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Porcine rotavirus vaccine, antigen for preparing vaccine and coding sequence of antigen

A porcine rotavirus and antigen technology, applied in the field of immunology, can solve the problems of ineffective prevention of rotavirus, and achieve the effects of low production cost, good immune effect and strong antigen specificity

Active Publication Date: 2020-08-25
山东信得动物疫苗有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

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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  • Porcine rotavirus vaccine, antigen for preparing vaccine and coding sequence of antigen
  • Porcine rotavirus vaccine, antigen for preparing vaccine and coding sequence of antigen
  • Porcine rotavirus vaccine, antigen for preparing vaccine and coding sequence of antigen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] The recombination of the antigen of embodiment 1 porcine rotavirus vaccine

[0026] In the present invention, by analyzing the VP6 gene of group A porcine rotavirus, the specific sequence fragments of highly hydrophilic regions, signal peptides and highly conserved regions are selected and combined to obtain a recombinant antigen with better immune effect, which completely contains The above-mentioned specific sequences ensure the high efficiency and specificity of the antigen to the greatest extent. The amino acid sequence of the antigen is shown in SEQ ID NO:1, and the nucleotide sequence encoding the antigen is shown in SEQ ID NO:2.

[0027] In order to increase soluble expression, SEQ ID NO:2 has been modified, and the sequence of the modified nucleotide fragment is shown in SEQ ID NO:3, that is, the cysteine ​​at the 93rd and 227th positions of SEQ ID NO:2 Acid codons were replaced with glycine codons.

[0028] The present invention also provides a method for pre...

Embodiment 2

[0034] The expression of embodiment 2 antigen

[0035] 1. Cloning of target gene and construction of recombinant expression plasmid

[0036] First, artificially synthesize nucleotide fragments of SEQ ID NO:6 and SEQ ID NO:7, add 10 μl of plasmid DNA containing artificially synthesized nucleotide fragments to E.coli Top10, mix in ice bath for 30 minutes, and then heat shock at 42°C for 90s , and then immediately ice-bathed for 2 to 3 minutes. Then spread evenly on LB plates containing ampicillin (100 μg / ml), and culture overnight at 37°C. Pick the positive colony and shake the bacteria, extract the plasmid, and identify it by PCR and double enzyme digestion (NcoI / XhoI). The correct recombinant plasmid is sent to Sangon Bioengineering (Shanghai) Co., Ltd. for sequencing. The recombinant cloning plasmid and the 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 ...

Embodiment 3A

[0041] Example 3 Purification, renaturation and subunit vaccine preparation of group A porcine rotavirus VP6 structural protein

[0042] Follow the steps below for purification and renaturation: (1) Take out the induced expression bacterial solution and put it in a 50ml centrifuge tube, centrifuge at 5000r / min for 10min at 4°C, and take the supernatant; (2) Add ammonium sulfate to the supernatant to 20% saturation, after fully mixing, place at 2-8°C, 12-16 hours, centrifuge at 7000r / min for 30 minutes, discard the precipitate, and collect the supernatant; (3) add ammonium sulfate to the supernatant to 50% saturation, fully After mixing, place at 2-8°C for 12-16 hours, centrifuge at 7000r / min for 30 minutes, discard the supernatant, and collect the precipitate; (4) After weighing the precipitate, add sterilized physiological saline to redissolve at a ratio of 1:10, 7000r / min Centrifuge for 30 minutes, discard the precipitate, and collect the supernatant; (5) Add ammonium sulfat...

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Abstract

The invention relates to the technical field of immunology, in particular to a porcine rotavirus vaccine, an antigen for preparing the vaccine and a coding sequence of the antigen. The invention provides the antigen with an amino acid sequence as shown in SEQ ID NO:1, and the antigen comprises a nucleotide sequence for coding the antigen. The invention further provides a method for preparing the antigen. The antigen for preparing the porcine rotavirus vaccine is relatively good in immune effect, the soluble expression quantity of the antigen in escherichia coli is obviously increased by modifying the nucleotide sequence encoding the antigen, the preparation process is simple, and the method is suitable for low-cost large-scale industrial production.

Description

technical field [0001] The invention relates to the technical field of immunology, in particular to a porcine rotavirus vaccine, an antigen for preparing the vaccine and its coding 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, the gene recombination between the attenuated vaccine and the wild strain causes back mutation, and the long-term detoxification of farmed animals pollutes 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 ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/15A61P31/14C07K14/14C12N15/46C12N15/70C12R1/19
CPCA61K39/12A61P31/14C07K14/005C12N15/70A61K2039/552C12N2720/12322C12N2720/12334
Inventor 马丽高江明王学波杜菲康正武孙玉明李朝阳
Owner 山东信得动物疫苗有限公司
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