Production of pB602L in Escherichia coli
The B602L gene of ASFV (Pig/HLJ/2018) was synthesized and cloned into
pGEX-6p-1 vector by Genscript Biotech Corporation (Nanjing, Jiangsu,
China). The recombinant plasmid pGEX-6p-1was transformed into E.
coli BL21(DE3) (Qiagen, Hilden, Germany) for pB602L expression. The
cells were cultured at 37℃ in LB medium containing 100 μg/ml ampicillin
and transferred to 18℃ when the optical density at 600 nm
(OD600) reached 0.5~0.6, then the
protein expression was induced by 0.3 mM IPTG for 16 h. The cells were
lysed using an ultrasonic crusher and then centrifuged for 40 min at 4℃
to remove the celluar debris. Glutathione Sepharose 4B (GE Healthcare,
Uppsala, Sweden) was used to purify the GST-tagged pB602L protein, which
was then treated with PreScission Protease to remove the GST taggs. The
purified pB602L protein was abtained by passing through anion-exchange
resin with Resource Q (GE Healthcare, Uppsala, Sweden), and identified
by SDS-PAGE and Western-blot.