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A recombined smallpox vaccine - SARS vaccine and preparation method thereof

A vaccine and vaccinia virus technology, applied in recombinant DNA technology, pharmaceutical formulations, medical preparations containing active ingredients, etc., can solve problems such as hidden safety hazards, and achieve good immunogenicity.

Inactive Publication Date: 2009-04-22
INST OF LAB ANIMAL SCI CHINESE ACAD OF MEDICAL SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Chinese application 200410044291.0 discloses a SARS vaccine comprising a SARS-associated coronavirus S gene and a eukaryotic expression plasmid, and Chinese application 200410044285.5 discloses a SARS vaccine comprising an adenovirus vector and a SARS-associated coronavirus S gene, but the existing SARS Vaccines have different levels of safety hazards, and their effectiveness needs further observation and verification

Method used

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  • A recombined smallpox vaccine - SARS vaccine and preparation method thereof
  • A recombined smallpox vaccine - SARS vaccine and preparation method thereof
  • A recombined smallpox vaccine - SARS vaccine and preparation method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Construction of pZC3d insertion vector: the pH5 promoter synthesized by DNA was introduced into the pLW7 vector (provided by Dr. Bernie Moss and Lynda Wyatt of NIH, refer to Development of areplication-deficient recombinant vaccine virus vaccine effectiveag ainstparainfluenza virus 3 infection in an animal model. Linda S. Wyatt.et.al.Vaccine.Vol.14, No.15, 1451-1456.1996), construct a new generation of pZC3d vector containing double promoters. In this vector, the S gene is under the strong synthetic promoter pSYN, the reporter gene GFP gene is under the weak promoter pH5 (Figure 1), the two genes are transferred into the same insert sequence, and GFP is used as a proxy marker to carry the S gene The recombinant MVA vector (provided by Dr. Bernie Moss and Lynda Wyatt of NIH, refer to Vaccine Protocols, Second Edition, August 2003, pps.51-68, ISBN: 1-59259-399-2Series: Methods in Molecular Medicine; Volume #: 87 ; by Caroline Staib and Gerd Sutter). Both promoters, pSYN ...

Embodiment 2

[0031] Construction and purification of ADS-MVA: The GFP gene and S gene were constructed into pZC3d by blunt-end ligation. The S gene is from the cDNA of the SARS-CoV HKU39849 strain, which is isolated from Hong Kong (the S gene is deposited in GenBank with the accession number AY278491). Recombinant ADS-MVA was produced in chicken embryo fibroblasts (CEF) by homologous recombination: First, CEF cells were infected with parental MVA (1 MOI). 90 minutes later, use Effectene (Qiagen Cat: 301427) to transfect the same group of cells with pZC3d; 48 hours later, the positive cell population was selected by the fluorescence emitted by GFP under a fluorescent microscope; the recombinant ADS-MVA was infected with CEF 8 times Cells were passaged to purify. Thus, using pZC3d, we recombined the wild-type SARS full-length S gene and GFP into the MVA III region to obtain ADS-MVA (see FIG. 1 ). For comparison, using the same method, we introduced the modified HCV E1E2 gene into the same ...

Embodiment 3

[0033] Immunofluorescence test: In order to detect the S glycoprotein expressed on the cell surface, we performed an immunofluorescence test. Simple steps to plate CEF cells on day 1 in 6-well plates (2 x 10 per well 6 cells), the plate was pretreated with Con A (100 μg / ml) for 30 minutes, and washed twice with PBS. On the second day, CEF cells were infected with recombined serially diluted ADS-MVA (1:10), and two hours later, the cell culture medium was replaced with DMEM containing 2% fetal calf serum (FCS), at 37°C 5% CO 2 After 48 hours of incubation, infected cells were incubated with heat-inactivated SARS patient serum (WH, 1:500) for 1 hour, and then incubated with AlexaFluor594-labeled goat anti-human IgG (H+L) (Molecular Probes, U.S.A.) for 30 minutes , the cells were washed three times with PBS, and the positive colonies were identified by immunofluorescence fiberscope, the results are shown in Figure 2A. CEF cells infected with control ADC-MVA were also stained a...

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Abstract

The invention provides a reorganization pox seedling virus vaccine SARS-CoV (ADS-MVA) for preventing or treating SARS, and also the method for carrying out preventive vaccine anti-SARS-CoV infection in the body of Chinese rhesus monkey infection models. By constructing SARS vaccine using modified attenuation virus vaccine (MVA) as vector, fundamental immunity and toxic material elimination are realized on rhesus monkey. The invention can screen ideal vaccines for the prevention and treatment of SARS.

Description

technical field [0001] The invention relates to a recombinant vaccinia-SARS vaccine and a preparation method thereof, in particular to a SARS vaccine ADS-MVA comprising a modified attenuated vaccinia MVA and a SARS coronavirus S gene and a preparation method thereof. Background technique [0002] Since SARS was first discovered and reported in Guangdong Province in November 2002, it quickly became popular around the world. WHO called it severe acute respiratory syndrome (Severe Acute Respiratory Syndrome, SARS). The disease is mainly transmitted through close-range air droplets and close contact, and has the characteristics of general population susceptibility, rapid transmission, acute onset and certain fatality rate. At present, there are 5,663 confirmed and suspected cases of SARS in the world, and 372 cases died as of May 15, 2004. In China, as of May 26, 2004, there were 5,316 cases, 315 cases died, 1,573 cases were suspected, and 2,742 cases were cured. . The widespr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/275A61P31/20C12N15/09C12N15/64
Inventor 秦川魏强高虹涂新明陈志伟张林琦何大一
Owner INST OF LAB ANIMAL SCI CHINESE ACAD OF MEDICAL SCI
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