Expression And Functional Analysis Of The Active Polypeptide

The expression and function of various active peptides were studied in this study,including several spider toxins and black carp interferon.The experiments contain heterologous expression and purification in E.coli,whole-cell patch-clamp experiments,real-time quantitative PCR detecting system,immunoblotting,immunofluorescence technique,antiviral experiments and so on.The results are as follows:1.We expressed and purified of neurotoxic Hainantoxin-? in E.coli,and the rHNTX-? has the similar activity to that of the natural one and it indicates that C-terminus amidation does not affect the electrophysiological activity and selectivity of HNTX-? to VGSCs.2.We expressed and purified of ProTX ?,GsAF ? and its mutants in E.coli,and the IC₅₀0 of rYC1 was determined to be 2.94?M for the rabbit Na_v1.3?rNa_v1.3?and the activity is between the ProTX ? and GsAF ?.3.The QPCR results showed that the mRNA transcription of some important genes in the RLR signaling pathway and the TLR signaling pathway were up-regulated?bcIFNb,bcMX1,bcTRAF3 etc?after Jing Zhao crude toxin stimulating.It suggested Jing Zhao crude toxin can active the innate immune signaling pathway of the black carp cell.4.bcIFNb gene was cloned and expressed through eukaryotic expression system:it consist 564 nucleotides and the predicted bcIFNb protein contains 188 amino acids,in which the first 20 amino acids represent a putative signal peptide.The calculated molecular weight of bcIFNb is 21 kDa and expressed in the cytoplasm.bcIFNb can against RNA virus GCRV and SVCV,which suggested that bcIFNb is an antiviral cytokine in the innate immune response.To sum up,we used the prokaryotic expression system to express and purify a variety of spider toxins.We firstly used spider toxin to stimulate the cells and studied the effects of RLR and TLR signaling pathway.Then the key gene bcIFNb in RLR signaling pathway was cloned and expressed in the eukaryotic expression system.These results provide a new idea for extract large amounts of active toxins and explore the relationship between toxins and cell immunity.