Human universal quick clone cdna 2

The DNA fragment coding residues 1–119 of Bombyx mori ß-1,3-glucan recognition protein (GRP) and the cleavage site of HRV 3C protease (3C) was amplified with polymerase chain reaction using KOD plus neo DNA polymerase (TOYOBO CO., LTD. Osaka, Japan) with the primers 5′-CATGCCATGGAGTACGAGGCACCACCGGC-3′ and 5′-GGAATTCCATATGCGGGCCCTGAAACAGCACTTCCAGAAATTCTACTCCTGGTGTTATTTCAGAG-3′ from pET-GRP-3C-His as a template^{41 (link)}. The DNA fragment coding hFGF5 residues 21–242 [hFGF5 (21–242)] (GenBank id: NM_004464.3) was amplified with the primers 5′-CACCCATATGCACGGGGAGAAGCGTCTCG-3′ and 5′-GCCCTCGAGAGGGCTAGGTGGCTTTTTCTTTTCAG-3′ from Human Universal QUICK-Clone cDNA II (Takara Bio USA, Inc., CA, USA) as a template. DNA fragments of GRP-3C and hFGF5 (21–242) were cloned into pET-21d ( +) (Merck KGaA, Darmstadt, Germany) to give pET-GRP-3C-hFGF5 (21–242)-His.

The DNA fragment coding an artificial signal peptide Secrecon-AA^{42 (link)} was amplified using the primer 5′-GGTTTGGTGTTATCGGCGGCGGCC-3′ from the oligonucleotide 5′-ATGTGGTGGCGCCTGTGGTGGCTGCTGCTGCTGCTGCTGCTGCTGTGGCCCATGGTGTGGGCCGCCGCC-3′. The DNA fragment coding hFGFR1 residues 142–356 [hFGFR1 (142–356)] (GenBank ID NM_023110.2) was amplified using the primers 5′-GCCGCCGCCGATAACACCAAACCAAACCGTATGCC-3′ and 5′-CGCCTCCGCCCCTCTCTTCCAGGGCTTCCAG-3′ from Human Universal QUICK-Clone cDNA II (Takara Bio USA, Inc.) as a template. The DNA fragment coding Gly-Gly-Gly-Gly-Ser-His-His-His-His-His-His [G4S-His] was amplified using the primers 5′-CCTGGAAGAGAGGGGCGGAGGCGG-3′ and 5′-GATCGAACCCTTTCAATGGTGATGGTGATGG-3′ from the oligonucleotide 5′-GGCGGAGGCGGAAGCCTGGAGGTGCTGTTCCAGGGCCCCCACCATCACCATCACCATTGA-3′ that was used as a template. The DNA fragment coding pcDNA was amplified using the primers 5′-CCATCACCATTGAAAGGGTTCGATCCCTACC-3′ and 5′-CGCCACCACATGGTGGTTCGATCCTCTAGAGT-3′ from pcDNA3.4 plasmid that was used as a template (Thermo Fisher Scientific, Waltham, MA, USA). Three amplified DNA fragments of Secrecon-AA, hFGFR1 (142–356), and G4S-His were cloned into pcDNA using in vivo assembly cloning^{43 (link)}. The resultant plasmid was named pcDNA-SecreconAA-hFGFR1 (142–356)-His.