Cloning,Sequence Analysis And Immune Evaluation Of Toxocara Canis Enolase Gene

Toxocara canis is a common parasitic nematode in canine animals.It can hinder thedevelopment of the affected hosts,can cause death of the host in severe infections;and its larvae can also infect humans,causing serious visceral and ocular larval migrans,and obvious immunopathological reactions.It can even cause blindness when it infects children,which seriously threatens children's health.It is called the most neglected parasitic disease and has important public health significance.However,to date,treatment and prevention of toxocariasis have been relied on drugs,and the progress of research on vaccines against toxocariasis has been slow.In this study,the infective larvae of T.canis were selected as the research object.The enolase gene specifically expressed in the infective larvae of T.canis was selected as the target gene for the study,and its cloning,sequence analysis and immune evaluation were preliminarily researched.In the first part of this study,enol-1 gene was amplified from the cDNA of T.canis.Then the fragment was cloned into the pMD18-T vector,and the positive clones were screened and sequenced.The correct nucleotide sequence was translated into amino acids and analyzed by bioinformatics analysis.The epitopes and functional sites were predicted,and the phylogenetic tree was established.The result revealed that the T.canis enol-1 gene was approximate 1311 bp in length,encoding 437 amino acids.Enol-1 protein was predicted to contain 18 ?-helix,7 ?-fold,10 hydrophilic regions,16 flexible regions and 9 linear B cell epitopes.In the second part of this study,the eukaryotic expression plasmid pVAX-Enol of the T.canis enol-1 gene was constructed,diluted to 100 mg/100 ?L with PBS,and the Balb/c mice were immunized with the diluted pVAX-Enol by intramuscular injection,and the control groups(pVAX I,PBS and blank Control)was injected with pVAX I,PBS or not injected.After the first immunization,the immunizations were performed at the 2nd and 4th week respectively,and the corresponding humoral and cellular responses were measured.Balb/c mice were infected with the infectious larvae of T.canis on the 7th day after the third immunization.After 4 days,the mice were euthanasized and their livers and lungs were collected.The larvae were collected using a Berman separation method,and the number of recovered worms was compared among the groups.The results showed that Balb/c mice immunized with the pVAX-Enol DNA vaccine failed to effectively induce humoral and cellular immune responses.There was no significant difference between the experimental group and the blank control group,PBS control group and empty plasmid control pVAX1 group(P<0.05).It is suggested that the the pVAX-Enol DNA vaccine induced no obvious immune protection.In this study,for the first time,the T.canis enol-1 gene was cloned,sequenced and expressed,and the molecular vaccine potential of the enol-1 gene was evaluated by constructing the nucleic acid vaccine.The results elucidated the sequence and structures of T.canis enol-1 gene and provided foundation for further functional studies of the T.canis enol-1 gene.