| The Spider-Venom peptides are rich in variety and have diverse functions in many fields,such as insecticide,bacteriostasis,medicine,etc.The Spider-Venom peptide can be used as pharmacological tools to study the mechanism of ion channels in basic research.The Heteropoda venatoria venom has toxic effects on insects,but not on rats.Therefore,the H.venatoria venom is a safer resource for drug development.The α2δ-1 was discovered as an auxiliary subunit of voltage-gated calcium channel.It plays a promoting role in the excitatory transmission of neurons,and is closely related to the occurrence of pathologic neuralgia and the drug resistance of analgesics.The interaction between voltage-gated calcium channel-assisted subunit α2δ-1 and H.venatoria venom peptides is an innovative research,which can promote the development of pharmacological mechanisms related to chronic pain and the development of new polypeptide drugs.The split-ubiquitin yeast two-hybrid system is a rapid and high-throughput screening technology for membrane protein interactions.However,the Spider-Venom peptide is a kind of secreted short proteins,and the α2δ-1 belongs to membrane proteins.The present split-ubiquitin yeast two-hybrid system cannot be directly applied to study the interaction between them.However,the optimal split-ubiquitin yeast two-hybrid system will greatly shorten the research cycle of the interaction between Spider-Venom Peptides and membrane proteins and promote the targeting research of peptide and receptor.The main work in this paper includes two parts.Firstly,the improved split-ubiquitin yeast two-hybrid system was made and verified for the application of the interaction between Voltage-gated ion channel and the Spider-Venom peptide.Secondly,the improved split-ubiquitin yeast two-hybrid system was employed to preliminarily screen the molecules targeted at the α2δ-1 in the H.venatoria venom peptides U5,U10,U13,U23,U25 and U28.The main results are as follows:1.The transmembrane sequence of TP was designed through bioinformatics analysis,and inserted into the C-terminus of the interesting protein by homologous recombination method.The improved vector is named p PR3-SUC-TP.The transformation of p PR3-SUC-TP into S.cerevisiae NMY51 showed that the transmembrane sequence TP had no toxic effect on NMY51.TP fused with m Cherry was successfully expressed and localized on the membrane of S.cerevisiae NMY51 cells.2.The mature peptide gene of JZTX-III was constructed into p PR3-SUC-TP,and the gene of sodium channel Na V1.5 was constructed into p BT3-N.The specific effect of JZTX-III on sodium channel Na V1.5 was verified by the improved split-ubiquitin yeast two-hybrid system,and the reliability of the improved system was verified by this experiment.3.The bait vector p BT3-SUC-YFP-α2δ-1 was successfully constructed by the homologous recombination method,and the yellow fluorescent protein was observed,indicating that α2δ-1 was successfully expressed in NMY51 cells.At the same time,the prey vectors related to U5,U10,U13,U23,U25 and U28 were constructed,and transformed into NMY51 to prove that the venom peptides have no toxic effect on NMY51.The peptides of U5,U23 and U25 show interaction with α2δ-1 through the improved split-ubiquitin yeast two-hybrid system.Based on the commercialized split-ubiquitin yeast two-hybrid system,this paper expands its application of the interaction between membrane proteins and secreted peptides.Three Spider-Venom peptides interacting with α2δ-1 were screened using the improved system,and these targeted peptides can be used for studying molecular functions of α2δ-1and related pharmacological research. |
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