Cloning And Bioinformatics Analysis Of Osteopontin Gene Of Echinococcus Alveolar

Objective Alveolar echinococcosis is a kind of invasive growth of human and animal diseases,most of which are primary in the liver.It has been found that OPN can promote the migration and metastasis of tumor stem cells.The OPN gene of Echinococcus multilocularis was cloned and its bioinformatics was analyzed.Methods Extraction of protoscolex of Echinococcus multilocularis from Meriones Unguiculatus infected with Echinococcus multilocularis.The protoscolex was cleaved with TRLZOL reagent,and the total RNA of Echinococcus multilocularis was extracted.The possible OPN gene conserved fragment(EMmg-13d08.q1k)was found from the NCBI gene database by blast.The PCR amplified target gene sequence and the Homologous Fragment of OPN gene of Echinococcus multilocularis were uploaded to the NCBI gene database again NCBI was used to identify,3 '-RACE PCR was used to synthesize the first strand of 3'-RACE cDNA with primers,Nest PCR was used as template,5 '-RACE PCR was used to synthesize the first strand of 5'-RACE cDNA,and 5 '-RACE cDNA was purified for Nest PCR.The software corrected and spliced the conserved sequence,3 '-RACE sequence and 5'-RACE sequence of OPN gene of Echinococcus.The amino acid sequence corresponding to open reading frame(ORF)of OPN was speculated,the secondary and tertiary structures of protein were predicted,and the in situ epitope analysis was carried out.Results Studies have shown that a full-length sequence of 1390 bp is obtained,which contains 411 adenines,297 cytosines,334 guanines,and 348 thymines,encoding 445 amino acids with a relative molecular mass of 50 363.75.(Pl)is 11.18.The entire peptide chain was searched for the full-length ORF of OPN,and it was found that it contained a complete ORF reading frame that was 849 bp in length and encoded 282 amino acids,starting at 96 bp and ending at 944 bp.The results of the predictive hydrophilicity analysis are considered to belong to hydrophilic proteins.The secondary structure predicted?-helix(h)accounted for 20.81%,?-sheet(e)accounted for 15.26%,?-turn(t)accounted for 10.08%,and random curl(c)accounted for 53.85%.Prediction of vesicular echinococcal osteopontin surface antigen by the jameson-wolf method.Possible epitope regions of the protein: 1-11,16-34,47-51,59-63,82-103,110-117,130-133,185-Amino acid residues.The non-uniform distribution of the hydrophilic region of vesicular bulb OPN mainly lies in: 1-10,16-35,48-51,61-73,68-93,97-117,169-176,204-266,231-252,263-297,312-320,326-346,355-361,372-379,386-394,408-424,451-463.At the same time,the OPN tertiary structure is predicted.Conclusion In this study,the OPN gene of Echinococcus multilocularis was cloned and analyzed by bioinformatics.At the same time,it was found that there was no good agreement between the hydrophilic site and the antigenic epitope in 227-234,447-453 amino acid residue.The full-length OPN gene sequence of Echinococcus multilocularis provides a new idea for the treatment,postoperative metastasis and recurrence of the patients in the late stage of Echinococcus multilocularis disease,and provides atheoretical basis for the endogenous factors and mechanism of the pluripotency of stem cells in the germinal layer of Echinococcus multilocularis.