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[Objective] The present paper aimed to construct the eukaryotic expression vector of rotavirus（Rotavirus, RV）non-structural protein1（NSP1）and 3（NSP3）and express it in human embryonic kidney epithelial HEK239 T cells, and then to study the preliminary effect of rotavirus NSP3 on the activity of IFN-β/ISRE reporter gene in vitro. [Method] First, the NSP1 and NSP3 coding genes of ape rotavirus SA11 strain were synthesized and cloned into the cloning vector pUCm-T, the target fragments of NSP1 and NSP3 were amplified by PCR, and then the eukaryotic expression plasmids specifically expressing recombinant proteins pRK-Flag-NSP1/pRK-HA-NSP1 and pRK-Flag-NSP3/pRK-HA-NSP3 were constructed by using eukaryotic expression vectors pRK-Flag and pRK-HA as skeletons. The plasmids were transfected into HEK293 T cells after restriction endonuclease digestion and sequencing. The expression of NSP1/NSP3 protein was identified by Western blot. Then, the recombinant plasmid pRK-Flag-NSP1/pRK-Flag-NSP3 and IFN-β-Luc/ISRE-Luc plasmid were co-transfected into HEK239 T cells. After stimulated by RIG-IN（RIG-I activation domain）, the effect of NSP1/NSP3 protein on the activity of IFN-β/ISRE reporter gene was determined by double luciferase reporter gene system. [Result] The full-length genes of NSP1 and NSP3 were cloned successfully, and the eukaryotic expression plasmids pRK-Flag-NSP1/pRK-HA-NSP1 and pRK-Flag-NSP3/pRK-HA-NSP3, of NSP1 and NSP3 protein of ape rotavirus strain SA11 were constructed. The successful expression of NSP1 and NSP3 protein was confirmed by transfection and expression of Western blot in HEK293 T cells. Recombinant plasmids pRK-Flag-NSP1 and pRK-Flag-NSP3 could significantly reduce the activity of double luciferase induced by RIG-IN. [Conclusion] NSP1 and NSP3 proteins can inhibit the activities of IFN-β and ISRE promoters stimulated by RIG-IN, which proves that NSP3 protein has the same function as NSP1 protein in inhibiting IFN-β signal pathway, which lays a theoretical foundation for further study on the mechanism of NSP3 protein regulating type I interferon signal pathway. [ABSTRACT FROM AUTHOR]

【Objective】This paper aimed to establish the 293 T and HT 1080 cell lines with CRISPR/Cpf1 gene by lentiviral vector and further to realize the high-efficiency gene editing. 【Method】AsCpf1 and LbCpf1 gene was amplified by PCR technique, which was inserted into the pLent-EF1 a-MF-CMV-GFP-P2 A-Puro vector. Monolayer of 293 FT cells were cotransfected with three plasmids psPAX2, pMD2.G and Cpf1 in pLent-EF1 a-MF-CMV-GFP-P2 A-Puro. The recombinant lentivirus expressing LbCpf1 and AsCpf1 was harvested, and the titer of virus was calculated by fluorescence microscope 48 hours later. The purified lentivirus was used to infect 293 T cell and the transduced cells were selected with puromycin and single cell colonies were isolated and verified for DNA, RNA and cDNA. Single cell colonies were transfected by sgRNA targeting the exons ABCG1 CDS regions to the functional domains of ABCG1 genes, and gene knockout efficiency was measured by DNA specificity and T7 EΙ digestion. 【Result】DNA sequnencing verified that Cpf1-pLent was constructed successfully, and the titer of recombinant lentivirus was greater than 10~6 TU/mL. Three cell lines expressing Cpf1 stably were obtained, two of which(AsCpf1-293 T-7 D and AsCpf1-HT1080 02-3 B) the targeted gene was knocked out successfully by T7 EΙ digestion. 【Conclusion】 293 T and HT 1080 cells stably expressing Cpf1 was successfully constructed, which could be a valuable tool for gene editing. [ABSTRACT FROM AUTHOR]