Cloning And Expression Of FABP And HSP Gene From Protoscolex Of Taenia Multiceps

Coenurosis caused by Coenurus cerebralis, the larval stage of the cestode parasite Taenia multiceps,is a fatal zoonosis. The larvainhabits the brain, spinal cord, skin, muscle and other parts of sheep, goats, cattle and other animals, while the adult worms inhabit in small intestine of dogs, wolves, foxes and other canids. Primarily, the diagnosis of Coenurosis mainly depend on clinlcal syptom and immunological diagnostic methods rely on native antigen. It is also wild spreaded in northwestern of China, and causes enormous economic loss in husbandry production.1. Cloning, Expression of FABP Gene from Protoscolex and Immunolocalization on Taenia multiceps.In this study, total RNA was extracted from protoscolex. Primers were designed based on FABP mRNA sequence of Taenia solium. Respectly, the fabp gene including a complete open reading frame were amplificated by RT-PCR technique. Tm-FABP sequence had84%identity to Echinococcus granulosus’FABP2gene (GenBank accession number AF321117.1)and96%identity to Taenia solium FABP(HQ259679.1)gene. The molecular weight and the isoelectric point of Tm-FABP were predicted to be15.1KDa and pI=8.61. The amplified fragments were inserted into pET-32a, and the recombinant plasmid were transformed into E.coli BL21(DE3).The recombinant proteins were expressed by E.coli BL21(DE3),and purified by passing the Ni+affinity chromatograph collumn. Western blotting demonstrated that the purified recombinant proteins could be recognized by sheep coenurosis positive serum. The antiserum of Tm-FABP produced by Tm-FABP vaccinated experimental rabbit was used in immunolocalization of FABP on collum segment, immaturate segment, maturate segment of Taenia multiceps. The results of immunolocalization of FABP demonstrated that Tm-FABP distributes in Cysticercus cerebralis, protoscolex cortex, cyst germinal layer, between cortex and parenchyma.2. Cloning and Expression of HSP Gene from Protoscolex of Taenia multiceps and Evaluation on the Applied Value of the Dot Immunogold Filtration Assay (DIGFA) for Rapid Detection of Coenurus cerebralis.In this study, total RNA was extracted from protoscolex. Primers were designed based on HSP mRNA or cDNA sequence of Taenia solium. Respectly, the fabp gene including a complete open reading frame were amplificated by RT-PCR technique. The molecular weight and isoelectric point of Tm-HSP was predicted to be14.7KDa and pI=4.88. The amplified fragments were inserted into pET-32a, and the recombinant plasmid were transformed into E.coli BL21(DE3).The recombinant proteins were expressed by E.coli BL21(DE3),and purified by passing the Ni+affinity chromatograph collumn. Western blotting demonstrated that the purified recombinant proteins could be recognized by sheep coenurosis positive serum. The purified Tm-HSP recombinant protein was used to develop a DIGFA for coenurosis diagnosis.The results of this experiment showed that the optimal concentration of recombinant is2mg/mL and the sensibility of DIGFA is83%. Unfortunately, we obtained the fact that Tm-HSP recombinant protein could take cross reaction with cysticercus tenuicollis and cystic echinococcosis postive serum.