Difference between revisions of "Team:Wageningen UR/Results/Viral Antigens"

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                             <li>Viral Antigens</li>
 
                             <li>Viral Antigens</li>
 
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                             <h1>Production of Viral Antigens</h1> </div>
 
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                            <p>In order to fully implement the point of care nature of our diagnostic, we developed a small battery powered device, which is easily brought into the field. The device is capable of measuring the fluorescence produced by our bacterial diagnostic system. The addition of a GPS sensor tags the location of the measurement for epidemiological analysis. By using 3D printing as well as the open source Arduino system, we were capable of producing an affordable product.
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                                <p>In order to safely produce antigens for phage display, two different appraoches were taken. The first focusses on the production of enveloped Virus-like-Particles (VLP). These particles are non infectious and a widely used as vaccines for various viral diseases. A second approach is to produce soluable <i>Strep</i>-tagged antigens. These antigens can be purified easier and can be direcly linked to <i>Strep</i>-tactin beads for antigen display in the phage-panning.
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                                <h2>Virus Like Particles</h2> </div>
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                                Three viruses were chosen for the production of antigens. Two alphaviruses (chikugunya and mayaro) and one flavivirus (Zika virus). These VLP's were generated using the invitrogen baculovirus expression system. The baculovirus expression system is widely used in biotechnology for recombinant protein expression by exploiting the life cycle of the virus in the host. Baculoviridae are DNA viruses which infect Lepidoteran insect species. During its infection cycle, two viral forms are recognized. The oclusion body derived virus (ODV) responsible for infection of the worm's midgut, and the budded virus (BV) responsible for the cell-to-cell spread of the virus. When insect cell lines are used in tissue culture, the ODV's are not necessary for infection, as no live worms are used. The proteins normally used for the occlusion bodies (polyhedrin and p10) can thus be used for recombinant protein expression in cell cultures. The advantage of using insect cells are the similar post translational modifications not possible in <i>E. coli</i>. To generate the VLP's, the cassette containing the structural proteins for the specific virus was placed in front of the polyhedrin or P10 promotor of the baculovirus, in this case Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV). This DNA is then used to transfect an <i>Spodoptera frugiperda</i> (<i>Sf-21</i>) cell line in order to produce the VLP's. <br>
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                                The Laboratory of Virology has contructs available for Zika virus (ZIKV) and Chikungunya virus (CHIKV), but not for Mayaro (MAYV). As the generation of such virus like particles is similar, only the generation of MAYV will be described below.
 
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                                                 RNA of MAYV was isolated from a Trizol sample. After the RNA was checked for degradation, cDNA was generated using Superscript RTII (see protocols). Specific primers were ordered to amplify the structural casette with attB1/2 overhanges (Gateway), after which phusion PCR was used to generate the MAYV inserts. <br>
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                                                 A BP Gateway reaction was used to insert the fragment in a pDONR207 vector, followed by an LR Gateway reaction to move the fragment to the pDEST8 vector introducing the required polyhedrin promotor suitable for the Bac-to-bac system. In paralel the fragment was directly cloned into pFastBacDual, another vector used in the bac-to-bac system, bypassing the gateway cloning, due to initial troubles with the gateway system. Both vectors  (pFBD and pDEST8) place the MAYV casette in front of a strong Polyhedrin promotor. Correctness of the vector was confirmed using restriction analysis, colony PCR and sequencing. The resulting expression clones are abreviated MAYV-D (pDEST8 derived) and MAYV-F (pFastBacDual derived).
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                                                Both contructs were used in the Bac-to-bac system, the resulting 136kb bacmids (plasmids containing AcMNPV DNA) were confirmed using colony PCR and isolated using a bacmid isolation protocol.<br>
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                                                Bacmids of MAYV-D and MAYV-F were used to transfect an Sf-21 at cell-line using ExpreSSion in a 6 wells plate, a healthy cell line was used as control/mock. The baculovirus infection was observed for 72 hours. The medium was then replaced and the cells were moved to a fresh T25 flask. After 48 hour the infection is clearly visible in figure A.
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                                            Working off the idea that we wanted a portable device quickly led us to a consumer 3D printer allowing us to do rapid prototyping. The printer we used is an Ultimaker 2+ provided to us by Ultimaker. We wanted a device that could show a fluorescent signal so we came we came up with our first designs. The fluorescent molecules produced by our system are excited by UV-LED’s with a specific wavelength. The emission spectrum is visible by the naked eye and this is how we planned to read out the signal. The membrane sealed sample vials containing our bacterial system are placed in the device from the top. Initially we started off with a device which fits one sample. However we soon realised that adding a positive and negative control makes out device more reliable. Once the button is pressed the UV-LED’s are turned on and the signal can be compared to the positive and negative control in order to determine the result. This approach was problematic since lighting conditions, background signal and observers are not a set variable. This prompted us rethink the way the device works and version 2 was on the way.
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Revision as of 14:04, 23 October 2017

Production of Viral Antigens

In order to safely produce antigens for phage display, two different appraoches were taken. The first focusses on the production of enveloped Virus-like-Particles (VLP). These particles are non infectious and a widely used as vaccines for various viral diseases. A second approach is to produce soluable Strep-tagged antigens. These antigens can be purified easier and can be direcly linked to Strep-tactin beads for antigen display in the phage-panning.

Virus Like Particles

Three viruses were chosen for the production of antigens. Two alphaviruses (chikugunya and mayaro) and one flavivirus (Zika virus). These VLP's were generated using the invitrogen baculovirus expression system. The baculovirus expression system is widely used in biotechnology for recombinant protein expression by exploiting the life cycle of the virus in the host. Baculoviridae are DNA viruses which infect Lepidoteran insect species. During its infection cycle, two viral forms are recognized. The oclusion body derived virus (ODV) responsible for infection of the worm's midgut, and the budded virus (BV) responsible for the cell-to-cell spread of the virus. When insect cell lines are used in tissue culture, the ODV's are not necessary for infection, as no live worms are used. The proteins normally used for the occlusion bodies (polyhedrin and p10) can thus be used for recombinant protein expression in cell cultures. The advantage of using insect cells are the similar post translational modifications not possible in E. coli. To generate the VLP's, the cassette containing the structural proteins for the specific virus was placed in front of the polyhedrin or P10 promotor of the baculovirus, in this case Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV). This DNA is then used to transfect an Spodoptera frugiperda (Sf-21) cell line in order to produce the VLP's.
The Laboratory of Virology has contructs available for Zika virus (ZIKV) and Chikungunya virus (CHIKV), but not for Mayaro (MAYV). As the generation of such virus like particles is similar, only the generation of MAYV will be described below.

RNA of MAYV was isolated from a Trizol sample. After the RNA was checked for degradation, cDNA was generated using Superscript RTII (see protocols). Specific primers were ordered to amplify the structural casette with attB1/2 overhanges (Gateway), after which phusion PCR was used to generate the MAYV inserts.
A BP Gateway reaction was used to insert the fragment in a pDONR207 vector, followed by an LR Gateway reaction to move the fragment to the pDEST8 vector introducing the required polyhedrin promotor suitable for the Bac-to-bac system. In paralel the fragment was directly cloned into pFastBacDual, another vector used in the bac-to-bac system, bypassing the gateway cloning, due to initial troubles with the gateway system. Both vectors (pFBD and pDEST8) place the MAYV casette in front of a strong Polyhedrin promotor. Correctness of the vector was confirmed using restriction analysis, colony PCR and sequencing. The resulting expression clones are abreviated MAYV-D (pDEST8 derived) and MAYV-F (pFastBacDual derived). Both contructs were used in the Bac-to-bac system, the resulting 136kb bacmids (plasmids containing AcMNPV DNA) were confirmed using colony PCR and isolated using a bacmid isolation protocol.
Bacmids of MAYV-D and MAYV-F were used to transfect an Sf-21 at cell-line using ExpreSSion in a 6 wells plate, a healthy cell line was used as control/mock. The baculovirus infection was observed for 72 hours. The medium was then replaced and the cells were moved to a fresh T25 flask. After 48 hour the infection is clearly visible in figure A.