B cell receptor accessory molecule CD79 gets involved in response against Streptococcus agalactiae infection and BCR signaling in Nile tilapia (Oreochromis niloticus)

CD3ε is involved in host immune response against challenges in Nile tilapia (Orechromis niloticus)

“…In order to obtain the open reading frame (ORF) of OnCD3ε , the predictive sequence of OnCD3ε (GenBank accession number: ) was obtained in NCBI, and the extracted spleen cDNA was used as template for PCR amplification (Wu et al, ). In a word, the procedure of PCR includes the following 95℃ for 5 min, then by 30 cycles of 95℃ for 15 s, 58℃ for 30 s, extend 72℃ for 30 s. The PCR products were prepared by 1% agarose gel electrophoresis and recovered by DNA gel kit (TaKaRa, Japan) linked to pMD18‐T, and then transferred into the E.coli cells (Wei et al, ).…”

“…The spleen leucocytes were segregated from Nile Tilapia by Histopaque ® 1077 (Sigma, USA) as previous reports (Mu et al, ; Wu et al, ). Simply, spleen was grinded in a flask, stacked on top of 1077 and centrifuged.…”

“…The procedure of qPCR is as follows: 95℃ for 5 min, then by 40 cycles of 95℃ for 15 s, 60℃ for 30 s, melt curve was obtained finally. The relative expression levels of OnCD3ε were calculated by using 2 −ΔCt method (Cuesta & Tafalla, ; Wu et al, ).…”

“…The spleen leucocytes were segregated from Nile Tilapia by Histopaque ® 1077 (Sigma, USA) as previous reports (Mu et al, 2017;Wu et al, 2019).…”

“…Samples were collected at time point. The relative expression levels of OnCD3ε were calculated by using 2 −ΔΔCt method (Livak & Schmittgen, 2001;Wu et al, 2019).…”