Cloning,Expression And Tissue Distribution Of Vitellogenin Gene In Toxocara Canis

Toxocariasis,a kind of parasitic disease,caused by Toxocara canis which parasited in the host small intestine.The distribution of the disease was worldwide,and the data showed that the infection rate of dogs was 0.9%~64.7%.The infected larvae not only infected dogs,but also could infect many kinds of animals and humans,which caused ocular larva migrans,nerve larvae migration,cryptic larval migration and visceral larva migrans etc,eventually leaded to severe pathological syndrome.Therefore,the disease was of great significance in veterinary public health.Vitellogenin is a member of the lipid transport protein family,usually synthetises in the extra ovarian tissue of non oviparous mammals,and enters the ovary through the endocytosis mediated by the vitellogenin receptor,which then splits into yolk protein,lipovitellin and phosvitin.Study found that Vg not only provided the nutrients for embryo development,also had the functions of promoting the growth and differentiation of animal oocytes,the carrier of particles,biomarkers,antioxidant activity,immune defense and so on.The discovery of vg gene in Caenorhabditis elegans which had promoted the research of Vg of Oesophagostomum dentatum,Haemonchus contortus,Trichostrongylus vitrinus,but there was no report about TcVg.In this paper,basing on the available transcriptome data of T.canis in the laboratory,the Tc-vg gene was cloned,the prokaryotic expression and tissue distribution of TcVg were studied.Results were summarised as follows:1.Bioinformatics analysis of Tc-vg geneThe molecular biology technique was used to analyze the Tc-vg gene.The results showed that the Tc-vg gene had a complete coding region of 5082 bp,contained a complete open reading frame and encoded 1694 aa.The molecular mass was 198 kDa and the isoelectric point was about 6.19.The first 18-amino-acid of N-terminal of Tc-vg was predicted as a signal peptide.The analysis showed the TcVg was hydrophilic in theprimary structure,and contained three functional domains: Vitellogenin_N,DUF1943(domain of unknown function)and vWD(von Willebrand factor type D domain).The two-dimensional structure contained alpha-helix,beta-cuttle,and irregular curls.The analysis of TcVg phosphorylation sites showed the presence of three kinds of amino acids:,phosphorylated threonine(pThr),phosphotyrosine(pTyr)and phosphorylation of serine(pSer)were 44,7,19,18 respectively.The subcellular localization of TcVg revealed potential roles in the biological processes of secretory pathway of T.canis.The functional prediction of TcVg showed its roles in lipid transport.According to the result of blast revealed that the similarity of Tc-vg with A.suum,N.americanus and C.briggsae were up to 100%.Phylogenetic analysis revealed that T.canis and A.suum(accession number: ERG83573)were closest,then following by other nematodes.2.Cloning of Tc-vwd and Tc-duf1943 genesIn this study,polymerase chain reaction was used to clone the two functional domain genes(Tc-duf1943 and Tc-vwd)of Tc-vg.Among them,the length of Tc-vwd was834 bp and the length of ORF was about 816 bp,which encoded 272 amino acids,the predicted molecular weight of the protein was 31.15 kDa and the isoelectric point was about 5.12.The TcvWD protein was mainly involved in the biological process of pathogenesis.The length of Tc-duf1943 was 882 bp and the length of ORF was about864 bp,which encoded 288 aa,the predicted molecular weight of the protein was 33.25 kDa and the isoelectric point was about 9.57.The TcDUF1943 protein was mainly in the biological process of lipid localization and organic matte.3.Construction the expression vector and prokaryotic expressionThe Tc-vwd/pMD19-T,Tc-duf1943/pMD19-T and pET28 a were extracted by Plasmid Extraction Kit from TaKaRa.Using the restriction enzyme for digestion,the target gene was linked to the pET28 a vector,then transformed the E.coli DH5? cells.After the plate culture,the single white colonies was identified by PCR,double enzyme digestion and sequencing.The correct recombinant plasmid Tc-vwd/pET28 a and Tc-duf1943/pET28 a were transformed into the E.coli BL21 cells.The single colony was cultured,when the OD600 valued to 0.6~1.0,adding IPTG for induced,then the optimization of IPTG concentration gradient and time gradient were simultaneously used to select the optimal induction conditions.The results showed that the recombinant protein TcvWD and TcDUF1943 were both in the form of inclusion bodies,the size ofTcvWD was about 40 kDa,and the TcDUF1943 was about 35 kDa.The highest expression of TcvWD was at 0.4 m M of IPTG for 6 h,the TcDUF1943 was at 0.4 m M of IPTG for 4 h.4.Polyclonal antibody preparation and detection by Western blotAfter a large expression of Tc-vwd/pET28a/BL21,the Inclusion bodies were collected by centrifugation,dissolved in 8 M urea,and purified by Ni-NTA affinity chromatography column.The recombinant protein TcvWD was used to immunize New Zealand white rabbits for 2-3 weeks and immunized four times,when the titer was more than 1:50000,the final blood samples were prepared,then the titer,concentration and purity were determined.The results showed that TcvWD with the high purity,the titer of antibody was 1:512000,the concentration was 0.82 mg/ml and the purity was qualified.Western blot showed that TcvWD was able to react with immune antiserum,but not with the pre immune serum,illustrating the specificity of TcvWD was good and could be used for immunohistochemistry.5.The specific transcription of Tc-vg and specific tissue distribution of TcVgReal-time fluorescent qPCR method was used to detect the tissue differential expression of Tc-vg.The results showed that Tc-vg expressed in intestinal tract,reproductive tract and body wall of female and male of T.canis,among them,the expression quantity of the intestinal tract was the highest,following by body wall and reproductive tract.Meanwhile,the distribution of TcVg was detected by immunohistochemistry.The results showed that TcVg was expressed in various tissues of female and male,intestinal with the highest expression,the genital tract and body wall,such as muscle cells,uterus and vas deferens with the weak expression.