| Trichinella is an important intestinal parasitic nematode in human and animal which can cause serious trichinellosis.In order to analyse the relationship between several Trichinella isolates from different areas in China,we amplified the COI gene from these isolates and obtained their sequences.We analyzed these sequences and established the genetic distance matrix and the phylogenetic tree by software ClusterX and Genedoc.The results showed that there was high similarity of COI between T1 and DB which the identity was 97.9%and the genetic distance was only 0.0081.The similarity of T2 and T5 was 95%and the genetic distance was 0.0412,they were more closer when compared with T3.And the distance between HB and YN was 0.0000. The difference was relatively greater in HN isolate compared to others which need to be further studied.Therefore,the COI sequence analysis could be a useful method to identify the relationship of different species and isolates in Trichinella.The genomic DNA of Trichinella was used as template to amplify the fragment of C.elegans lin-28 homologus gene from 5 isolates.The sequences were analyzed by Genedoc and showed the similarity was above 93%which indicated that there was lin-28 homologus gene in Trichinella.The whole-mount in situ hybridization was used to detect the distribution of lin-28 and other three heterochronic genes in T. spiralis.The results showed that lin-28,let-7,lin-4,lin-14 mRNA were all distributed extensively in oocyte and early embryos,and decreased with the cell number increased in the embryo,let-7 and lin-4 mRNAs presented cortex distribution around the midterm and late embryos.All the four genes mRNAs were not detected in the newborn larvae.Besides above,lin-28 and lin-4 mRNAs were also detected in the head and pharynx of adult worm while the let-7 and lin-14 mRNAs can be detected not only in the head and pharynx but also in the tail of adult worm.We also found that let-7 and lin-4 mRNAs presented in the muscle larvae of Trichinella spiralis.P49 is a protective antigen which presents in excretory-secretory products.In order to provide the foundation of the diagnosis of Trichinellosis as well as for the development of anti-Trichinella vaccine,the gene encoding P49 antigen was amplified by 3'RACE PCR from Trichinella spiralis.The cDNA of p49 is about 1100bp and the cDNA product was cloned initially into pMD18-T vector then transformed into E.coli DH5αstrain and cultured on LB plus ampicillin(100μg/ml) plates.Colonies containing the recombinations were selected by PCR and the plasmids DNA were extracted and digested with enzymes.Plasmids containing the right insert were sequenced to confirm their identities,and then the right recombinants were digested and cloned into the expression vector pGEX-4T-3 then transformed into E.coli BL21(DE3) strain.Bacterial lysates from cultures induced by IPTG (1mmol/L) were directly loaded onto SDS-PAGE gel,and a distinct 60KD band can be detected which was consistent with the fuse protein molecular weight.The purification of recombination protein and the immunization of mice were undertaken and the antibody was obtained.The expresstion of P49 in muscle larvae was more than in adult by western blot analysis.Furthermore,a p49-L4440 RNAi vector was constructed and used to RNAi in C.elegans.The lifespan of C.elegans by P49 RNAi was obviously shortened than control worms.A series of primers were designed according to the EST library of T.spiralis and the genomic information of C.elegans,and we got three cDNA fragments of C. elegans homologus genes from T.spiralis by 3' RACE PCR.Three proteins sequences were translated and analyzed by several softwares.The results showed that these genes were homolgus to C.elegans CDC42,RNP,MRP-L28 proteins respectively and the similarity were 71%,40%and 57%respectively.We also found a CDC42 conserved domain in Trichinella CDC42 protein and a RNA recognition motif in RNP protein of T.spiralis.
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