| Coenuriasis is caused by Coenurus cerebralis,the metacestode stage of Taenia multiceps,which usually inhabits the central nervous system and subcutaneous and intramuscular tissues of cloven hoofed herbivores,especially cattle,sheep and goats.This disease often causes the death of the host,which leads to huge economic losses in developing countries.In this study,annexin genes of T.multiceps including ANXB2,ANXB3 and ANXB12 were isolated.The relative transcription level and structure prediction were analyzed by q RT-PCR and bioinformatics methods,respectively.A prokaryotic expression was used to express the recombaint proteins Anx B2 and Anx B3.Then two proteins were analyzed by Western blotting and immunofluorescence localization.Meanwhile,the r Tm16 and r Tm GST were respectively made into genetic engineering subunit vaccine to evaluate the protection of goat coenurosis,hoping to provide a new direction for vaccine research of coenurosis.1.Expression and characteristic analysis of annexin(B2,B3 and B12)in Taenia multiceps.Annexin is a class of structure related calcium dependent phospholipid binding protein superfamily,which is widely found in all kinds of eukaryotic cells.There were 3 T.multiceps annexin(Anx B2,Anx B3 and Anx B12)were cloned and expressed.The result showed that the sizes of Tm Anx B2,Tm Anx B3 and Tm Anx B12 were 1065 bp,933 bp and 957 bp,respectively.Among them,r Tm Anx B12 was unstable.Bioinformatics analysis showed that all of annexins had four repeated domain.By the way,there was a lack of calcium binding sites in the Anx B2 structure,which is replaced by a KDG sequence.The westeron blot showed that both r Tm Anx B2 and r Tm Anx B3 can be identified by the positive sera of the goats infected with T.multicep.The indirect immunofluorescence localization showed that Tm Anx B2 was mainly distributed in the larval stage,while Tm Anx B3 was distributed in both the adult and the larvae.A high transcription level of Tm Anx B3 was found in the scolex and immature segment by a quantitative PCR,while Tm Anx B2 and Tm Anx B12 were mainly transcribed in the oncosphere and coenurus.2.Immune protective effect elvaluation of r Tm16 and r Tm GST protein against Coenurus cerebralis in goat.The 16 k Da gene of T.multiceps is one of member in taeniidae 16 K gene family,which has the preferably immune protective.Glutathione S-transferase is widely found in all kinds of organisms and is recognized as vaccine candidates.To explore the immune protective effect of recombinant Tm16 and GST protein against Coenurus cerebralis in goat.In this study,r Tm16,r Tm GST,and r Tm16 + r Tm GST protein were immunized 3 times in each group of goats.The serum was collected regularly.After two weeks of the final vaccination,each goat was orally challenged 5500 eggs of Taenia multiceps.After105 days,all animal were slaughtered humanistic and the number of cysts was recorded.In the result,liken to the control group,the cysts of goat in group Tm16+Tm GST reduced by 87.5% and got the best protective effect(P < 0.05).Result of i ELISA showed that the level of r Tm GST-Ig G was not significantly compared with r Tm16-Ig G in combined group(P>0.05).While the antibody induced by r Tm GST in Tm GST-immunised group was significantly lower than that induced by r Tm16 in Tm16-immunised group(P<0.05).This revealed that the combined use of r Tm16 and r Tm GST can induce higher antibody levels in goats,thus obtaining significant protective immunity. |
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