Preliminary Study On The Expression Of Shark Hepatic Peptide In Silkworm And Its Oral Absorption

The diabetes is a kind of serious threat to human health,and the incident of diabetes has increased year by year.The insulin is an effective drug for diabetes,but the insulin is not ideal.It was reported that active peptide from shark liver(APSL)could repair damage in islet,and promote recovery of islet function.As the source of the natural shark liver peptide drugs can not solve,we had the means through genetic engineering and used the silkworm bioreactor to prepare large-scale rAPSL for preliminary research on oral administration.APSL gene was obtained from the chiloscyllium plagiosum regenerated liver cDNA library.APSL gene contained an ORF of 342 bp,encoding 112 amino acids with a predicted molecular mass of 12.42 kD.To study the function of protein,we inserted its ORF into the enzyme site of the expression vector pET-28a(+),thus successfully constructing the prokaryotic expression vector pET-28a(+)-APSL.Then it was transformed into the conpetent celll E.coli BL21(DE3).After induced with IPTG,the analysis of SDS-PAGE showed that the fusion protein His-APSL was high level expressed in BL21,which accounted up to 41%of germ proteins.The most recombinant protein was insoluble incision body and only little fraction of recombinant protein of His-APSL was soluble.After rAPSL was purified with Ni-NTA,purity of the protein was about 94.6%.Two male New Zealand rabbits were immunized with fusion protein to prepare polyclonal antibody,whose titer was over 1:25600 measured by ELISA.Western blotting analysis showed that the specific reaction of the antibody was well.At the same time,Bac-to-Bac Baculovirus Expression System was used,and the ORF of APSL was inserted into the enzyme site of the transfer vector pFastBac HTA,and then the transfer plasmid pFastBac HTA-APSL was constructed successfully.Then the pFastBacHTA-APSL was transformed into competent E.coli DH10Bac cells.The transposition was occurred between pFastBac HTA-APSL and bacmid.By the blue-white screening and PCR identification,the recombinant bacmid(rBacmid-APSL)was obtained.The recombinant bacmid DNA was transfected into BmN cells by lipofectin-mediated method,and the recombinant baculovirus(rBv-APSL)was obtained.The recombinant virus was amplified and P4 recombinant virus was got after passaging through three generations.P4 virus infected silkworm larvae,and the expressed rAPSL was detected by Western blotting.Prepare two rAPSL oral formulations to carry out preliminary study on oral administration.The presence of rAPSL in serum of ICR mouse orally administered rAPSL was detected by ELISA.The ELISA results indicated rAPSL could be absorbed into ICR mouse after oral administration of the two formulations,making foundation for study on oral formulations of rAPSL.Pharmacodynamic research on oral administration of rAPSL to STZ-induced diabetic mouse is still in progress.