The Effect of TLR9 Agonist CpG Oligodeoxynucleotides on the Intestinal Immune Response of Cobia (Rachycentron canadum)

Table 1

Primer name, sequence, product size, and target gene details used in this study.

(a) Primers used for cloning of TLR9 isoform B

Primer name	Primer sequence (5′-3′)	Application
TLR9F1^a	ATCTCAGCCACAATCAGATC	Partial cloning
TLR9R2^a	AGTTTGGGAAACATCTCATC
TLR9B1 RacF^b	CTGTGAATACCCTGAGTCTCAACAGGG	RACE
TLR9B RacR^b	GTGATGCTGTGAATCAGTACCAGCC
Universal primer mix (UPM)	Long 0.2 μM CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT; Short 0.4 μM-CTAATACGACTCACTATAGGGC
Nested universal primer mix (NUP)	AAGCAGTGGTATCAACGCAGAGT
TLR9B1-NGSP1	GCCTTTATGTCCTGCCCAAAGCACC
TLR9B-NGSP2	CTTCCTCGTCTGTTCCATCTTGGCTGT

(b) Primers used for gene expression studies

Primer name	Primer sequence (5′-3′)	Product size (bp)	Target gene	Gene bank accession number	Application
B3-5^c	ACAGACTGTTCCTCCTCCCC	532	3′UTR	HM754627	cDNA quality
B3-2^c	GAAACCTCCAACAGCGGG
RAF	AAGGACCTGTACGCCAACA	330	β-Actin-3	HM754627
RB1	TGGCGTCTCGCATCGTTT
TLR9A-RC F	TCTGTTCCATCTTGGCTGTG	160	TLR9A	KC180322	qRT-PCR
TLR9A-RC R	CTGGTTTCTGGTGTCAAACA
TLR9B-RC F	GCCTTCCTCGTCTGTTCCAT	178	TLR9B	JX073035.1
TLR9B-RC R	ACAGCCTGGTTTCTGGTGTC
MyD88-F1Q	GAGGTGTAAGAGGATGGTGGT	183	MyD88	KF018033.1*
MyD88-R2Q	GGTGGGGAATGGCTTTGTCAT
RCIL-1βF1	CAGGCAGAACAACCACTGAC	170	IL-1β	AY641829
RCIL-1βR2	TTCCAAGTCCAGTCCTTTGG
RCMxF1	TGGACATAGCAACCACAGAG	157	Mx	AY834185
RCMxR2	TTCTTCAGGTGGATGACCTC
RCIgM-F1	AGACAGCCTGCAGGGAAAAG	182	IgM	JX025102
RCIgM-R2	TGTTCCTTTCCCCCAGTAGT
CC1eF	ATTACAATAAGAACCCTGTGC	185	CC	JF975593
CC1eR	TCTTTCCTGGGATGGATTTG
β-Actin RC-F	ACAGACTGTTCCTCCTCCCC	160	β-Actin-3	HM754627
β-Actin RC-R	AAAATCCTGAGTCAAGCGCC

aPartial sequence primers.
^bTouchdown primers for 5′ and 3′ designed from TLR9 isoform B partial sequence.
^cPrimers designed from 3′UTR of cobia β-actin-3.
*Partial sequence cloned in this study.