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Human rotavirus Delta VP8* subunit recombinant protein and application thereof

A recombinant protein and rotavirus technology, applied in the field of animal molecular biology and genetic engineering, can solve the problem of low immunogenicity, achieve strong neutralizing antibody titers, improve immune efficacy, and overcome potential risks

Active Publication Date: 2014-12-10
HEILONGJIANG BAYI AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the immunogenicity of the △VP8* protein alone is low, and the immunogenicity of the △VP8* protein needs to be further improved

Method used

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  • Human rotavirus Delta VP8* subunit recombinant protein and application thereof
  • Human rotavirus Delta VP8* subunit recombinant protein and application thereof
  • Human rotavirus Delta VP8* subunit recombinant protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Construction of an expression vector containing a fragment of interest.

[0038]The material used was the Wa strain of human rotavirus with genotype G1P[8]. The Wa strain of human rotavirus is a classic strain, which is cultured with primary African green monkey kidney (AGMK) cells. The medium used was EMEM, in which trypsin was added to a final concentration of 0.5 μg / ml, penicillin 100 IU / ml, streptomycin 100 μg / ml, amphotericin B 2.5 μg / ml. The formula of EMEM medium is shown in Table 1:

[0039] Table 1 EMEM medium formula

[0040] serial number

Compound name

Content (mg / L)

serial number

Compound name

Content (mg / L)

1

Anhydrous Calcium Chloride.2H2O

265.00

18

L-serine

42.00

2

Ferric nitrate.9H2O

0.10

19

L-threonine

95.00

[0041] 3

potassium chloride

400.00

20

L-tryptophan

16.00

4

Anhydrous Magnesium Sulfate

97.67

21 ...

Embodiment 2

[0051] Example 2 Expression of human rotavirus subunit recombinant protein

[0052] The expression plasmid pET28a-P2-P[8]ΔVP8* was transformed into E.coli BL21(DE3)pLysS competent cells by heat shock method. Pick a single clone from the agar plate and inoculate it in LB liquid medium containing 50 μg / ml kanamycin (add 2% w / v glucose, 1% v / v ethanol). When the absorbance value at 600 nm reached 0.5, IPTG was added to a final concentration of 1 mM, and the protein expression was induced overnight at 18°C. SDS-PAGE electrophoresis results such as image 3 shown. Then, the recombinant E.coli cells were collected by centrifugation at 10,000 g at 4°C for 15 minutes, and stored at -80°C for later use. After testing, the yield of soluble protein obtained by this method is 46 mg / L.

[0053] The present invention further adopts the second method to induce recombinant protein expression, the steps of which are the same as the above method, the difference is that glucose and ethanol a...

Embodiment 3

[0055] Embodiment 3Western blot method detects protein

[0056] Proteins were analyzed on 4-12% NuPAGE gels and transferred to nitrocellulose membranes (Whatman). The membrane was incubated overnight at 4°C with hyperimmune guinea pig anti-Wa(P[8]) serum (1:50). After washing three times, the membrane was incubated with peroxidase-labeled goat anti-guinea pig IgG (H+L) (KPL) for 1 h at room temperature. 3,3'Diaminoblue aniline (DAB) (Sigma) was used for color development. The result is as Figure 4 shown.

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Abstract

The invention relates to human rotavirus Delta VP8* subunit recombinant protein and application thereof. The human rotavirus Delta VP8* subunit recombinant protein comprises a T cell epitope P2 in tetanus toxin and a rotavirus Delta VP8* subunit. By the recombinant protein disclosed by the invention, the immune efficacy of a Delta VP8* subunit vaccine can be greatly improved; faster and stronger neutralization antibody titer can be induced; moreover, anti-p[4] genotype specific rotavirus cross neutralization antibody of high titer can be induced; simultaneously, the potential risk of inducing intussusception by taking attenuated rotavirus vaccine orally can be overcome; therefore, the recombinant protein is applicable to preparing a rotavirus vaccine.

Description

technical field [0001] The invention relates to the fields of animal molecular biology and genetic engineering, in particular to a human rotavirus ΔVP8* subunit recombinant protein and its application. Background technique [0002] Rotavirus gastroenteritis, also known as rotavirus diarrhea, is the most important acute intestinal infectious disease caused by diarrhea in infants and young animals worldwide. It is characterized by diarrhea and dehydration, and has a high mortality rate. It has caused great harm to human health and the development of animal husbandry. About 110 million children under the age of 5 suffer from rotavirus gastroenteritis every year in the world, of which 25 million children need to see a doctor, 2 million children must be hospitalized, and about 453,000 children die of rotavirus diarrhea every year, among which the development Deaths in China and less developed countries accounted for more than 85% of the total. Taking China as an example, about ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/63C12N1/21C12N15/70A61K39/15A61K48/00A61P31/14
Inventor 闻晓波孔凡志冉旭华张峣曹殿军朱战波张春山
Owner HEILONGJIANG BAYI AGRICULTURAL UNIVERSITY
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