Characterization Of Ddi1-like And Drug Availability Of Its Micromolecular Inhibitors In Taenia Solium

Parasitic worms,such as cysticercosis,have a serious impact on the development of human health and livestock breeding,and are widely co-infected with immunosuppressive diseases,for instance the disease caused by HIV.The current treatment against helminthiasis or co-infection of HIV and helminths is hightly limited,and there are serious drug interactions and side effects.DNA damage-inducible protein 1-like(Ddi1-like)protein,together with HIV proteinase,belongs to retroviral aspartyl proteases.The protein plays a fundamental and important role in eukaryotes and has been shown to be used as a target of HIV protease inhibitors(HIVPIs)for the treatment of co-infection of HIV and protozoa.Our previous studies have shown that there is a single Ddi-like protein coding gene in the tapeworm and trematode genome.In this study,we performed the cloning,eukaryotic expression and function study of Ddi1-like gene from Taenia solium(TsDdi1-like)to validate TsDdi1-like as a target for HIVPIs and explore the possibility of HIVPIs as a new drug for treatment of tapeworms or a single therapeutic agent for the co-infection of HIV and worms.We have achieved the following results:1.According to gene annotations of T.solium genome,the cDNA of Ddi1-like gene was amplified by RACE(rapid-amplification of c DNA ends)technique using the specific 5' and 3' primers,followed by the bioinformatic analyses.The results revealed that the full ORF of Ddi1-like gene was successfully obtained,which was 1173 bp in length,encoding 390 amino acids.Its coding protein contained the conserved motifs of retroviral aspartyl proteases(RVP)and ubiquitination-like(UBA).2.The recombinant plasmid pPIC9K-TsDdi1-like was successfully constructed.After induced by methanol.The recombinant protein was expressed in pichia pastoris,the molecular weight of which was about 55 kD.Enzyme kinetic experiments show that TsDdi1-like could not degade fluorescent substrates RE-(EDANS)-SQNYPIVQK-(DABCYL)-R and Bz-RGFFL-4M?NA,indicative of no hydrolytic activity.This result is similar to the case in most of the Ddi1 homologous proteins.3.By immunization of rabbits with specific synthetic peptide sequences and purification in eukaryotic expression,we obtained two specific polyclonal antibodies after four rouds of immunizations.The antibody titer of the anti-serum could both reach to 1: 12800,and both antibodies were highly specific in Wsetern-blotting analysis,implying that they do not cross-react with other proteins in the tapeworm.The results of immunohistochemistry showed that the results based on different polyclonal antibodies were highly consistent,that TsDdi1-like protein was expressed in all parts of scolex from Taenia solum,suggesting that TsDdi1-like may be involved in the process of important basic physiology.4.Making use of retro-complementation analysis for DDI1-knockout derivative Y16141,we demonstrated that TsDdi1-like was the key factor affecting the secretion of 4742 strains of S.cerevisiae.We futher proved that HIVPIs could effectively inhibit the activity of TsDdi1-like protein,and TsDdi1-like was one of the target molecules of HIVPIs.Meanwhile,by cocultivation of proscolex with HIVPIs,we found that HIVPIs nelfinavir could significantly inhibit the activity of the proscolex and lead to its death in vitro.