一种流感病毒纯化的新方法

有学者利用离子交换层析和分子筛层析纯化甲型流感病毒。整个过程操作方便，可操作性强，避免了密度梯度离心等操作。

Hemagglutinin Stability

Stability of HA activity was investigated by titrating inactivated supernatant to different pH using 1M sodium hydroxide or hydrochloric acid while stirring in a beaker (pH was measured with an electrode and recorded for each reaction). Titrated supernatants were incubated for 2.5, 4.5 or 23h at room temperature and neutralized immediately afterwards. Neutralized supernatants were analyzed for HA activity. Testing the stability with respect to salt, 200μl of phospate buffer (10mM, pH 7.3) containing different concentrations of sodium chloride (NaCl) were mixed with 100μl of SEC-purifieed virus (150mM NaCl, pH 7.3) in microtiter plates. Plates were incubated for 2h at room temperature. After incubation, 100μl of each reaction were transferred into a sample tube and diluted 1:10 in appropriate phosphate buffer to reduce the concentration of NaCl to 150mM. Diluted reactions were anaylzed for HA activity.

Batch Adsorption

Batch adsorption was conducted in microtiter plates. Unused Sepharose Q XL resin (GEHealthcare Bio-Sciences, Sweden) was washed with water three times and adjusted to an approximate volume fraction of 62.5% in a graduated cylinder. 80μl of resin slurry (approximately 50μl of packed volume) were mixed with 120μl of phosphate buffer (10mM, pH 7.3) containing different concentrations of NaCl.

In the next step, 100μl of SEC-purified virus (dissolved in150 mM NaCl, 20mM phosphate buffer, pH 7.3) were added to each well resulting in a final volume of 300μl. Plates were incubated in a Thermomixer Comfort (Eppendorf, Germany) for 2h at room temperature shaking with 1000rpm. After incubation, 100μl of each reaction were transferred into a sample tube and diluted 1:10 in appropriate phosphate buffer to reduce the concentration of NaCl to 150mM. Diluted samples were anaylzed for HA activity and hcDNA content.

Chromatography

An ÄKTAexplorer 100 was used for column chromatography. SEC media (Sepharose CL-2B,Sepharose 4FF and Sepharose 6FF and Superdex 200p.g.) were packed into XK 16/40 or Tricorn 10/30 columns. AEC media were packed into Tricorn 10/15 columns. First experiments with ion-exchangers were conducted with HiTrap screening columns (all from GE Healthcare Bio-Sciences, Sweden). Void volumes were determined with BSA-coated latex beads of 100nm diameter (Postnova, Germany) or estimated from the virus-containing peak. The packed bed of each column was tested regularly by determination of the asymmetry and HETP with acetone (2% v/v dissolved in eluent). Specifications of columns are summarized in Table I. Columns were sanitized with 0.5M sodium hydroxide for at least one hour before and after experiments. Columns were stored in 20% (v/v) ethanol at room temperature. The feed material (i.e. virus concentrates or pooled SEC eluate fractions) was injected using a 50 or 150ml superloop (GE Healthcare Bio-Sciences, Sweden).

Eluates were either fractionated by a Frac-950 fraction collector (GE Healthcare Bio-Sciences, Sweden) or using the outlet valve of the chromatography SEC runs were conducted at a constant flow rate of 60cm h-1 using phosphate buffer (20mM, pH 7.3) containing up to 0.65M NaCl as eluent. Complete batches were processed automatically by multiple injection-elution cycles. The product fractions from each run were pooled and loaded onto an anion-exchange chromatography column. Presentation over Kav = (elution volume – void volume) / (column volume – void volume) instead of elution volume or retention time was preferred in scouting experiments for better comparibility between chromatography media. Area normalized concentrations (i.e. density distributions) instead of absolute concentrations were used for plotting HA activity, DNA and total protein such that areas below the curves can be compared between different analytes. AEC runs were operated at a constant flow rate of 200cm h-1.

The same eluent as for SEC was used to equilibrate columns and wash out unbound feed material. Virus was collected in the flowthrough. Adsorbed hcDNA and other impurities were desorbed by a salt gradient up to 1.5M NaCl. Columns were cleaned afterwards by a mixture of 0.1M hydrochloric acid and 2M sodium chloride followed by a mixture of 1M sodium chloride and 1M sodium hydroxide (5cv/23min contact time and 7cv/32min contact time, respectively).