Sarcoptes Scabiei: Cloning, Prokaryotic Expression And Localisation Of A Gene Encoding Tropomyosin

Sarcoptic mange is a common and highly contagious human zoonotic parasitic disease, highly contagious, which can cause itching and various types of dermatitis, and is a stubborn, contagious skin disease. Over the years, medicines are still the main control of Sarcoptic mange, yet can be used for prevention and treatment of vaccine immunization. The traditional treatment of scabies is usually drug, it is not only ineffective, but also there are drug residues and environmental pollution. Although the development of parasite vaccine is difficult, especially genetic engineering vaccine, but the scabies treatment of genetic engineering vaccine will become the most promising methods with the continuous development of molecular biology, gene library of scabies mites continue to improve.Here scabies mites from the rabbit were collected, and a pair of primers was designed referencing GenBank. The 855 bp DNA fragment was amplified by polymerase chain reactions,encoding a 285 amino acid residue polypeptide. By blasting the homologous sequence in GenBank databases, the gene which encoded a predicted protein with 87.60%,85.26% and 85.03% identity to the Dermatophagoides farinae allergen Der p10, Dermatophagoides pteronys sinus allergen Der p10 and Psoroptes ovis allergen P. ovis 10 respectively. The molecular mass of polypeptide was 50.90 kDa, its isoelectric point was 4.43, also has the strong hydrophilicity and antigenicity. The derived chemical formula is C1391H2318N412O4878S10, with a negative charge. There is no signal peptide cleavage site and no transmembrane domain in the protein which is an extracellular protein.Linking the open reading frame of tropomyosin and the pET32a (+) expression vector, and positive clones were picked and sequenced.The correct recombinant plasmid pET32a (+)-Tm was transformed into E. coli BL21 (DE3), and induced expression of tropomyosin with the concentration of 1.0mM/L of IPTG, SDS-PAGE analysis showed that the molecular weight is about 50kDa, which was consisted of a 30.9 kDa protein of the Tm gene and pET-32a(+)(20kDa).The amount of proteinum was to peak at 4 h after IPTG induced. By Western-blotting, the Tm gene was evaluated by immunoblot analysis using recombinant proteins.For the accurate positioning of tropomyosin's position in the scabies mite,the purified recombinant protein was emulsified with Freund's adjuvant to inject rabbits with vaccine subcutaneous which obtain hyperimmune serum.The EnVision TM +System-HRP(DAB) (DAKO) was used, according to the manufacturer's instructions, for detection of the rabbit antibodies.The tropomyosin-encoding gene of S.scabiei was characterized and the tissue distribution of the proteins encoded by the gene was determined by immunolocalization. Result showed that tropomyosin gene present in the mouthparts, limbs, and internal organs, the epidermis of somato and internal organs, indicating that the gene exists in scabies mite body widespread.