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Bovine rotavirus VP8* subunit recombinant chimeric protein and application thereof

A bovine rotavirus and chimeric protein technology, applied in the fields of molecular biology and genetic engineering, can solve problems such as increased vaccine production costs, difficulties in vaccine development and production, and achieve the goal of improving immune efficacy, low cost, and reducing economic losses Effect

Active Publication Date: 2015-04-01
HEILONGJIANG BAYI AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Conventional vaccine development is mainly to develop bivalent vaccines to prevent and control G6P[5] and G10P[11] rotavirus strains at the same time, which brings certain difficulties to vaccine development and production, and is bound to lead to increased vaccine production costs

Method used

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  • Bovine rotavirus VP8* subunit recombinant chimeric protein and application thereof
  • Bovine rotavirus VP8* subunit recombinant chimeric protein and application thereof
  • Bovine rotavirus VP8* subunit recombinant chimeric protein and application thereof

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Experimental program
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Effect test

Embodiment 1

[0029] Embodiment 1 Contains the construction of the expression vector of target fragment

[0030] The material used was bovine rotavirus of genotype G5P[6], which was 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 (high glucose type) is shown in Table 1.

[0031] Table 1 EMEM medium formula

[0032]

[0033] (1) Primer design

[0034] According to the bovine rotavirus VP4 gene sequence published in GenBank (gene accession number: JF693062.1), primers were designed using Oligo7.0 and DNAStar software, and the primer sequences are shown in Table 2. Among them, the annealing of the first pair of primers VP8*-ab-0-F and VP8*-ab-0-R is used as the template for subsequent amplification, a and b represent aa 1-11 and aa 218-235 respectively), the underlined part in italics...

Embodiment 2

[0056] Example 2 Expression of bovine rotavirus VP8* subunit recombinant chimeric protein

[0057] Using the heat shock method, the expression plasmid pET28a-P2-VP8*-(ab) 3 , pET28a-P2-VP8*-ab, pET28a-VP8*-(ab) 3 , pET32a-VP8*-ab, pET32a-VP8*-a and pET28a-VP8* were transformed into E.coli BL21(DE3) competent cells. Pick a single clone from the agar plate and inoculate it in LB liquid medium containing 50 μg / ml kanamycin (pET28a vector) or 50 μg / ml ampicillin (pET32a vector) (add 2% glucose, 1% ethanol and 2 %glycerin). When the absorbance value at 600 nm reached 0.5, IPTG was added to a final concentration of 0.5 mM, and the protein expression was induced overnight at 18°C. Then centrifuge at 10000g at 4°C for 15min to collect the recombinant E.coli cells, and store them at -80°C for later use (recombinant protein SDS-PAGE electrophoresis results are as follows: Figure 6 A and Figure 6 As shown in B, P2-VP8*-(ab) 3 The amino acid sequence is shown in SEQ ID No.4).

Embodiment 3

[0058] The Western blot detection of embodiment 3 recombinant protein

[0059] After 15% SDS-PAGE electrophoresis, the expressed recombinant protein was transferred to PVDF membrane, blocked with PBST containing 5% skim milk at 37°C for 1 hour, washed 3 times with PBST, and anti-His-tag monoclonal antibody diluted 1:2000 Incubate overnight at 4°C, wash with PBST three times, dilute HRP-labeled goat anti-mouse IgG at 1:5000 and incubate at 37°C for 1 hour; wash with PBST three times, develop color with DAB ( Figure 7 ).

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Abstract

The invention provides a bovine rotavirus VP8* subunit recombinant chimeric protein. According to the bovine rotavirus VP8* subunit recombinant chimeric protein, a T cell epitope polypeptide P2 in a tetanus toxin is introduced into an epitope vaccine of a bovine rotavirus VP8* subunit multi-copy chimeric gene recombinant protein, so that the immune efficacy of the epitope vaccine can be greatly enhanced, cross neutralizing immune protection can be induced, higher neutralizing antibody titer can be induced, and a high-titer anti-P[5] and P[11] genotype-specific rotavirus neutralizing antibody can be induced. The bovine rotavirus VP8* subunit recombinant chimeric protein provided by the invention can be used for preventing and controlling both G6 and G10 type rotaviruses which are mainly pandemic within a world range, and is suitable for preparing a safe and low-cost bovine rotavirus vaccine; in addition, the bovine rotavirus vaccine can be used for effectively reducing the economic loss brought by rotavirus gastroenteritis to cattle rearing by being matched with other rotavirus vaccines researched and developed at present, and has wide market prospects.

Description

technical field [0001] The invention belongs to the fields of molecular biology and genetic engineering, in particular, relates to bovine rotavirus VP8* subunit recombinant chimeric protein and application thereof. Background technique [0002] Rotavirus is a member of the Reoviridae family and the genus Rotavirus, and is the main pathogen that causes gastroenteritis in many newborn animals and infants. Bovine rotavirus diarrhea is characterized by depression, anorexia, diarrhea, and dehydration. Calves within 7 days of age are most susceptible to infection, with a morbidity rate of 90% to 100% and a mortality rate of 10% to 50%. If secondary Escherichia coli sepsis can lead to a further increase in mortality, seriously endangering the economic benefits of the cattle industry. [0003] Rotavirus particles consist of three capsids, the outermost capsid consists of VP7 and spike protein VP4, both of which can independently induce neutralizing antibodies. Studies have shown t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/70A61K39/15A61P31/14
Inventor 闻晓波朱光怡冉旭华苗艳张峣曹思倪宏波朱战波
Owner HEILONGJIANG BAYI AGRICULTURAL UNIVERSITY
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