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Application of rotavirus non-structural protein 4 in improving immune function of recombinant rotavirus subunit vaccine

A subunit vaccine, non-structural protein technology, applied in the field of vaccine preparation, to avoid the risk of intussusception, improve protection, and enhance the effects of immunogenicity

Pending Publication Date: 2022-07-22
INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In contrast, whether the immune response of these candidate vaccines can be further enhanced by other viral components, especially those with immunostimulatory potential, remains to be tested

Method used

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  • Application of rotavirus non-structural protein 4 in improving immune function of recombinant rotavirus subunit vaccine
  • Application of rotavirus non-structural protein 4 in improving immune function of recombinant rotavirus subunit vaccine
  • Application of rotavirus non-structural protein 4 in improving immune function of recombinant rotavirus subunit vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Four antigens were designed below. The expression vectors for the four antigens were the loop2 loop and / or the loop3 loop surface loop of the protruding domain of Norovirus VA387 (genome II, cluster 4 [GII.4]).

[0027] The partial nucleotide sequence of the Norovirus VA387 expression vector containing the surface loops of loop2 and loop3 is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2.

[0028] (1) PP-VP8: The VP8 antigen gene is inserted into the surface loop of loop2 for expression, and the insertion site is between T369-D372 of the amino acid sequence shown in SEQ ID NO: 2.

[0029] (2) PP-NSP4-VP8 (tandem expression): both NSP4 gene and VP8 antigen gene are inserted into the loop2 surface loop for expression, and the insertion site of NSP4 gene and VP8 antigen gene is T369 of the amino acid sequence shown in SEQ ID NO: 2 -D372, and the NSP4 gene precedes the VP8 antigen gene.

[0030] (3) PP-VP8-NSP4 (parallel expression): insert th...

Embodiment 2

[0035] Expression and purification of recombinant proteins

[0036] The expression vectors of the four antigens constructed in Example 1 were introduced into Escherichia coli (BL21, DE3) to obtain transformed cells. Expression of recombinant protein was induced in E. coli (BL21, DE3) with 0.4 mM isopropyl-β-D-thiogalactoside (IPTG) at 16°C overnight. Take out the induced bacterial solution, centrifuge at 5000rpm at 4°C for 15min, discard the supernatant, resuspend the pellet with 100mL PBS, then perform ultrasonic disruption, ultrasonicate for 10s, stop for 50s, the total ultrasonic time is 1.5h, and then centrifuge again at 13000rpm , 1h, 4 ℃, the supernatant was collected for purification. Pierce for recombinant proteins TMGlutathione agarose (Thermo Fisher Scientific) was used for purification. Take 2 mL of mixed GSTAgarose and add it to the purification column, then add 50 mL of supernatant, mix and combine at room temperature for 2 hours, and discharge the flow-through...

Embodiment 3

[0038] Validation of Purified Antigens

[0039] 1. Verification by polyacrylamide gel electrophoresis: use serially diluted bovine serum albumin (BSA) of different concentrations to conduct SDS-PAGE electrophoresis simultaneously with the sample. The result is as figure 1 shown in the left image.

[0040] 2. Western Blot analysis and verification: After the protein was electrophoresed by 10% SDS-PAGE, it was transferred to 200 mA for 80 min. -P on transfer membrane (Millipore). Detection was performed with rabbit anti-GST antibody (1:5000, Rockland Immunochemicals, Inc., USA) and secondary antibody (1:5000, goat anti-rabbit IgG-HRP, Multi Sciences Biotech, China). After adding an enhanced chemiluminescence substrate (Multi Sciences Biotech, China), the corresponding software Image Lab 5.2.1 was used by Molecular ChemiDoc TM Images were taken by the XRS+ imaging system. The result is as figure 1 shown in the figure on the right.

[0041] 3. The morphology of the purif...

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Abstract

The invention provides an application of a rotalike non-structural protein 4 in improving the immunologic function of a recombinant rotavirus subunit vaccine, which is characterized in that a rotalike non-structural protein 4 gene and a rotavirus antigen gene are expressed in different surface rings on norovirus P particles to obtain a recombinant protein, the recombinant protein is the recombinant rotavirus subunit vaccine. The immunologic function of the recombinant rotavirus subunit vaccine is improved by using the NSP4 protein, and the NSP4 gene and the VP8 antigen gene or the NSP4 antigen gene are expressed in parallel, so that the specific IgG antibody titer of the NSP4 antigen and the VP8 antigen can be improved, the neutralizing capacity of the VP8 antigen on rotavirus can be improved, and the protective effect of the NSP4 antigen and the VP8 antigen on diarrhea mice induced by a rotavirus SA11 strain can be improved.

Description

technical field [0001] The invention relates to the technical field of vaccine preparation, in particular to the application of a rotavirus non-structural protein 4 in improving the immune function of a recombinant rotavirus subunit vaccine. Background technique [0002] Rotavirus is one of the most common viral pathogens causing infectious gastroenteritis, with high morbidity and mortality. Although in developed countries, commercial rotavirus vaccines, such as GlaxoSmithKline's and Merck's Iso-rotavirus live attenuated vaccines are highly effective (80-90%) in reducing severe rotavirus-related disease, but less effective (40-70%) in developing countries. Thus, even with current vaccines, rotavirus infection is still the cause of 24 million outpatient visits, 2.3 million hospitalizations, and approximately 200,000 deaths globally each year. In addition, a small risk of intussusception (1:50,000 or less) remained after the first dose of these live attenuated vaccines in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/39A61P37/04A61K39/15A61P31/14A61P1/12B82Y5/00B82Y40/00
CPCA61K39/39A61P37/04A61K39/12A61P31/14A61P1/12B82Y5/00B82Y40/00C12N2720/12334A61K2039/54
Inventor 刘存宝曹晗吴晋元栾宁王云飞
Owner INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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