Construction of Gallus gallus RIPK2 Gene Lentiviral Interference Vector and Screening of HD11 with Stably Low Expressing RIPK2
SUN Hong-Yan1,2,*, CHEN Ting-Hong1, WU Yu-Xiang1, SUN Chang-Hua3, LI Huan3,*
1 College of Animal Science and Technology, Yangzhou University, Yangzhou 225009; 2 Joint International Research Laboratory of Agriculture and Agri-Product Safety of Ministry of Education of China, Yangzhou 225009; 3 College of Biological and Chemical Engineering, Yangzhou Polytechnic College, Yangzhou 225012
Abstract：In a previous study, by high-throughput sequencing, receptor interacting serine/threonine kinase 2 (RIPK2) was demonstrated to be highly up-regulated in the immune and inflammatory response of chickens (Gallus gallus). To investigate the function of RIPK2-mediated NOD/RIPK2 signaling pathway in chicken macrophage cell line (HD11) for immunity and inflammation, a chicken HD11 stably interfering with RIPK2 gene was established by using Lentivirus-mediated RNA interference approach. According to the sequence of chicken RIPK2 gene, three RNA interference target sequences and one negative control sequence were designed for RIPK2 gene, recombined with the pLVshRNA-EGFP(2A) Puro interference vector, and then transiently transfected into HD11. Interference efficiency of each target on RIPK2 was tested by qRT-PCR and the recombinant vector with high interference efficiency was packaged with Lentivirus to transfect HD11. qRT-PCR and Western blot were used to detect the expression changes of RIPK2 and the downstream key genes IKKα (component of inhibitor of nuclear factor kappa B kinase complex), IKKβ (inhibitor of nuclear factor kappa B kinase subunit beta), NFκB (nuclear factor kappa B subunit 1) and IL1β (interleukin 1 beta) of NOD/RIPK2 signaling pathways in different experimental groups. Meanwhile the RIPK2 overexpression vector (pcDNA3.1-RIPK2) was constructed for the rescue experiment of RIPK2 gene. Three recombinant plasmid of RIPK2-shRNA1 (small hairpin RNA 1), RIPK2-shRNA2 and RIPK2-shRNA3 were successfully constructed. The recombinant plasmids of RIPK2-shRNA1 and RIPK2-shRNA3 had the most obviously inhibitory effect of (77.4±0.61)% and (90.21±0.68)%, respectively on the RIPK2 gene. The RIPK2-shRNA3 recombinant plasmid was packaged into retrovirus with a virus titer of 2×108 TU/mL. After the RIPK2-shRNA3 Lentivirus was transfected into HD11, the results of qRT-PCR and Western blot showed that RIPK2 mRNA and protein expression level in the interference group were significantly low compared to control group (P<0.05). The expression of downstream key genes IKKα, IKKβ, NFκB and IL1β of the NOD/RIPK2 signaling pathway were significantly down-regulated (P<0.05). The interference group was transfected with RIPK2 overexpression vector (pcDNA3.1-RIPK2), and the expression level of RIPK2 was recovered. In this study, a lentiviral vector expressing shRNA targeting chicken RIPK2 was successfully constructed, which can effectively silence the expression of RIPK2 gene in HD11. Moreover, HD11 stably expressing RIPK2-shRNA3 can interfere with NOD/RIPK2 signal transduction. The results will provide a theoretical basis for further research on the function of chicken RIPK2 gene and its mediated NOD/RIPK2 signaling pathway.
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