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Novel coronavirus virus-like particle vaccine based on vaccinia virus vector
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A technology of vaccinia virus, virus, applied in the field of biomedicine
Active Publication Date: 2022-08-09
INST OF PATHOGEN BIOLOGY CHINESE ACADEMY OF MEDICAL SCI +1
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Due to the high morphological similarity between virus-like particles and true viruses, the effectiveness of vaccines designed to target virus-like particles may exceed that of vaccines targeting a single component of the virus, such as the S protein
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Embodiment 1
[0040] Example 1 Construction of recombinant vaccinia virus F4L-EM expressing new coronavirus E and M proteins
[0044] 293T cells were seeded in six-well plates (5×10 5 ), cultured overnight, and when the cells grew and spread 60% to 70%, the recombinant plasmid pF4L-EM with the coding gene sequences of the new coronavirus E and M was transfected. After 4 hours, the wild-type virus was infected; after 2 hours, the supernatant was discarded and replaced with fresh complete medium; after 48 hours, the cells were harvested, and freeze-thawed three times to obtain a virus suspension.
[0045] 1.1.2. Purification by virus plaque formation experiment
[0046] Vero (1 × 10 6 ) cells were cultured overnight, and when the cells grew to 80% to 90% of the plate, the collected virus suspension was diluted 10-fold into a six-well plate for infection. After 2 h, the cells were wash...
Embodiment 2
[0056] Example 2 Construction of recombinant vaccinia virus TK-SN / F4L-EM expressing new coronavirus S, N, E, M proteins simultaneously
[0060] 293T cells were seeded in six-well plates (5×10 5 ), cultured overnight, and when the cells grew and spread 60% to 70%, the recombinant plasmid pTK-SN with the S and N coding sequences of the new coronavirus was transfected. After 4 hours, the recombinant virus F4L-EM expressing E and M was infected; after 2 hours, the supernatant was discarded and replaced with fresh complete medium; after 48 hours, the cells were harvested and freeze-thawed three times to obtain a virus suspension.
[0061] 1.1.2. Purification by virus plaque formation experiment
[0062] Vero (1 × 10 6 ) cells were cultured overnight, and when the cells grew to 80% to 90% of the plate, the collected virus suspension was diluted 10-fold into a six-well p...
Embodiment 3
[0073] Example 3 Construction of TK and F4L-deleted vaccinia virus
[0074] 1. Method
[0075] 1.1. Construction of TK-deficient vaccinia virus
[0076] 293T cells were transfected with the recombinant vector pTK (SEQ ID NO: 4) with the homology arms (J1R, J3R) of the TK region of vaccinia virus, and the wild-type virus was infected after 4 hours; after 2 hours, the fresh complete medium was replaced; after 48 hours , cells were harvested, and freeze-thawed three times to obtain a virus suspension. Multiple rounds of screening and purification were performed using the virus plaque formation experiment, and the purified recombinant virus was amplified and identified by PCR. The specific methods are the same as those in Examples 1 and 2.
[0077] 1.2. Construction of vaccinia virus lacking TK and F4L
[0078] 293T cells were transfected with the recombinant vector pF4L (SEQ ID NO: 3) with homology arms (F3L, F5L) in the F4L region of vaccinia virus, and 4 hours later, the abov...
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Abstract
The invention provides a novel coronavirusvirus-like particle vaccine based on a vacciniavirus vector. The recombinant vaccinia virus is constructed by integrating coding gene sequences of a new coronavirus envelope protein E and a membrane protein M at the position of an F4L gene in a vacciniavirus Tian Tan strain genome and integrating coding gene sequences of a new coronavirusspike protein S and a nucleocapsid protein N at the position of a TK gene; wherein the coding gene sequences of the protein E and the protein M are connected in a manner that 5'ends are opposite, and are respectively connected to the downstream of different promoters; the coding gene sequences of the proteins S and N are connected in a manner that 5'ends are opposite, and are respectively connected to the downstream of different promoters. The recombinant virus is used for immunizing mice, high S and N specific antibody titers are detected in serum of the mice, and antigen-specific T cell immune response can be induced after immunization. The novel coronavirus vaccine provided by the invention has important significance on prevention and control of novel coronavirus.
Description
technical field [0001] The invention relates to the field of biomedicine, in particular to a novel coronavirus virus-like particle vaccine based on a vaccinia virus vector. Background technique [0002] Vaccinia virus (Vaccinia virus) is a large, enveloped DNA virus that has been used as a vaccine against smallpox in the early days. Due to the large genome size of vaccinia virus, the large capacity of foreign genes, and the long-term use history, vaccinia virus is currently used in the form of vectors for the construction of vaccines against infectious diseases, as well as for therapeutic tumor vaccines and oncolytic viruses. Vaccinia virus Tiantan strain (VTT) is a vaccine strain independently developed by China and successfully used to prevent smallpox, and has played an important role in the eradication of smallpox in my country. [0003] The new coronavirus (SARS-CoV-2) belongs to the β genus of the Coronaviridae family, as well as SARS-CoV-1 and MERS, which threatened ...
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Application Information
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