SARS vaccine and its preparation method

A vaccine and coronavirus technology, applied in the field of virus disease vaccines and their preparation, can solve the problems of long development cycle, high safety, hidden dangers, etc., and achieve the effect of good immune protection effect and high safety.

Inactive Publication Date: 2007-08-01
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Inactivated vaccines are easy to prepare, but for highly pathogenic viruses such as SARS-Cov, there are high safety hazards in the production and use of inactivated vaccines; attenuated vaccines are relatively safe, but the development cycle is long. difficult emergency

Method used

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  • SARS vaccine and its preparation method
  • SARS vaccine and its preparation method
  • SARS vaccine and its preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] The preparation of embodiment 1, SARS vaccine

[0028] Adopt the Bac-to-Bac in vitro transposition system (article number: 10359-016, 10608-016) of Invitrogen Company and construct SARS vaccine of the present invention according to instructions, the flow chart of preparation method is as shown in Figure 1, and specific method comprises the following steps :

[0029] 1. Subcloning of the extracellular region cDNA of SARS coronavirus S protein

[0030] According to the cDNA full-length sequence of SARS coronavirus S protein gene (SEQ ID No. in the sequence table: 1) design primer, PCR amplification S protein extracellular region cDNA fragment, primer sequence is as follows:

[0031] Fs-12: 5'-ggg agtggtagtgaccttgacc-3' (the base in the box is the recognition site of the restriction endonuclease BamHI)

[0032] Rs-1190: 5'-attggg gttgctcatattttcccaattc-3' (the base in the box is the recognition site of restriction endonuclease Not I)

[0033] Using the plasmid conta...

Embodiment 2

[0038] The identification of embodiment 2, S-gp64 and S-vsvG recombinant virus surface S protein

[0039] 1. Western blot and immunofluorescence identification results

[0040] After infecting Sf9 cells with the S-gp64 and S-vsvG recombinant viruses obtained in Example 1, the S protein expressed in Sf9 cells and the purified S-gp64 and S-vsvG recombinant viruses were preliminarily analyzed by Western blot and immunofluorescence methods. For detection, the wild-type baculovirus was used as a control. The Western blot detection results of S protein expressed in Sf9 cells after 38 hours and 48 hours after infection are shown in Figure 3 (wt Bac is wild-type baculovirus), and in Sf9 cells after 38 hours and 48 hours after infection. The S protein band of about 175KDa can be detected, and the expression level of S protein is significantly increased after 48 hours after infection. The Western blot detection results of the S protein and the gp64 protein of purified S-gp64, S-vsvG r...

Embodiment 3

[0044] Mouse immunity and neutralizing activity test of embodiment 3, S-gp64 and S-vsvG recombinant virus

[0045] 0.1 mL of purified S-gp64 and S-vsvG recombinant viruses were injected subcutaneously into 6-week-old Balb / c female mice. On the 11th day after the initial immunization, a booster immunization was given at the same dose. On the 25th day, the serum neutralization The activity of antibody, neutralizing activity is to measure by measuring the infection inhibitory effect of mouse antiserum to a kind of S protein-HIV pseudovirus, the mouse serum immune with wild-type baculovirus is negative control (wt Bac), its There is no inhibitory effect on pseudovirus infection; the positive control (Positive, with a dilution ratio of 1: 200-3200 in the experiment) is the positive control (Positive) with the serum of a SARS recovered patient, which has obvious inhibitory effect on pseudovirus infection. The neutralizing activity detection result of low concentration pseudovirus in...

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Abstract

The invention discloses a SARS vaccine and preparing method, which comprises the following steps: 1) constructing external baculoviral surface display carrier of S protein of SARS coronary virus; 2) transmitting the carrier into susceptive cell with baculoviral genome plasmid Bacmid; obtaining recombinant baculoviral genome plasmid; 3) using the plasmid to infect insect cell; purifying the recombinant plasmid; obtaining the product.

Description

technical field [0001] The invention relates to a vaccine for viral diseases and a preparation method thereof, in particular to a SARS vaccine and a preparation method thereof. Background technique [0002] Severe Acute Respiratory Syndrome (SARS), also known as atypical pneumonia, is an acute respiratory infectious disease that became a global pandemic in 2003. It has been confirmed that the disease is caused by a mutated coronavirus, which the World Health Organization named SARS coronavirus (SARScoronavirus, SARS-CoV). There is no specific drug for SARS virus at present, so it is necessary to develop an effective virus vaccine as soon as possible. Traditional virus vaccines include inactivated vaccines and attenuated vaccines. Inactivated vaccines are easy to prepare, but for highly pathogenic viruses such as SARS-Cov, there are high safety hazards in the production and use of inactivated vaccines; attenuated vaccines are relatively safe, but the development cycle is lo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/215A61P31/14A61P11/00
Inventor 陈建国冯倩刘莹莹
Owner PEKING UNIV
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