Mapping of IgE-binding regions on recombinant Cyn d 1, a major allergen from Bermuda Grass Pollen (BGP)

Table 1 Sequence of primers used for cloning rCyn d 1 and of Cyn d 1 fragments and deletion mutants in expression vector pQE30Xa.

Primer Name	Sequence (5' – 3')
λgt11Fwd	GGTGGCGACGACTCTGGAGCCCG
λgt11Rev	TTGACACCAGACCAACTGGTAATG
pQEFwd	GTGAGCGGATAACAATTTCACACAG
pQERev	GTTCTGAGGTCATTACTGGATC
F1Rev	CCATCCTTAAGCTT GAAGATGGGTTCGTTG
F2Rev	GATGAGGACAAGCTT GGGCTCGCCGGAGC
F3Rev	CTCCTCTCCAAGCTT CTTCTTGGCCCATGGC
F4Rev	GCCATCGCCAAGCTT GGCAGCGTACTTCAC
F5Fwd	GTGAAGTACAGCGCT GCCGGCGATGGCAAC
F6Fwd	CTGCGCAAGAGCGCT GGCGAACTGATG
F7Fwd	GCAAGGAGCCCAGCGCT GAGTGCTCCGGC
F8CynFwd	GGCGCATGGAGCGCT ATGGGCGACAAGCCG
F8CynRev	GCATCAATGCAAGCTT TCAGAACTGGATC
D120Fwd	CTCCTCTCCGGATCC CTTCTTGGCCATGGC
D170Fwd	GTGAAGTACGCTGGATCC GCCGGCGATGGC
D224Rev	GACATCGTCCTGAAGCTT TTCGACATGGCC

The sequences of the 7 overlapping fragments of varying lengths covering the entire rCyn d 1 allergen molecule (previously cloned from the cDNA expression library) were amplified using specific primers designed to introduce restriction sites at both ends and were inserted as Stu1/Hind III-fragments in the correct reading frame downstream from the 6× His tag encoding gene sequence of the pQE30Xa vector. The deletion mutants were cloned using the Bam H1 and Hind III restriction sites on the expression vector. Restriction enzyme sites are italicised and in bold.

ISSN: 1476-7961