Establishment And Identification Of Cell Lines Transcribing Shrna Targeted To Jiv Gene

Classical swine fever (CSF), caused by classical swine fever virus (CSFV), is an acutefebricity contagious disease, which is widespread, serious and has an important effect ondevelopment of pig industry. The protein Jiv which is a cellular molecular chaperone of pig,plays an important role in CSFV multiplication.To further study the influence of Jiv on viralRNA replication, search gene segment which can regulate CSFV multiplication, and provideexperimental material and theoretical basis for the study of transgenic pigs against CSF, wedesigned four pairs of shRNA targeted to Jiv, constructed lentiviral vectors. Then PK-15celllines transcribing shRNA targeted to Jiv were established to research its function in resistingCSFV. Based on the results obtained on PK-15cells, the fetal fibroblast cell line transcribingshRNA for Jiv gene was established, and provide cytoblast for nucleus transplantation. Theresult as follows:(1) The detective method of CSFV by real-time PCR was established using a pair ofprimers and an internal TaqMan fluorogenic probe derived from the NS2region of CSFVgenome. The varieties of conditions were optimized and the standard curve was achieved byusing quantitative concentration of serial10fold dilutions of recombined plasmid DNA. Thelinear range was from1.0×109copies/μL to1.0×101copies/μL. This method has highsensibility, specificity and good reproducibilities, which can be applied to detectconcentrations of CSFV.(2) Four pairs of RNAi fragments targeted to Jiv were designed, and according to thesesequences, four lentiviral vectors P1, P2, P3and P4which harboring RNAi sequences andcontrol vector NC were constructed. These vectors were transformed into PK-15cells,respectively and the cell lines transcribing shRNA for Jiv were established by screening andidentifying. The result of real-time PCR showed that, compared to the control groups, themRNA expression of Jiv gene in four cell lines which transformed with lentiviral vectorsdecreased in varying degrees, the cell line P2PK-15decreased about95%particularly.Whencells infected with CSFV after72h, the result of real-time PCR showed that CSFV RNA infour cell lines decreased, and the cell line P2PK-15decreased about85%compared to thecontrol groups, indicating that the lentiviral vector P2could interfere the replication of CSFVRNA and could be chosen as a priority vector for resisting CSFV.(3) Fetal fibroblast of pig was set up from pig embryos at40d of gestation by using tissue culture method. After transformed with lentiviral vectors P2, the fetal fibroblast cellline transcribing shRNA for Jiv was established by screening and identifying, and positiverate was99%. These positive cells were used to produce transgenic pigs.