Editing Expression of HA NA using Baculovoirus (section)

== Generation of Recombinant Baculovirus ==

=== Cloning into baculovirus transfer-plasmids<br> ===

Clone H7 HA and N9 NA ectodomain (or any other HA or NA gene) into modified pFastBac transfer plasmids (see introduction). Vectors for HA containing a C-terminal trimerization domain and a hexahistidine tag, as well as for NA containing an N-terminal tetramerization domain and hexahistidine tag<br>

<br> Explanatory note: HA ectodomains expressed without a trimerization domain will be folded incorrectly, leading to loss of conformational neutralizing epitopes, especially in the stalk domain. Similarly, the NA enzymatic function relies on proper tetramerization.<br> <br>

=== Generation of bacmids<br> ===

<br>Transform baculovirus transfer-plasmids containing HA and NA genes into DH10Bac bacteria according using a baculovirus expression system per manufacturer's instructions. Isolate and purify bacmids using a mini or midiprep kit.<br>

=== <br> Rescue of recombinant baculovirus and verification of protein expression<br> <br> ===

Explanatory note: The following steps have to be performed aseptically in a laminar flow hood Perform this procedure independently for HA and NA (or any other gene of interest). <br>

Insect cells are usually grown at 28 °C without CO2. Sf9 cells for this protocol are grown in full TNM-FH insect cell media supplemented with 10% FBS, 1% Pen-Strep* and 0.1% Pluronic F68 solution, if not indicated otherwise, and passaged 1:4 twice a week. High Five cells are grown in SFX serum free media and passaged 1:15 twice a week.<br> <br>

* Addition of antibiotics should be considered optional throughout the protocol and adjusted according to the practices used in each laboratory.<br> <br>

1. Plate Sf9 insect cells in 6-well plates at a density of 2 x 105 cells/cm2 and let sit for 20 min in a 28 °C incubator (without CO2).<br> <br>

2. Mix 2 μg (conc. 0.5-2 μg/ul) of recombinant bacmid with 100 μl of TNM-FH medium (serum free, 1% Pen-Strep). Mix 6 μl of transfection agent with 100 μl of TNM-FH medium (serum free, Pen-Strep). Combine the two volumes, mix gently and let sit for 20 min at room temperature (transfection mixture). Include one mock-transfection as control. <br> <br>

3. Remove supernatant from plated Sf9 cells (cells will now stick to the plate) and replace with 2 ml of serum free TNM-FH medium containing Pen-Strep (1%). Add the complete volume of the transfection mixture (from step 1.2.2) and rock the 6-well plate carefully for 5 sec. Incubate in a 28 °C incubator without CO2 for 6 hr.<br> <br>

4. Replace media with fresh full TNM-FH media (containing 10% FBS, 1% Pen-Strep and 0.1% Pluronic F68). Incubate in a 28 °C incubator without CO2 for 6 days. Check cells occasionally under a microscope. Infected cells usually appear bigger, rounder, have enlarged nuclei, and start to detach. <br>

=== <br>Harvest of cells and confirmation of protein expression<br> ===

<br> 1. Harvest cells 6 days post transfection by centrifugation at 2,000 x g for 5 min at room temperature. The supernatant contains the recombinant baculovirus - this will be used to generate working stocks right away, or can be stored at 4 °C for years.<br>

<br> 2. To check protein expression collect pellets and resuspend in 500 μl of PBS. Mix 50 μl of the suspension with 50 μl of SDS-PAGE loading buffer (containing a reducing agent). Run SDS-PAGE and perform a Western blot analysis with an anti-hexahistidine antibody to confirm gene expression. Include cells from the mock transfection as negative controls.<br>

=== <br> Preparation of working stocks<br> ===

<br> 1. Plate 5 x 105 Sf9 cells/cm2 in a T175 cm2 flask and let sit for 20 min allow them to attach to the dish. Replace complete TNM-FH media with TNM-FH media containing 3% FBS (and 1% Pen-Strep, 0.1% Pluronic F68).<br>

<br> 2. Infect cells with 100 μl of the rescue supernatant. Incubate in a 28 °C incubator without CO2 for 6 days and check occasionally if cells get infected.<br>

<br> 3. Spin at 2,000 x g at room temperature for 5 min and collect supernatant. This is now your P2 stock. This stock can be stored indefinitely at 4 °C. Repeat the infection procedure using 100 μl of the P2 stock to infect cells. The resulting stock is called P3 or working stock, which can be stored at 4 °C or used directly for protein expression. The P3 stock usually contains 107-109 plaque forming units/ml.

<br>