Porcine rotavirus delta VP8* subunit recombinant protein and applications thereof

Abstract

The invention relates to a porcine rotavirus delta VP8* subunit recombinant protein and an encoding gene of the protein. The invention further provides a recombinant protein formed after increasing tetanus toxin T cell epitope P2 into the recombinant protein, and an encoding gene. The delta VP8* protein is 64th-site to 223th-site amino acid in the VP8* and can effectively stimulate an organism to produce specific serum antibody, humoral immune response is good, the problem that the VP4 gene can not conduct prokaryotic expression due to overlarge fragment can be overcome, the protein can be expressed as a soluble protein in vitro, so that the problem that the VP8* is expressed as an inclusion body in vitro can also be overcome; the T cell epitope P2 (830th-site to 844th-site amino acid of TT) in the tetanus toxin is induced into the delta VP8* subunit recombinant protein, so that the immunity efficacy of the protein can be greatly improved, the faster and stronger neutralizing antibody titer can be induced, and high-titer rotavirus cross neutralizing antibody can also be induced.

Description

technical field

[0001] The invention belongs to the field of animal molecular biology and genetic engineering, and relates to a porcine rotavirus ΔVP8* subunit recombinant protein and application thereof. Background technique

[0002] Rotavirus (RV) is a non-enveloped, segmented, double-stranded RNA virus belonging to the Reoviridae family, and is one of the main pathogens causing viral diarrhea in infants and various young animals. Piglet rotavirus diarrhea is an acute infectious disease characterized by diarrhea, anorexia, vomiting, and dehydration caused by porcine rotavirus (PRV). The disease mostly occurs in late autumn, winter and early spring, and pigs of all ages can be infected, and the infection rate can reach as high as 90% to 100%. Susceptible to secondary bacterial infection, leading to increased mortality of piglets, resulting in serious economic losses. Porcine rotavirus infection is distributed worldwide and has brought great harm to the pig industry. [0...

Examples

Embodiment 1

[0053] This example is used to illustrate the construction of the expression vector containing the target fragment.

[0054] The material used is porcine rotavirus OSU strain. Porcine rotavirus OSU strain 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:

[0055]

[0056]

[0057] Porcine rotavirus OSU strain RNA 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 porcine rotavirus OSU strain VP4 (GenBank accession number: X13190.1). The primers for amplifying ΔVP8* are as follows:

[0058] Upstream primer: 5'-GATC CATATG TTATTAGATGGCCCATACCAACC-3' (SEQ ID No.3), the underlined ...

Embodiment 2

[0064] This example is used to illustrate the expression of porcine rotavirus subunit recombinant protein.

[0065] Single colonies of positive recombinant bacteria and empty vector control bacteria were picked respectively, inoculated in 3 mL of LB liquid medium containing 30 μg / mL of kanamycin, and cultured with shaking at 37°C for 12 to 16 hours. Then transfer the bacterial liquid to 10 mL of fresh LB liquid medium (containing 30 μg / mL kanamycin) at a ratio of 1%, shake culture at 37°C and 200 rpm until OD600≈0.6, and take out 1 mL of bacterial liquid as an induction For the former control, add IPTG to the remaining bacterial solution to a final concentration of 0.5mM, lower the temperature to 22°C, and induce expression overnight. Take 1mL post-induction bacterial solution and 1mL pre-induction bacterial solution respectively, collect the bacterial cells by centrifugation, resuspend the bacterial cells in 0.01M PBS (pH7.4), and then ultrasonically disrupt them in an ice ba...

Embodiment 3

[0067] This example is used to illustrate the purification of recombinant proteins.

[0068] 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 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 BCA Quantification Kit was used to determine the concentration of the purified recombinant protein. The purified recombinant protein was stored at -8...

technical field

[0001] The invention belongs to the field of animal molecular biology and genetic engineering, and relates to a porcine rotavirus ΔVP8* subunit recombinant protein and application thereof. Background technique

[0002] Rotavirus (RV) is a non-enveloped, segmented, double-stranded RNA virus belonging to the Reoviridae family, and is one of the main pathogens causing viral diarrhea in infants and various young animals. Piglet rotavirus diarrhea is an acute infectious disease characterized by diarrhea, anorexia, vomiting, and dehydration caused by porcine rotavirus (PRV). The disease mostly occurs in late autumn, winter and early spring, and pigs of all ages can be infected, and the infection rate can reach as high as 90% to 100%. Susceptible to secondary bacterial infection, leading to increased mortality of piglets, resulting in serious economic losses. Porcine rotavirus infection is distributed worldwide and has brought great harm to the pig industry. [0...

Examples

Embodiment 1

[0053] This example is used to illustrate the construction of the expression vector containing the target fragment.

[0054] The material used is porcine rotavirus OSU strain. Porcine rotavirus OSU strain 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:

[0055]

[0056]

[0057] Porcine rotavirus OSU strain RNA 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 porcine rotavirus OSU strain VP4 (GenBank accession number: X13190.1). The primers for amplifying ΔVP8* are as follows:

[0058] Upstream primer: 5'-GATC CATATG TTATTAGATGGCCCATACCAACC-3' (SEQ ID No.3), the underlined ...

Embodiment 2

[0064] This example is used to illustrate the expression of porcine rotavirus subunit recombinant protein.

[0065] Single colonies of positive recombinant bacteria and empty vector control bacteria were picked respectively, inoculated in 3 mL of LB liquid medium containing 30 μg / mL of kanamycin, and cultured with shaking at 37°C for 12 to 16 hours. Then transfer the bacterial liquid to 10 mL of fresh LB liquid medium (containing 30 μg / mL kanamycin) at a ratio of 1%, shake culture at 37°C and 200 rpm until OD600≈0.6, and take out 1 mL of bacterial liquid as an induction For the former control, add IPTG to the remaining bacterial solution to a final concentration of 0.5mM, lower the temperature to 22°C, and induce expression overnight. Take 1mL post-induction bacterial solution and 1mL pre-induction bacterial solution respectively, collect the bacterial cells by centrifugation, resuspend the bacterial cells in 0.01M PBS (pH7.4), and then ultrasonically disrupt them in an ice ba...

Embodiment 3

[0067] This example is used to illustrate the purification of recombinant proteins.

[0068] 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 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 BCA Quantification Kit was used to determine the concentration of the purified recombinant protein. The purified recombinant protein was stored at -8...

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