0 (Table 4). The PCR cycling eFT-508 manufacturer conditions for amplifying EV71 vp1s, EV71 vp4s and CA16 vp4s consisted of 4 min at 94°C, followed by 35 cycles of 94°C 30 s, 52°C 30 s, 72°C 1 min, and then 72°C for 7 min. The steps for amplifying EV71 vp4s were the same as those for amplifying the other 3 protein genes except for annealing temperatures at 55°C for 30 s. Agarose gel electrophoresis and EasyPure Quick Gel Extraction Kit (Trans Gen Biotech, China) were used to purify those amplified products.

The purified products were ligated to pGEM-T cloning vector (Promega, USA) for transformation into competent DH5α cells. Positive clones were identified by White-Blue colony selection and sequencing (Invitrogen Co). Table A769662 4 Primers used for cloning and sequencing primers sequences fragments (bp) EV71-VP1-1F 5′-TGAAGTTRTGYAAGGATGC-3′   EV71-VP1-1R 5′-CCACTCTAAAATTRCCCAC-3′ 993 EV71-VP4-1F 5′-CTACTTTGGGTGTCCGTGTT-3′   EV71-VP4-1R 5′-GGGAACTTCCAGTACCATCC-3′ 655 CA16-VP1-1F 5′-ACTATGCAAGGACACWGAG -3′   CA16-VP1-1R 5′- check details CAGTGGTGGAAGAGACTAAA-3′ 1076

CA16-VP4-1F 5′- GGCTGCTTATGGTGACAA-3′   CA16-VP4-1R 5′- CATGGGAGCTATGGTGAC-3′ 1090 F referred as forward primer and R referred as reverse primer. Olopatadine Expression and Purification of VP1s and VP4s The pET-30a vector with an N-terminal His·Tag/thrombin/S·Tag™/-enterokinase configuration plus an optional C-terminal His·Tag sequence with endonuclease sites of BamH׀and Xho׀and the pGEX-4T-1 vector with an N-terminal GST (glutathione S-transferase) ·Tag/thrombin configuration with endonuclease sites of EcoR׀

and Xho׀were used for expressing VP1s and VP4s, respectively. The virus isolates selected for expression were s67 (for VP4 of EV71), s108 (for VP1 of EV71), s390 (for VP1 of CA16) and s401 (for VP4 of CA16). The genes were purified with agarose gel electrophoresis and EasyPure Quick Gel Extraction Kit after being amplified by PCR with corresponding primers (Table 5). The cycling condition for amplifying VP1s of EV71 and CA16 consists of 95°C for 4 min, followed by 35 cycles of 95°C 30 s, 55°C 30 s, 72°C 1 min, and then 72°C for 7 min. The steps for amplifying VP4 of EV71 and CA16 were the same as those for amplifying the VP1s, except that the annealing temperatures were 50°C and 57°C respectively.