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Nucleic acid constructs

A technology of nucleic acid constructs and constructs, applied in the fields of molecular biology and immunology, can solve problems such as the reduction of the expression level of early promoters of hCMV

Inactive Publication Date: 2008-04-02
POWDER JECT VACCINES INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Chapman et al (1991) mentioned above reported that the expression level of the hCMV immediate early promoter was reduced when hCMV intron A was not included

Method used

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  • Nucleic acid constructs
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  • Nucleic acid constructs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0520] Embodiment 1. Construction of hepatitis B virus surface antigen (HBsAg) carrier series

[0521] Many plasmid expression vectors expressing HBsAg have been constructed.

[0522] raw material

[0523] (i) pWRG7128 (Roy, M, et. al. Vaccine (2001) 19 :764-778), which contains the hCMV immediate early promoter sequence, the first exon, the first intron and part of the second exon of the hCMV major immediate early gene, with flanking regions (derived from HBV preS2 5' UTR sequence and 3' post-transcriptional response element) HBsAg coding sequence and bovine growth hormone polyadenylation region (BGHpA).

[0524] (ii) pJV7284, a derivative of pWRG7128, which replaces BGHpA with the rabbit globin polyadenylation region (RBGpA).

[0525] (a) pPJV7384 (CMV (intronless), HBV preS2 5'UTR and RBGpA)

[0526] Using standard conditions, pWRG7128 was amplified with JF93 (SEQ ID NO: 15) and F110 (SEQ ID NO: 16), Sal 1 and BamH 1 cuts to isolate the CMV promoter, exon 1 and pa...

Embodiment 2

[0534] Embodiment 2. Construction of herpes simplex virus glycoprotein D antigen (HSVgD) vector series

[0535] A number of plasmid expression vectors expressing HSV gD have been constructed.

[0536] raw material

[0537] (a) In pPJV7334, a derivative of pWRG7284 (pPJV 7284), the HBsAg coding sequence was replaced by in-frame Nhe1 immediately downstream of the ATG start codon, followed by a stuffer fragment with BamH1, followed by 5 of HBC Enh (enhancer). 'end.

[0538] (b) pWRG7202, a derivative of pGem3Z (Promega) with a stuffer fragment allowing for the fusion of the coding sequence to the human tissue plasminogen activator (TPA) signal peptide downstream of the Nhe1 site.

[0539] (a) pPJV7392 (CMV (native intron), HBsAg 3'UTR, HBsAg 5'UTR and RBGpA)

[0540]Use primer DS1 (SEQ ID NO: 21) and DA1 (SEQ ID NO: 22) from virus DNA stock (AdvancedBiotech, Inc, Columbia, MD) PCR amplifies the coding region of HSV2gD, and cut with Nhe 1 and EcoR1, with An insert is for...

Embodiment 3

[0550] Example 3. Construction of Flu M2 antigen carrier series

[0551] (a) pPJV7450 (CMV (intronless), HBsAg 5'UTR and RBGpA)

[0552] The Flu M2 coding region was PCR amplified from plasmid pFL-M2 (Joel Haynes, pPJV) using primers JF301 (SEQ ID NO:23) and JF302 (SEQ ID NO:24) and cut with Nhe1 and Bgl2 to create an insert fragment. Nhe 1 and Bgl 2 cut pPJV7401 to form a vector fragment into which the insert was ligated, resulting in pPJV7450.

[0553] (b) pPJV7452 (CMV (intronless), HBsAg 3'UTR, HBsAg 5'UTR and RBGpA)

[0554] The 3'UTR fragment was PCR amplified from pPJV7389 using primers JF84 (SEQ ID NO: 25) and JF225 (SEQ ID NO: 26), Bsp120I cut, T4 DNA polymerase fill in, Bgl 2 linker (cat#1036, New England Biolabs )connect. This fragment was then cut with Bgl2 and EcoR1 to isolate the insert containing the HBsAg 3'UTR and RBGpA region. Bgl2 and EcoR1 cut pPJV7450 to form a vector fragment into which the insert is ligated, resulting in pPJV7452.

[0555] ...

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Abstract

A nucleic acid construct comprising a chimeric promoter sequence and a cloning site for inserting a coding sequence operably linked to the chimeric promoter, wherein the chimeric promoter sequence includes: (a) hCMV immediate early promoter sequence (b) exon 1 and at least a part of exon 2 of the hCMV major immediate early gene; (c) replacing a heterologous intron in the intron A region of the hCMV major immediate early gene.

Description

technical field [0001] The invention relates to the fields of molecular biology and immunology, and mainly relates to reagents for nucleic acid immunization technology. More specifically, the present invention relates to nucleic acid constructs expressing polypeptides, especially antigenic polypeptides, especially influenza antigens, and nucleic acid constructs expressing adjuvant polypeptides, as well as nucleic acid immunization methods using such reagents. Background technique [0002] Gene therapy and nucleic acid immunization are promising approaches for the treatment and prevention of acquired and hereditary diseases. Such techniques enable the transfer of a desired nucleic acid into a subject for subsequent in vivo expression. Transfer can be accomplished by in vitro transfection of cells or tissues from a subject, and reintroduction of the transformed material into the host. Alternatively, the nucleic acid can be administered directly into the subject. [0003] Ea...

Claims

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Application Information

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IPC IPC(8): A61K39/245A61K39/145A61K39/39A61K9/16C12N15/85C12N15/44C12N15/38C12N15/31C12N15/36
CPCC12N15/85A61K2039/55544C12N2760/16122C12N2830/15C12N2830/42C12N2830/00A61K2039/53C12N2730/10122C12N2830/60C12N2710/16722A61K48/00C12N2710/16622C07K14/005A61P31/16A61K39/145A61K9/16A61K39/39
Inventor J·富勒
Owner POWDER JECT VACCINES INC
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