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A virus gene, vector vaccine technology, applied in the field of biomedicine
Inactive Publication Date: 2021-03-26
翁炳焕
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However, there is no report on the preparation of a new coronavirus vaccine using artificially constructed stem cells with RNA interference function as a carrier
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Embodiment 1
[0031] Example 1. Preparation of Novel Coronavirus Gene Silenced Immortal Lung Stem Cells
[0032] 1. Collection of cell samples
[0033] 1.1. Collection of amniotic fluid fibroblasts
[0034] According to the prenatal diagnosis process, the amniocytes of pregnant women to be tested are collected, cell culture and prenatal diagnosis are carried out, and the spindle-shaped amniocytes growing on the wall are screened from the remaining amniocytes under an inverted microscope.
[0035] 1.2. Collection of lung mesenchymal stem cells
[0036] The lung tissue of the aborted fetus was aseptically collected, mechanically separated, digested with 0.25% trypsin, filtered through gauze with a pore size of 100 μm, centrifuged at 1 000 r / min for 5 min, discarded the supernatant, and added DMEM culture medium (0.1 μmol β-mercaptoethanol, 100 UI / mL penicillin, 10% fetal bovine serum). At 37°C, 5% CO 2 cultivated under conditions. Change the medium after 45 minutes to remove unattached ...
Embodiment 2
[0135] Example 2. Preparation of Gene Silenced Immortal Lung Stem Cell Vector Vaccine
[0136] 1. Gene assembly of gene silencing immortalized lung stem cells for SARS-CoV-2 antibody production
[0137] 1.1. Synthesis of RBD gene
[0138] The amino acid sequence of the S protein of SARS-CoV-2 was analyzed with reference to the GenBank accession number (MN908947.3). The RBD amino acids were Gly319~Asn541, and amino acid codon synonymous substitutions were used to synthesize the whole genome of the nucleic acid corresponding to the RBD peptide. . Codon optimization is performed before synthesis, the purpose is to increase the G+C% content of the genome, increase the stability of mRNA in mammalian cells or the rate of mRNA input into the cytoplasm, avoid the loss of rare tRNAs, enhance protein expression efficiency, and improve immunogenicity. sex. The specific method is as follows: Statistical analysis of the usage frequency of NC_045512.2 strain virus codons and human codons...
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Abstract
The invention discloses a novel coronavirus gene-silencing immortalized lung stem cell vector vaccine, which is characterized in that amniotic fluid fibroblast stem cells are separated from amniotic fluid cells remained after prenatal diagnosis, an immortalized amniotic fluid stem cell line is constructed by an SV40LT and/or hTERT transfection method, an immortalized lung stem cell line is screened out, RNA interference sequences shRNAs of M, N, E and/or S genes of the novel coronavirus are inserted into the DNA of the immortalized lung stem cell line to prepare a gene-silencing immortalized lung stem cell line capable of interfering with the replication of the novel coronavirus in the lung stem cell line through shRNA targeting, and then a novel coronavirus antibody generation gene S1-RBDis inserted into the DNA of the gene-silencing immortalized lung stem cell line to prepare a gene-silencing immortalized lung stem cell vector vaccine which can be industrially expanded in vitro, interfere with the replication of the novel coronavirus in the lung stem cell line and generate a neutralizing antibody against novel coronavirus.
Description
technical field [0001] The invention relates to a novel coronavirus gene silencing immortal lung stem cell vector vaccine, which belongs to the infectious disease prevention and control technology in the field of biomedicine. Background technique [0002] The novel coronavirus (SARS-CoV-2) has posed a serious threat to global public health. The main structure of SARS-CoV-2 includes single-stranded positive-strand nucleic acid (ssRNA), spike protein (S), membrane protein (M), envelope protein (E) and nucleocapsid protein (N). Among them, the S protein is cleaved by host proteases into the N-terminal S1 subunit and the C-terminal S2 subunit during the infection process. The S1 subunit is composed of an N-terminal domain (S1-NTD) and a receptor binding domain (S1-RBD). -RBD is responsible for recognizing and binding the host cell surface receptor angiotensin-converting enzyme 2 (ACE2), and the S2 subunit mediates the fusion between the viral envelope and the host cell membrane...
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