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Novel porcine rotavirus vaccine VP6 antigen

A porcine rotavirus and antigen technology, applied in the field of porcine rotavirus VP6 gene, can solve the problem that vaccines cannot effectively prevent rotavirus

Inactive Publication Date: 2018-02-23
青岛宏昊生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, in the research of rotavirus recombinant antigen subunit vaccines currently used, due to the diversity of rotavirus genomes, the prepared vaccines cannot effectively prevent rotaviruses with different genetic backgrounds.

Method used

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  • Novel porcine rotavirus vaccine VP6 antigen
  • Novel porcine rotavirus vaccine VP6 antigen
  • Novel porcine rotavirus vaccine VP6 antigen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1: Screening and sequence analysis of VP6 gene

[0021] In recent years, the gene sequence analysis of porcine rotavirus (RV) shows that there are differences between the genome sequences of different strains, and there are obvious "drift" and "transformation" phenomena at the same time, resulting in the existence of multiple serotypes of RV. One reason for this phenomenon is that when two or more RV viruses invade a host cell at the same time, the gene segments interact, and exchange, crossover or rearrangement occur during transcription and replication, causing virus mutations. Thus, segmental rearrangements among different strains and mutations within the genome lead to the generation of mutant strains. Mutant strains have a certain probability of reducing the immune effect of existing vaccines or having no effect at all. The serum cross-neutralization test can be used to detect whether the existing vaccine antiserum can effectively neutralize the screened m...

Embodiment 2

[0075] Example 2: Expression of VP6 antigen

[0076] 1. Cloning of target gene and construction of recombinant expression plasmid

[0077] Add 10 μl of artificially synthesized plasmid DNA to E.coli Top10 and mix in ice bath for 30 minutes, then heat shock at 42°C for 90 seconds, and then immediately ice bath for 2‐3 minutes. Then spread evenly on LB plates containing ampicillin (100 μg / ml) and culture overnight at 37°C. Pick the positive colonies and shake the bacteria, extract the plasmid, and identify the correct recombinant plasmid by PCR and double enzyme digestion (NcoI / XhoI), and send it to Sangon Bioengineering (Shanghai) Co., Ltd. for sequencing. The recombinant cloning plasmid and expression vector pGEX-4T-1 were digested with NcoI and XhoI respectively, the gene fragment and the pGEX-4T-1 vector fragment were recovered, ligated overnight at 16°C with T4 DNAse, and the ligated product was transformed into Escherichia coli Top 10 In the process, positive colonies we...

Embodiment 3

[0080] Example 3: Neutralizing effect of VP6 antigen obtained antibody serum on virus strains

[0081] According to the method described in Example 2, the VP6 recombinant protein (amino acid sequence is SEQ ID NO: 3) numbered as Sequence ID: AFD33516.1 in NCBI is recombinantly expressed, and the amino acid sequence prepared in Example 2 is SEQ ID NO: 2. The VP6 antigen protein was used as an immune antigen to immunize mice to prepare antibody serum, and the serum was subjected to neutralization experiments to detect immune performance.

[0082] The purified recombinant protein was absolutely quantified by spectrophotometry according to the following steps:

[0083] (1) Add 0.15ml of PBS for protein measurement and 2.85ml of Coomassie Brilliant Blue staining solution for protein quantification in the cuvette as a control, and perform zero adjustment;

[0084] (2) Add 0.14ml of PBS for the protein to be tested, 0.01ml of the protein solution to be tested and 2.85ml of Coomassie...

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Abstract

The present invention provides a porcine rotavirus VP6 antigen gene, the amino acid sequence of which is SEQ ID NO: 2; a nucleotide fragment encoding the above antigen, a sequence of which is SEQ ID NO: 1. The invention obtains a novel porcine rotavirus VP6 gene through screening, and constructs an Escherichia coli BL21 (DE3) host bacterium capable of expressing porcine rotavirus VP6 gene 1 by using the pET28a(+) expression vector. After SDS‑PAGE analysis, the recombinant target protein was expressed. The recombinant protein is purified to prepare a genetically engineered subunit vaccine, which has a significant immune effect on porcine rotavirus.

Description

technical field [0001] The invention belongs to the technical field of antigen preparation, and in particular relates to a novel antigen of a porcine rotavirus vaccine, that is, a porcine rotavirus VP6 gene with a mutated amino acid sequence. Background technique [0002] Porcine rotavirus (RV) causes pig diarrhea and piglet death, which brings huge economic losses to farmers. The application of vaccine immunization has become an important way to reduce the related morbidity, mortality and economic burden. Animal farms such as pig farms are the main foci of the disease. At present, attenuated vaccines are mainly used clinically for epidemic prevention, but due to the polymorphism of rotavirus; and the gene recombination between attenuated vaccines and wild strains, causing back mutation; long-term detoxification of farmed animals, polluting water sources and food; The prevention and control of the disease poses great difficulties. In recent years, foreign countries have c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/14C07K16/10C12N15/46C12N15/70A61K39/15A61P31/14
CPCC07K14/005A61K39/12C07K16/10C12N2720/12322C12N2720/12334
Inventor 陈艺燕周佩佩
Owner 青岛宏昊生物科技有限公司
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