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Recombinant vaccine using flavivirus as vector

A yellow fever virus and carrier technology, applied in the field of virology and immunology, can solve the problems of high cytotoxicity, affecting the effect of vaccines, and potential safety hazards

Inactive Publication Date: 2007-03-14
上海天甲生物医药有限公司 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0013] (1) Most people (55%) have adenovirus antibodies in their bodies. When vaccinated, the antibodies combine with the adenovirus vector, which seriously affects the vaccine effect;
[0014] (2) When the titer is high, the cytotoxicity is greater;
[0015] (3) The carrier has the possibility of returning to the wild type, which has potential safety hazards
(c) The cleavage effect of the proteolytic cleavage site used is not very satisfactory

Method used

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  • Recombinant vaccine using flavivirus as vector
  • Recombinant vaccine using flavivirus as vector
  • Recombinant vaccine using flavivirus as vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0092] Preparation of full-length cDNA clone of yellow fever virus vaccine

[0093] The construction process is shown in Figure 1, Figure 2, and Figure 3:

[0094] 1. The full-length yellow fever virus vaccine cDNA is integrated into the pRS424 plasmid (Plasmid), and the yellow fever virus vaccine Yellow Fever virus Strain 17D (YF 17D) and pRS424 plasmid (ATCC#: 77105) are purchased from American Type Culture Collection (ATCC).

[0095] a) Convert the RNA of yellow fever virus YF 17D into three yellow fever virus cDNA fragments (5' end cDNA, 3' end cDNA and middle cDNA) by RT-PCR method. Add Not I restriction site and Sp6 enhancer sequence at the 5' end; EcoR I restriction site of YF cDNA at the 3' end of the 5' end cDNA fragment; add Xho at the 3' end of the 3' end fragment I restriction site; The two ends of the middle fragment include part of the same gene sequence as the 3' end of the 5' end cDNA fragment and the 5' end of the 3' end cDNA fragment.

[0096] Pri...

Embodiment 2

[0101] Prepare the SARS vaccine (the insertion site is 4572-4573nt of the yellow fever virus genome) modified by the gene fragment (2A) of the foot-and-mouth disease virus between NS2B and NS3 of the yellow fever virus cDNA

[0102] The construction process is shown in Figure 4.

[0103] 1. the full-length yellow fever virus cDNA clone that embodiment 1 obtains cuts out an about 2.2kb long fragment with Kpn I endonuclease and Nhe I endonuclease, and this fragment comprises the 3445th of yellow fever virus cDNA sequence The base at position -5576.

[0104] 2.以黄热病病毒cDNA克隆为模板,分别以GGATACAAGGTTCAGACGAAC(SEQID NO:10)和AACCATCGATTCGGGGCCAGGGTTGGACTCGTCTCCCGCAAGCTTAAGAAGGTCA AAATTCAACAGCTGCATATGCCACAAGACATCCCCACTTCTC(SEQ ID NO:11)为一对引物;并以AACCCTGGCCCCGAATCGATGGTTCGAGGCGCGCGACGCAGCGGTGACGTACTCTGGGATAT TCCCACTCCTAAGATCATC(SEQ IDNO:12)和CGCTGCCCAACCTCTAGCGGC(SEQ ID NO: 13) is another pair of primers, and then two DNA fragments are produced by PCR, and a 2A gene sequence is introduced into i...

Embodiment 3

[0115] Prepare the SARS vaccine (the insertion site is 2453-2454nt of the yellow fever virus genome) modified by the gene fragment (2A) of the foot-and-mouth disease virus between E and NS1 of the yellow fever virus cDNA

[0116] The construction process is shown in Figure 5.

[0117] 1. Cut out a fragment about 1 kb in length from the full-length yellow fever virus cDNA clone with Pst I. This fragment includes positions 1959-2782 of the yellow fever virus cDNA sequence.

[0118] 2.以黄热病病毒cDNA克隆为模板,分别以GTCAAGAACCCAACTGACACT(SEQ ID NO:18)和AAGGTCAAAATTCAACAGCTGAAAGAAGATGGCGCATCCTTG(SEQ ID NO:19)为一对引物;并以CAGCTGTTGAATTTTGACCTTCTTAAGCTTGCGGGAGACGTCGAGTCCAACCCTGGCCCCAACATGACAATGTCCATGAGC(SEQ ID NO:20)和CCAGACCCGGTTTGAAAACGG(SEQ ID NO: 21) is another pair of primers, then use the PCR method to produce two DNA fragments, in which a 2A gene sequence is introduced, and the 2A gene sequence is the gene fragment of foot-and-mouth disease virus (SEQ ID NO: 1 ). The above-mentioned two DNA f...

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Abstract

The invention provides a flavivirus carrier, wherein exogenous polypeptide expresxion components inserted into the genome of the flavivirus vector. The said expression components has in order from 5' to 3': (a) 5' end releasing components; (b) genes components for coding the exogenous polypeptide; and (c) 3' end releasing components. The said releasing components are selected from: nucleotide sequences for encoding self-hydrolase of foot-and-mouth disease viruses, nucleotide sequences for encoding a signal peptide hydrolase substrate, and their combinations. The carrier can provide antigen peptides of the virus and tumor in the host cells, thereby triggering an immune response against viruses and tumor.

Description

technical field [0001] The invention relates to the fields of virology and immunology, and more specifically relates to a yellow fever virus vector expressing foreign polypeptide (such as an antigen) and a recombinant vaccine containing the vector. Background technique [0002] Yellow fever virus (yellow fever vaccine, YFV) is spherical, with a diameter of 40-50nm. It is composed of an envelope and a nucleocapsid. The nucleocapsid is composed of a capsid protein (C) and nucleic acid. The protein (E) spikes, and there is an inner membrane protein (M) in the envelope, which participates in the assembly of the virus. The virus genome is a single-stranded positive-strand RNA with a total length of 11kb, which encodes structural proteins C, M, E and non-structural proteins NS1-NS5 in sequence from the 5' to 3' end. Viral RNA directly acts as mRNA in the cytoplasm and translates out Structural and nonstructural proteins. [0003] Yellow fever (yellow fever, YF) caused by yellow ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/63C12N15/86C12N15/33A61K48/00C12N7/01
CPCY02A50/30
Inventor 庞小伍顾心彬
Owner 上海天甲生物医药有限公司
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