Recombinant Expression And Antibacterial Activity Of Four Spider Toxins

Most spiders have venom glands and can secrete venom rich in bioactive substances.Because of its high specificity and selectivity,spider toxins are widely used in the research and development of new drugs and new-type biological insecticides.However,the natural spider toxin content is extremely low,and the chemical synthesis route is costly,which makes it impossible to be mass-produced.Therefore,the large-scale production of spider toxins by genetic engineering has become one of the new research hotspots in the field of biotoxins in recent years.In this study,representative genes U1,U3,U4 and U27 of four families were screened from the c DNA Library of venom gland of Heteropoda venatoria,and they were recombined and expressed by eukaryotic and prokaryotic expression systems respectively.Saccharomyces cerevisiae S78 was used as the host cell for eukaryotic expression,while Escherichia coli BL21(DE3)was used as the host bacteria for prokaryotic expression.And the biological activity of recombinant spider toxin was further explored.The specific results are as follows:(1)Target genes U1,U3,U4 and U27 were obtained from the DNA Library of the venom gland of the spider by PCR.Eukaryotic expression vectors p VT102U/α-U1,p VT102U/α-U3,p VT102U/α-U4,p VT102U/α-U27 were successfully constructed and transformed into Saccharomyces cerevisiae S78.After expression,recombinant spider toxins U1,U3,U4 and U27 were obtained.(2)Using eukaryotic expression vector as template,the target genes U3,U4 and U27were obtained,and the prokaryotic expression vectors p ET32a-U3-HIS,p ET32a-U4-HIS and p ET32a-U27-HIS were successfully constructed and transfected into the host strain E.coli BL21(DE3).The fusion proteins Trx-HIS-U3,Trx-HIS-U4,Trx-HIS-U27 were successfully expressed by IPTG.And the fusion protein was affinity-purified by Ni~+column using the HIS-tag in the fusion protein,and the fusion partner Trx-HIS was separated from the target protein at the formate cleavage site DP between HIS-tag and the target protein by formic acid.The recombinant spider toxins U3,U4 and U27 were successfully obtained by affinity purification with Ni~+column once again.(3)The inhibitory effects of four recombinant spider toxins expressed in eukaryotic cells on the growth of Staphylococcus aureus,Aeromonas hydrophila,Escherichia coli and Salmonella were different.Among the four eukaryotic expressed recombinant spider toxins,U3 and U27 showed stronger antimicrobial activity than that of U1 and U4.Among the four tested strains,S.aureus is the most sensitive to the recombinant spider toxins,while Salmonella is the least sensitive one.The three recombinant spider toxins through prokaryotic system did not show inhibition on the four tested strains of S.aureus,A.hydrophila,E.coli and Salmonella.