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Porcine rotavirus VP6 and VP7 combined vaccine

A vaccine adjuvant and subunit vaccine technology, applied in the field of vaccine preparation, can solve the problems of high porcine rotavirus infection rate and insufficient research and development of porcine rotavirus vaccine

Inactive Publication Date: 2018-04-06
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

[0004] Moreover, the research and development of porcine rotavirus vaccine in my country is not enough, and the infection rate of porcine rotavirus in China is very high, so the domestic research and development process of porcine rotavirus needs to be accelerated

Method used

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  • Porcine rotavirus VP6 and VP7 combined vaccine
  • Porcine rotavirus VP6 and VP7 combined vaccine
  • Porcine rotavirus VP6 and VP7 combined vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1: Screening and sequence analysis of VP6 gene

[0024] 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

[0079] Example 2: Expression of VP6 antigen

[0080] 1. Cloning of target gene and construction of recombinant expression plasmid

[0081] 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

[0084] Example 3: Neutralizing effect of VP6 antigen obtained antibody serum on virus strains

[0085] 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.

[0086] The purified recombinant protein was absolutely quantified by spectrophotometry according to the following steps:

[0087] (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;

[0088] (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 invention provides a porcine rotavirus VP6 and VP7 combined vaccine, which comprises vaccine antigen proteins and a vaccine adjuvant. The antigen proteins are a VP6 protein having an amino sequence represented by SEQ ID No.2 and a VP7 protein having an amino sequence represented by SEQ ID No.5. On the basis that a novel VP6 antigen gene is obtained through screening, the VP6 antigen protein isrecombinant expressed, and the expressed VP6 antigen protein and existing VP7 protein are used to produce a gene engineering subunit vaccine. The gene engineering subunit vaccine has a good immune effect on conventional porcine rotavirus and newly screened strains of rotavirus.

Description

technical field [0001] The invention belongs to the technical field of vaccine preparation, in particular to a porcine rotavirus VP6 and VP7 combined vaccine. 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 carried out in-depth research on new rotavirus vaccines and made great progress. Amo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/15A61K39/295A61K39/39A61P31/14C07K14/14C12N15/46
Inventor 陈艺燕周佩佩
Owner 青岛宏昊生物科技有限公司
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