Roles of the highly conserved amino acids in the second receptor binding site of the Newcastle disease virus HN protein

Liu, Yaqing; Chi, Miaomiao; Liu, Ying; Wen, Hongling; Zhao, Li; Song, Yanyan; Liu, Na; Wang, Zhiyu

doi:10.1186/s12985-019-1273-y

Table 1 Mutant primer sequences

From: Roles of the highly conserved amino acids in the second receptor binding site of the Newcastle disease virus HN protein

Name	Sequence (5′-3′) ^a
Vector1-FP	CTATCGTCTTGAGTCCAACCCGGTA
Vector1-RP	TACCGGGTTGGACTCAAGACGATAG
Vector2-FP	CTGGGTGAGCAAAAACAGGAAGGC
Vector2-RP	GCCTTCCTGTTTTTGCTCACCCAG
T167A-FP	CGGCACCCGCTACAGGATCAGGTTGCACTC
T167A-RP	CTGATCCTGTAGCGGGTGCCGGGATAAAATTC
G171A-FP	CTACAGGATCAGCTTGCACTCGGATACCCTC
G171A-RP	CGAGTGCAAGCTGATCCTGTAGTGGGTG
C172A-FP	GATCAGGTGCTACTCGGATACCCTCATTCGAC
C172A-RP	GTATCCGAGTAGCACCTGATCCTGTAGTGGGTG
R174A-FP	GTTGCACTGCTATACCCTCATTCGACATG
R174A-RP	GAGGGTATAGCAGTGCAACCTGATCCTG
C196A-FP	CTGGTGCTAGAGACCATTCACACTCAC
C196A-RP	GAATGGTCTCTAGCACCAGAAAATATCAC
D198A-FP	GTTGCAGAGCTCATTCACACTCACATCAG
D198A-RP	GTGTGAATGAGCTCTGCAACCAGAAAATATC
S202A-FP	CATTCACACGCTCATCAGTATTTAGCAC
S202A-RP	CTGATGAGCGTGTGAATGGTCTCTGC
R516A-FP	CATAACCGCTGTAAGTTCAAGCAGAAC
R516A-RP	GAACTTACAGCGGTTATGCGACTGCG
Y526A-FP	CAAGGCAGCAGCTACGACATCAACGTG
Y526A-RP	GATGTCGTAGCTGCTGCCTTGGTTCTGC
E547A-FP	CATTGCAGCTATATCCAATACCCTCTTTG
E547A-RP	ATTGGATATAGCTGCAATGCTGAGGC
Ch1-FP	CCGGGATTATTAGCTATGCCAACGACTGTTGATGGCTGTGTTAGAATACCCTCATTCG
Ch1-RP	TGGCATAGCTAATAATCCCGGCCCCGGCATTAACCTTATTTTCGCAGAGGGATAG
Ch2-FP	GGTTGCCAGGATATAGGAAAATCATATCATCAGTATTTAG
Ch2-RP	TCCTATATCCTGGCAACCTCGAGTAATTAGATTGTGAGTATAACAG
Ch3-FP	CGAAACAAAACACTCTCAGCTGGATATACAACATCAACG
Ch3-RP	TGAGAGTGTTTTGTTTCGGATGGCCAGTATGCGACTG
Ch4-FP	AATCATAAAAGCTTAGACACATTCAGGATCGTC
Ch4-RP	TAAGCTTTTATGATTTATTTCTGCAATGCTGAGGCAATAGG
De1-FP	TCCCTCTGCGATACCCTCATTCGACATG
De1-RP	GAGGGTATCGCAGAGGGATAGAATGATG
De2-FP	CTCACAATCATCAGTATTTAGCACTTGG
De2-RP	TACTGATGATTGTGAGTATAACAGTAGTGGG
De3-FP	GTCGCATAACATCAACGTGTTTTAAAG
De3-RP	CGTTGATGTTATGCGACTGCGGGATATG
De4-FP	GCATTGCAAGGATCGTCCCTTTACT
De4-RP	GACGATCCTTGCAATGCTGAGGCAATAG

The 10 amino acids in the second receptor binding site were mutated to A by over-lapping PCR. EcoRI-digested pBSK-HN was used as the template to create one PCR fragment with primers mutations-FP (forward primer) and Vector-RP (reverse primer). The same plasmid digested by XbaI was used as the template to generate the other PCR fragment with primers mutations-RP and Vector-FP. Two PCR products at each mutation point with homologous ends were purified and then transformed into DH5α competent cells to obtain the desired mutants. The similar methods were applied in constructing the Ch and De mutants with the primers shown in the above table. In addition, primers Vector1-FP/RP were used in obtaining mutants T167A, G171A, C172A, R174A, C196A, S202A, Ch1, Ch2, De1 and De2. Primers Vector2-FP/RP were used in gaining other mutants
^areplacement fragments are underlined in the primer sequences

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