The other three fragments (E3, E4 and E5 corresponding to nucleotide 690-3101, 3090-5437 and 5425 to the 3′-end) were produced by PCR with primer

pairs E3/E3′, E4/E4′, E5/E5′. Cycling parameters for three PCRs were as follows: initial denaturation at 94°C for 1 min, 30 cycles of 98°C for 20 s, 68°C for 3 min, and then 72°C for 10 min. The E3, E4 and E5 amplicons were cloned into the M-pSK vector with XbaI/PstI, PstI/EcoRI, and EcoRI/NotI sites, the resulting positive plasmids were designated pSKE3, pSKE4, and pSKE5, respectively. ABC294640 The M-pSK vector derived from pBluescriptSK (+) by removed T7 promoter and modified some restriction enzyme sites in the vector sequence, was synthesized by GenScript Biotech Company (Nanjing, China). To introduce the genetic tags into the genome of Asia1/JSp1c8, recombinant plasmid pSKE3Δ, which contained two synonymous mutations (1185A→G, 1185T→C) to eliminate the EcoRI site in the E3 fragment, were constructed by oligonucleotide-directed mutagenesis with PCR amplification of the parent plasmid pSKE3 using p1/p1′primer pair. PCR amplification was carried out for 18 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 1 min, and extension at 68°C for 8 min. All recombinant plasmids were confirmed by complete DNA sequencing.

learn more Primers used to construct full-length cDNA clones of Asia1/JSp1c8 are listed in table 5. Figure 5 Strategy used to construct FMDV Asia1/JSp1c8 full-length cDNA clone, pRDD. The location of restriction enzyme cleavage sites used to assemble the subcloned RT-PCR fragments (E1, E2, E3, E4, E5 and E12) are shown (numbered relative to nucleotide position in the virus genome). Thick lines and an open box represent the untranslated regions ADAMTS5 and the open-reading frame for the viral polyprotein, respectively. The thin line represents the vector sequence. FMDV cDNA is under the control of the T7 promoter. Table 5 Sequences of the primers used for the construction of a full-length cDNA clone and mutants of FMDV Asia1/JSp1c8 Name Nucleotide Sequence (5′→3′) Nucleotide Position (nt) E1 CAGGATCC TAATACGACTCACTATAGGGTTGAAAAGG GGCGCTAGGGTC 1-21 E1′ TAAAACTTAGGGGGGGGGGGGGGGGGGGGTGAAAG

361-390 E2 TTTCACCCCCCCCCCCCCCCCCCCCTAAGTTTTAC 362-391 E2′ CCTCTAGA CCTGGAAAGACCAGGC 677-700 E3 AGGTCTAGAGGGGTGACATTTTGT 690-713 E3′ GTCTGCAGCAGAAAGGTAAGGGAT 3078-3101 E4 CTGCTGCAGACTATGCTTACACTG 3090-3113 E4′ AAAGAATTC AATTGCTGCCTCATG 5414-5437 E5 AATTGAATTCTTTGAGGGAATGGTGCAC 5425-5452 E5′ TTGCGGCCGCTTT(38) 3′end P1 ACAAGGAAAGATGGAGCTCACACTTCACAAC 1168-1198 P1′ GTTGTGAAGTGTGAGCTCCATCTTTCCTTGT 1168-1198 TR1 ACTGCATTCATTCTGAGTGGGA 2960-2984 TR1′ GGCAAGATCACCACGCCGCGAGGA 3679-3703(D→G) TR2 TCCTCGCGGCGTGGTGATCTTGCC 3679-3703(D→G) TR2′ 5′-GAAGAAACTCGAGGCGACTTTGAC-3′ 4342-4366 TR3 TCCTCGCGGCGTAGTGATCTTGCC 3679-3703(D→S) TR3′ GGCAAGATCACTACGCCGCGAGGA 3679-3703(D→S) Nucleotide positions of primers used for cloning are shown: numbering according to Asia1/JS/CHA/05 (Genbank Accession: EF149009).