Enhanced expression of picornavirus proteins

A protein and virus technology, applied in antisense single-stranded RNA viruses, positive-sense single-stranded RNA viruses, viruses, etc.

Inactive Publication Date: 2015-12-30
NOVAVAX
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Fusion proteins can be used to diagnose diseases and induce immune responses

Method used

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  • Enhanced expression of picornavirus proteins
  • Enhanced expression of picornavirus proteins
  • Enhanced expression of picornavirus proteins

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0088] Preparation and expression of FMDV polyprotein

[0089] The FMDVP1 polyprotein was cloned into two vectors BV1168 and BV1169 ( figure 1). The vector was identical except that BV1169 contained the N-terminal A / Indonesia / 5 / 05 signal peptide sequence (SEQ ID NO: 3; MEKIVLLLAIVSLVK). In both cases, the P1 polyprotein contains an N-terminal His6 tag. The protein and nucleotide sequences encoded by BV1168 are shown in Figure 4C and 4D . The protein and nucleotide sequences encoded by BV1169 are shown in Figure 4A and 4B .

[0090] Sf9 cells were infected with three clones of BV1168 and BV1169 at an MOI of 0.5 ffu / cell and incubated at 27°C, 150 rpm for ~70 hours. Crude harvests (cells and media) were analyzed by SDS-PAGE and Western blot, as figure 2 displayed in . Lanes are as follows: M-Marker. Lanes 1-3 show FMDVP1 protein expressed from BV1168 with a hexahistidine tag and no signal peptide. Lanes 4-6 show FMDVP1 protein expressed from BV1169 with a hexahist...

Embodiment 2

[0093] Enhanced Expression of Single FMDV Polypeptides

[0094] Expression of individual non-secreted proteins is enhanced when fused to a signal peptide. Sf9 cells were infected with baculoviruses expressing FMDVvp0 or FMDVvp1 proteins with and without signal peptides, respectively. Cells were harvested 70 hours after infection. Harvested crude material (ie cells and media) was analyzed by SDS-PAGE and by Western blotting with monoclonal antibodies specific for the recombinant protein. image 3 , panel (a), the left panel shows the expression of FMDVvp0. image 3 , panel (b), the right panel shows the expression of FMDVvp1 protein. In each case, the "+" recombinant protein contained a signal peptide and the "-" recombinant protein did not have a signal peptide. Arrows indicate expressed vp1 protein.

Embodiment 3

[0096] Expression of FMDV polypeptides containing single and multiple proteins

[0097] Signal peptides increase the expression of single proteins and proteins in tandem. Figure 5 shows increased expression with various constructs. Sf9 cells were infected with recombinant baculovirus (BV) expressing proteins 1, 2, 3, and / or 4 with (+) or without (-) signal peptide, and harvested ~65 hours post infection. Crude samples (ie cells and media) were harvested and analyzed by SDS-PAGE and Western blot. Expressed recombinant proteins are indicated by dots. BV1 expresses FMDVvp1. BV2 expresses FMDVvp0vp3. BV3 expresses FMDV vp1, vp0 and vp3. BV4 expresses the FMDVP1 polyprotein (ie vp0-vp3-vp1). Figure 5A Total protein staining is shown. Figure 5B and 5C Binding of vp1 and vp2 antibodies is shown, respectively. Note that the vp0 protein contains the vp2 protein. In each case, increased protein expression was obtained.

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Abstract

The disclosure provides fusion proteins containing N-terminal signal peptides fused to immunogenic polypeptides. The immunogenic polypeptides may be from viruses, bacteria, or fungi. The disclosure also provides elevated expression of the immunogenic polypeptides using the N-terminal signal peptide. The N-terminal signal peptides enhance synthesis of the protein, particularly where the protein is neither a secretory nor a transmembrane peptide. The fusion proteins may be used to diagnose disease and to induce immune responses.

Description

[0001] Cross References to Related Applications [0002] This application claims priority to US Provisional Application Serial No. 61 / 790,114, filed March 15, 2013. Its content is hereby incorporated in its entirety for all purposes. [0003] Cross Reference to Sequence Listing [0004] This application incorporates by reference the contents of the 24 kb Sequence Listing entitled "NOVV_52_SeqList.txt" created on March 14, 2013. Background of the invention [0005] Humans and animals suffer from diseases and conditions caused by pathogens such as bacteria, fungi and viruses. The use of vaccines to induce immunity against pathogenic infection is well known in the art. Various methods have been used historically to prepare vaccines, including killed virus and live attenuated vaccines. However, killed virus and live attenuated virus can be associated with adverse effects, and in some cases killed virus is not effective or can actually exacerbate disease. [0006] The efficien...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62C07K7/08C07K14/09C12N5/10A61K39/135
CPCC07K2319/02C07K2319/21C07K2319/40A61K39/12C12N7/00C12N2760/16122C12N2770/32122C12N2770/32134C12N2770/32151A61K47/646C07K14/005A61K39/135
Inventor M·J·马萨尔
Owner NOVAVAX
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