| Coenurosis is caused by larval stage of the cestode parasite Taenia multiceps in livestock animals. This parasite also causes zoonotic infections in humans, although the condition is believed to be relatively rare. The disease has caused huge economic losses. With the development of molecular biology techniques, it have necessitated the search for vaccination is one of the best control strategies.In this study, we aimed to research Taenia multiceps for vaccine candidates 1 pair of gene special primers were designed based on the published amino acid motifs of the 45W gene of Taenia ovis,Taenia solium and Taenia saginata. Total RNA isolated from activated oncosphere of Taenia multiceps, was used as template to generate complementary DNA by reverse transcription, the 813 bp DNA fragment was amplified by polymerase chain reaction, and clones it into pMD18-T vector. DNA sequencing confirmed the inserted fragment was Tm45W gene. The complete ORF is 765 bp, encoding a 255 amino acid residue polypeptide. The molecular mass of polypeptide was 27.623 kDa, its isoelectric point was5.08, also has the strong hydrophilicity and antigenicity. The sequence produced in this study was deposited in Genbank (Accession No. FJ514241). By blasting the homologous sequence in GenBank databases, the sequence of Tm45W gene of Taenia multiceps is 90.60% percent identity to Taenia multiceps of xin jiang,87.24% percent identity to Taenia saginata,84.05% percent identity to Taenia ovis and 78.17% percent identity to Taenia solium.By the technology of DNA recombination, the Tm45W gene was cloned into expression plasmid pET-32a(+), and was transformed to E.coli BL21. The recombinant bacteria were induced to expression through the changes of IPTG.. The results showed that a new protein band was found in SDS-PAGE with molecular mass of about 45 kDa, which was consisted of a 27.623kDa protein of the Tm45W gene and pET-32a(+)(20 kDa). The amount of proteinum was to peak at 8 h after IPTG induced. There were almost no difference to amount of proteinum in different IPTG concentration, all be high. By Western-blotting, the Tm45W was evaluated by immunoblot analysis using recombinant proteins. Sera from sheep with Taenia multiceps also reacted with Tm45W, did not react with negative control.Thirty mice were randomly divided into control groups (FCA group and PBS group) and another group which was immune to Tm45W recombinant protein, ten mice for each group. The 1st group was injected by S.C with recombinant protein (pET-32a(+)-Tm45W) thoroughly mixed with Freund's complete adjuvant. The 2nd group was injected by S.C with Freund's complete adjuvant. The 3rd group was injected by S.C with PBS (pH 7.5). Those injections were conducted for third times at a dose 0.1mL per 1 mouse. The interval between each injection was 14d. At 0d,7d, 14d,21d,28d,35d and 42d after the first immunization, the mice tails were cuted off to collect blood. And the blood at 4℃over a night and then centrifugated at 5000 r/min for 10min to collect serum which was storeed at-20℃. The results of ELISA to detect the antibody in the serum showed:there was a extremely significant difference between control groups and immune group (P<0.01), and no significant difference between group FCA and group PBS (P>0.05); the difference between IgG and IgG1 was insignificant (P>0.05), and IgG/IgG1 and IgG2a was significantly different (P<0.05); Immunized group,FCA group and PBS group between the three groups IgM/IgE antibody levels was no significant(P>0.05).
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