Deficient of glycosylation site in the Envelop protein attenuated Zika virus replication in mosquito cells
Description
western blotting
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Total RNA was extracted from tissue samples using Trizol (Solarbio, 15596-018) and purified by phenol-chloroform extraction (P.C.I). The RNA concentration and purity were measured using Nanodrop 2000, and RNA integrity was assessed by agarose gel electrophoresis. cDNA was synthesized using the StarScript III RT Kit (GenStar, A232-10) according to the manufacturer's instructions. Quantitative PCR was performed using the TB Green® Premix Ex Taq™ II (Tli RNaseH Plus) (TaKaRa, RR820A) on an Applied Biosystems QuantStudio system, with specific primers for the Envelope gene and the housekeeping gene GAPDH. The thermal cycling conditions were: 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds, and 60°C for 1 minute. Gene expression was calculated using the ∆∆Ct method and normalized to the housekeeping gene GAPDH. The sequences of the ZIKV Envelope primers were as follows: sense, 5’-CAATCAAGTCCTAGGCTTCCA-3’; antisense, 5’-ATCCAGCCAGAGAATCTGGAGT-3’. The sequences of the GAPDH primers were as follows: sense, 5’-AGAAGGCTGGGGCTCATTTG-3’; antisense, 5’-AGGGGCCATCCACAGTCTTC-3’. The following amplification program was used: reverse transcription at 42°C for 5 minutes with an incubation at 95°C for 10 seconds, followed by 40 cycles of 95°C for 5 seconds and 60°C for 20 seconds. Information collection and melt curve analysis were done following the instrument’s operation manual.