Recombinant measles viruses expressing novel coronavirus proteins and application thereof

A virus and protein technology, applied in the field of vaccines, can solve the problems of affecting the immune effect, slow immunity, and low human pathogenicity

Pending Publication Date: 2021-10-08
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Its advantage is that it is safe, there is no danger of loose poison, it is convenient for storage and transportation, and it produces a high level of antibody; its disadvantage is that it has a weak ability to induce cell-mediated immunity, and the immunity is slow to develop, and it usually takes 2 weeks after vaccination to obtain good immunity. immunity
The advantage of adenovirus vector vaccine is that it has a wide range of hosts, low pathogenicity to humans, can continue to proliferate in the body after immunization, is safe, and does not integrate into the human genome; the disadvantage is that there are pre-existing antibodies in the body that affect the immune effect

Method used

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  • Recombinant measles viruses expressing novel coronavirus proteins and application thereof
  • Recombinant measles viruses expressing novel coronavirus proteins and application thereof
  • Recombinant measles viruses expressing novel coronavirus proteins and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0207] Example 1: Amplification of the recombinant measles virus infectious cloning plasmid pYES2-rMeV-SC2-ST target fragment expressing the new coronavirus fusion pre-S trimer protein

[0208] The Hu191 strain used in the experiment was rescued from the measles Hu191 reverse genetics system (Yilong Wang et al., Virology. 2018; 518:210-20.). The schematic diagram of the amplification of the target fragment of the recombinant measles virus infectious cloning plasmid pYES2-rMeV-SC2-ST expressing the pre-fusion S trimer protein of the new coronavirus is as follows figure 1 shown. In short, using the full-length cDNA of measles Hu191 strain as a template, nine pairs of overlapping primers were used to amplify the full-length genome of the Hu191 vaccine strain. The nine fragments are N, P, M, F, H, L1, L2, Y1, Y2. The S protein codon-optimized sequence (as shown in SEQ ID No: 3) synthesized with the amino acid sequence of the S protein before the fusion of the new coronavirus as ...

Embodiment 2

[0211] Example 2: Splicing of pYES2-rMeV-SC2-ST full-length infectious clone

[0212] Using the method of yeast transformation, the amplified 10 fragments N, P, S, M, F, H, L1, L2, Y1, Y2 and pYES2 plasmids (purchased from Wuhan Miaoling Biotechnology Co., Ltd.) were completed in yeast stitching. The reaction system is as follows:

[0213]

[0214] Then the yeast plasmid was extracted by the yeast plasmid extraction kit, and then electrotransferred into TOP 10 Escherichia coli competent cells.

Embodiment 3

[0215] Example 3: Verification of full-length infectious clones of pYES2-rMeV-SC2-ST

[0216] Randomly pick individual clones, inoculate them into 5 mL of LB liquid medium containing ampicillin (100 μg / mL) resistance, culture in a shaker at 37 °C for 12 h, and extract the picked clones according to the instructions of the AxyPrep Plasmid DNA Mini Kit. Then obtain the pYES2-MeV-SARS-CoV-2-S full-length infectious clone plasmid, perform PCR amplification on the full-length infectious clone, and verify whether the molecular weight of the PCR product meets expectations by nucleic acid electrophoresis; when the molecular weight of the PCR product is correct , the correctness of the sequence was verified by gene sequencing.

[0217] PCR sequencing primers adopt the same primers in Table 1, take 5 μ L of PCR product and detect by electrophoresis in 1% agarose gel, all gene fragments of pYES2-rMeV-SC2-ST full-length infectious clone can be detected (results such as Figure 4 shown). ...

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Abstract

The present invention relates to recombinant measles viruses expressing novel coronavirus proteins, such as S proteins or trimers or functional fragments thereof, vaccines comprising the recombinant measles viruses, and the use of such vaccines for the prevention or treatment of novel coronavirus infections.

Description

[0001] priority claim [0002] This application claims priority to Chinese Patent Application No. 202011112686.5 filed on October 16, 2020, which is hereby incorporated by reference in its entirety. technical field [0003] The present invention relates to the field of vaccines, in particular, the present invention relates to a recombinant measles virus expressing a novel coronavirus protein, a vaccine comprising the recombinant measles virus, and the application of such a vaccine in preventing or treating a new coronavirus infection. [0004] sequence listing [0005] This application contains a Sequence Listing, which is incorporated herein by reference. Background technique [0006] Novel coronavirus (SARS-CoV-2) is the causative agent of novel coronavirus pneumonia (COVID-19). Effective treatment and preventive measures against 2019-nCoV are urgently needed. [0007] Prophylactic vaccination remains the most effective way to prevent viral infection. The new coronavir...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/165C12N7/01C12N15/50A61K39/215A61P31/14
CPCC07K14/005C12N7/00A61K39/12A61P31/14C12N2770/20022C12N2770/20034C12N2760/18421
Inventor 黄耀伟徐令东陈相波宗明瑞王斌杨永乐梅小强覃盼彭蕾
Owner ZHEJIANG UNIV
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