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Human rotavirus P[8]deltaVP8*-P[6]deltaVP8* recombinant chimeric protein and application thereof

A rotavirus and chimeric protein technology, applied in the fields of molecular biology and genetic engineering, to achieve the effect of overcoming intussusception, improving immune efficiency and overcoming potential risks

Inactive Publication Date: 2013-09-18
HEILONGJIANG BAYI AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Our study found that P[8]△VP8* can induce specific neutralizing antibodies against various rotaviruses of P[8] genotype, and can also induce high titers of anti-P[4] genotype-specific polyclonal antibodies. However, there is still a lack of an effective vaccine that can cover the three genotypes of P[8], P[4], and P[6] to improve the efficiency of the vaccine after injection

Method used

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  • Human rotavirus P[8]deltaVP8*-P[6]deltaVP8* recombinant chimeric protein and application thereof
  • Human rotavirus P[8]deltaVP8*-P[6]deltaVP8* recombinant chimeric protein and application thereof
  • Human rotavirus P[8]deltaVP8*-P[6]deltaVP8* recombinant chimeric protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] This example is used to illustrate the construction of the expression vector containing the target fragment.

[0050] The materials used were Wa strain of human rotavirus with genotype G1P[8]; 1076 strains with genotype G2P[6]. All are classic strains, which are 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:

[0051] Table 1 EMEM medium formula

[0052]

[0053] The RNA of human rotavirus Wa strain (G1P[8]) and 1076 strain (G2P[6]) was extracted by TRIzol-LS (Invitrogen), and the extracted RNA was reverse-transcribed into cDNA by RT-PCR. The primers used were designed according to the gene sequence of RNA fragment 4 of human rotavirus Wa strain (GenBank accession numbers: FJ423116 (Wa) and M88480 (1076) respectively).

[0054] The primers...

Embodiment 2

[0070] This example is used to illustrate the expression of human rotavirus subunit recombinant protein.

[0071] Using the heat shock method, transform the recombinant plasmid pETite-P[8]ΔVP8*-P[6]ΔVP8* into HI-control BL21(DE3) expression host cells, pick a single clone from the agar plate and inoculate it in a medium containing 50 μg / ml kanamycin LB liquid medium (add 2% glucose, 1% ethanol). When the absorbance at 600 nm reaches 0.5, add IPTG to a final concentration of 1 mM, and induce at 18° C. for 16-20 h. SDS-PAGE electrophoresis results such as Figure 4 shown. 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.

Embodiment 3

[0073] The examples serve to illustrate the purification of recombinant proteins.

[0074] The cell wall of E. coli cells was disrupted with BugBuster Master Mix (Novagen) containing protease inhibitor cocktail (Roche), and the soluble product was collected and stored at -80°C for future use. Each protein was purified by ProBond nickel-NTA agar affinity chromatography (Invitrogen) according to the manufacturer's protocol. After the cellular protein was washed away with wash buffer containing different concentrations of imidazole (20-100mM), the recombinant protein was eluted with 250mM imidazole. The SUMO tag at the N-terminus of P[8]ΔVP8*-P[6]ΔVP8* was removed with SUMO-expressing protease (Lucigen) according to the manufacturer's guidelines. The purity of the recombinant protein was analyzed by SDS-PAGE, and imidazole in the solution was removed with a centrifugal filter device (Millipore). According to the BCA Quantification Kit (Thermo) manufacturer's instructions, the B...

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Abstract

The invention relates to a human rotavirus P[8]deltaVP8*-P[6]deltaVP8* recombinant chimeric protein, wherein the amino acid sequence of the protein is shown in SEQ ID NO.2. Furthermore, the invention provides a coding gene and a preparation method of the protein as well as an application of the protein in preparation of a rotavirus vaccine. Two segments P[8]deltaVP8* and P[6]deltaVP8* are connected in serial to be expressed into the human rotavirus P[8]deltaVP8*-P[6]deltaVP8* recombinant chimeric protein; the protein can simultaneously induce neutralizing antibodies of major P genotypes P [8], P[4] and P[6] of the human rotavirus, overcome the shortcoming of an existing vaccine which can induce the generation of an antibody only capable of resisting one genotype and greatly enhance immune efficiency; and meanwhile, the protein can avoid a potential risk that oral administration of attenuated vaccine rotavirus possibly induces intestinal invagination, and is applicable to preparation of rotavirus vaccine.

Description

technical field [0001] The invention belongs to the fields of molecular biology and genetic engineering, and relates to a human rotavirus P[8]△VP8*-P[6]△VP8* recombinant chimeric 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, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/63A61K39/15A61P31/14C12R1/93
Inventor 冉旭华朱光怡闻晓波张峣曹思张春山
Owner HEILONGJIANG BAYI AGRICULTURAL UNIVERSITY
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