The overall goal of this procedure is to generate correctly folded purified influenza, hemagglutinin and neuraminidase surface glycoprotein for immunological assays and vaccination experiments. This is accomplished by first cloning the recombinant BMid carrying influenza genes, followed by generation of recombinant baula viruses. The second step is to use these recombinant baula viruses to express hemagglutinin and neuraminidase proteins in insect cells.
Next, the recombinant proteins are purified from the cell culture supernatant using affinity chromatography. The final step is to characterize these proteins to ensure good quality and biological functionality. Ultimately, the recombinant proteins are used to investigate immune responses to the influenza virus surface glycoproteins.
The main advantage of this technique over existing methods like bacterial expression of HA one, for example, is that the purified antigens are corrective folded and remain biologically active also, as opposed to using whole virus preparations for immunological assays. These reagents allow for measuring immune responses against the hemagglutinin and non neuraminidase proteins. Only Begin this protocol with generation of recombinant baula virus as described in the text protocol working aseptically in a laminar flow hood plate, SF nine insect cells in six well plates at a density of 200, 000 cells per square centimeter.
And let's sit for 20 minutes in a 28 degrees Celsius incubator without CO2. Then mix two micrograms of recombinant back mid with 100 microliters of Trico Leia knee medium formulation HINK or TNM fh. Then mix six microliters of the transfection agent with 100 microliters of TNM FH insect medium.
Combine the two volumes mix gently and let the transfection mixture sit for 20 minutes. At room temperature include one mock transfection as a control, which contains the transfection reagent but no DNA. Remove the SNAT from the plated SF nine cells and replace with two milliliters of serum free TNM FH medium containing 1%penicillin streptomycin solution.
Add the complete volume of the transfection mixture to the cells and rock the six well plate carefully for five seconds. Incubate in a 28 degrees Celsius incubator without CO2 for six hours. Replace the media with fresh full TNM FH media before incubating the plate under the same conditions for six days.
Check the cells occasionally under a microscope. Infected cells usually appear bigger. Rounder have enlarged nuclei and start to detach.
Harvest cells six days post transfection by centrifugation at 2000 G for five minutes at room temperature. The supernatant contains the recombinant baula virus and will be used to generate working stocks right away, or can be stored at four degrees Celsius for years. Check protein expression via SDS page and western blot and prepare the working stocks as described in the text protocol.
SF nine cells support growth of macular virus very well. However, their ability to secrete large amounts of recombinant protein is limited. Therefore, we use another insect cell line.
High five cells for the expression of secreted recombinant proteins. These cells do not support growth of balo virus to high titers, but they have a very high secretory capacity. Grow high five cells in SFX insect cell culture medium in T 175 square centimeter flasks passage every other day.
Splitting them at a one to three ratio. Grow five confluent flasks for a 200 milliliter expression culture to harvest and infect the high five cells. First, detach high five cells by tapping the flasks.
Collect the cell suspension and transfer into 50 milliliters centrifugation tubes. Spin the tubes at 1200 G for seven minutes at room temperature. Remove the supernatant by decanting and unite the pellets by resus, suspending them in 15 milliliters of P three stock.
Let the cells sit for 15 minutes in the flow hood. Transfer 200 milliliters of high clone SFX culture medium into a clean sterile 1000 milliliters shaker flask. Then transfer the P three cell suspension into the shaker flask seal with sterile aluminum foil before shaking at 70 RPM and 28 degrees Celsius for 72 to 96 hours.
To harvest recombinant protein from high five cell expression supernatants at 72 to 96 hours post-infection. Transfer culture supernatants into 500 milliliters centrifugation buckets. Spin at 5, 500 G for 20 minutes at four degrees celsius.
In the meantime, rinse the shaker flask three times with double distilled water. Next pipette. Three milliliters of nickel resin slurry into a 50 milliliter tube with 45 milliliters of PBS per 200 milliliters of culture.
Shake well before spinning for 10 minutes at 3000 G and room temperature following centrifugation. Decant the PBS and keep the pellet. Mix the insect cell supernatant with the nickel resin pellet and transfer into the already rinsed shaker flasks.
Incubate at four degrees Celsius for two to four hours while shaking. Next, mount a 10 milliliter polypropylene column on a clamp to a support stand. Remove both caps and place a beaker below the column to collect the flow through.
Following incubation of the shaker flask, start to transfer the supernatant slurry mixture onto the column. It will retain the nickel resin and only the supernatant will flow through. Pass the entire volume over the column.
Wash the resin four times with 15 milliliters of washing buffer. Let the washing buffer drain from the column and close the lower side with the cap. Add two milliliters of elucian buffer and let's sit for five minutes.
Open the column and collect the eit, keeping it on wet ice whenever possible. Repeat this three more times. An EIT that contains high concentrations of protein typically shows foam when shaken to exchange the buffer and concentrate the protein first, free-spin 15 milliliter ultra filtration centrifugation units with PBS at 3000 G and four degrees Celsius for 20 minutes.
Discard the flow through and load the EIT onto the spin column. Fill the column up with five milliliters of sterile ice cold PBS before spinning under the same conditions for 60 minutes. Discharge the flow through again, refill with 15 milliliters of ice cold PBS and spin for an additional 60 minutes.
Repeat this procedure one more time, then collect the buffer exchanged Concentrate which represents the highly concentrated recombinant protein. Keep the concentrate on ice at all times. Rinse the spin column with 400 microliters of sterile ice cold PBS and add this to the concentrate.
The protein can then be characterized as described in the text protocol. The HA and NA proteins expressed by the ENUE and Shanghai strains showed moderate expression levels ranging from 0.2 milligrams per liter culture for influenza A collected in Shanghai to 0.7 milligrams per liter for influenza. A collected in enue, neuraminidase or NA molecules of both strains showed moderate expression levels ranging from 0.2 milligrams per liter culture for influenza A collected in Shanghai to 0.7 milligrams per liter for influenza A collected in enue.
All four protein preparations had very little to no impurities with ha's being of higher purity than nas, which can be explained by the expression level. Influenza A collected in Shanghai HA was further analyzed using ELI SA with the broadly neutralizing stalk reactive human monoclonal antibody CR 9 1 1 4. As shown here in blue, this monoclonal antibody binds to a sensitive confirmational epitope in the stock domain and is used here as a probe for correct folding of this domain.
The antibody shows good binding as evidenced by increased absorbance at 490 nanometers, which gives confidence that the HA is indeed folded correctly. A second, Eliza with anti hemagglutinin of subtype seven mouse polyclonal serum was performed to confirm the identity of the obtained protein as hemagglutinin of subtype seven origin. After watching this video, you have a good understanding of how to express recombinant influenza virus, hemagglutinin, and neuraminidase proteins in your laboratory.
This process might also be useful for the expression of other viral or cellular membrane proteins. I wish you good luck with your experiments.
