIVPSDVLGNASPEEMRSALPADVLTAFKAKLTTVASVVGRALNPNDSAHAPSSGTVLGPLAIENKAQSKPKPVSDLWIAA*...

Embodiment 3

[0074] Embodiment 3 Transformation of recombinant expression vector PEB03-CotC-VP4-NS38

[0075] In this example, the constructed recombinant plasmid PEB03-CotC-VP4-NS38 was electrotransformed into Bacillus subtilis WB600, and the specific steps were as follows:

[0076] 1) Inoculate WB600 in 3ml LB medium, culture overnight at 37°C, 250rpm;

[0077] 2) Transfer 2.6ml of bacterial liquid into 40ml of electroporation A medium, shake at 37°C and 250rpm until the OD value is 0.8-0.9;

[0078] 3) Bacterial solution in an ice-water bath for 10 minutes; centrifuge at 5000g at 4°C for 5 minutes;

[0079] 4) Use 50ml of pre-cooled electroporation B medium to blow the suspended bacteria; centrifuge at 5000g, 4°C for 5min; repeat this step 4 times;

[0080] 5) Suspend the bacteria in 1ml electroporation B medium, aliquot into 10 tubes, and aliquot 100μl in each EP tube;

[0081] 6) Add 5 μl (about 1 μg) of recombinant plasmid PEB03-CotC-VP4 to 100 μl WB600 competent cells, and let st...

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Abstract

The invention discloses a type II grass carp reovirus VP4-NS38 fusion protein gene, expression vector, bacterial strain and application thereof. In the present invention, the VP4 and NS38 proteins capable of causing grass carp to produce different immune responses are connected with a flexible linker by way of fusion PCR to construct a recombinant bacillus capable of expressing the GCRVVP4-NS38 protein in the spores of Bacillus subtilis. Oral immunization of grass carp with the recombinant Bacillus can stimulate fish to produce mucous membrane antibodies and serum antibodies with neutralizing activity, and can induce grass carp to produce humoral and cellular immune responses; inflammatory response in the process. It has good safety, does not have a significant impact on the growth of grass carp, and does not cause pathological damage to the tissues and organs of grass carp, and can be used in the research of oral vaccines for grass carp hemorrhagic disease.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to type II grass carp reovirus VP4-NS38 fusion protein gene, carrier, bacterial strain and application thereof. Background technique [0002] Grass carp is the most important freshwater aquaculture fish in my country. According to statistics, the output of aquaculture grass carp in my country reached 5.3456 million tons in 2017. However, grass carp hemorrhagic disease caused by grass carp reovirus (GCRV) can cause diseased grass carp Congestion and hemorrhage symptoms of various organs and tissues in different degrees can lead to the death of the fish in severe cases, and the fatality rate is as high as 85%. At present, grass carp hemorrhagic disease is widely prevalent in my country's Hubei, Hunan, Guangdong, Guangxi, Jiangsu, Zhejiang, Anhui, Fujian, Shanghai, Sichuan and other grass carp main breeding areas. huge economic losses. At present, there is no specific and...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/75C12N1/21A61K39/15A61P31/14C12R1/125
CPCC07K14/005C12N15/75A61K39/12A61P31/14C12N2720/12022C12N2720/12034C07K2319/00A61K2039/523A61K2039/575
Inventor 尹纪元王庆王英英李莹莹吴斯宇石存斌张德锋
Owner PEARL RIVER FISHERY RES INST CHINESE ACAD OF FISHERY SCI