Clone And Characterization Of DNase Ⅱ-T3223-7 Gene From Trichinella Spiralis

Trichinella spiralis is a typical zoonotic parasite nematode.Since the discovery of Trichinella spiralis,the interaction between Trichinella spiralis and the host innate immune system has been focusing for decades,especially in the field of the immune escape mechanism of Trichinella spiralis.In recent years,Trichinella spiralis genome research results show that Trichinella has a huge amount of genes that encode the DNase Ⅱ family proteins,which are almost 125 parts,and the half of those genes has been considerd to encoding Excretion/Secretion（ES）.However,although Trichinella has such a large amount of DNase Ⅱ family proteins,but its function and role is still unknown.The present study confirmed that the ES products of Trichinella spiralis in different periods had nuclease activity,while the nuclease activity in adult and neonatal larvae was significantly stronger than that in muscle larvae.Our lab primary study has identified 28 period-specific DNase Ⅱ genes,which were obtained from the Trichinella gene pool in different periods by gene probe,subsquently classified them into three gene families,the Newborn larva（NBL）stage T31D4,Muscle larva（ML）stage P43,and Adult worm（Ad）stage T3223,respectively.In this study,we selected the adult stage T3223 family member T3223-7 as our target protein.We expressed and identified the T3223-7 of Trichinella spiralis by means of prokaryotic expression,meanwhile,verified the enzyme activity of the expressed product.First of all,we did bioinformatics analysis for the Trichinella spiralis T3223-7 gene sequence by using the data which is in GenBank.According to the gene sequence,designed a pair of specific primer for T3223-7 gene,then,used the RT-PCR technologie to cloning the T3223-7 gene,subsquently,constructed the recombinant expression plasmid of pET-28a-T3223-7 and convert it into E.coli Rosseta（DE3）.So we got recombinant proteins by IPTG induction expression.The molecular weight is 39.7 kDa which is consistent with the theoretical value.We purified the recombinant proteins in His Trap and rubber cutting purification to get high purity protein.Secondly,we used the purified rT3223-7 protein for 1 D enzyme spectrum to validate nucleic acid enzyme activity.We choose pH 5.0 solution buffer as the best liquid reaction incubation conditions.In order to determine whether the enzyme activity depends on bivalent cation,so the reaction incubation fluid was used two kinds of circumstances: Ca²⁺ exists and EDTA exists.The enzyme activity test results show that T3223-7 proteins under acid condition（pH=5.0）has the ability to degradate DNA,and the nucleic acid enzyme activity works well both in the two reaction solution which contain Ca²⁺ or EDTA.The results confirmed that DNase Ⅱ of the Trichinella spiralis adult has a nucleic acid enzyme activity,and its also challenge the previous prediction that Trichinella spiralis DNase Ⅱ are considered having no enzymatic activity as it does not have typical histidine active site.Finally,we stimulated rabbits with rT3223-7 proteins to obtainpolyclonal antibody from rabbits which may be specific binding to rT3223-7,through ELISA,we measured it titer is up to 1:32 0000.To identified the specificity of polyclonal antibody,we measured it by using the western blot to detect rT3223-7,natural worm body protein and ES,and the results show that the recombinant protein antiserum T3223-7 can not only identify rT3223-7 protein,but also can be specific identification and combining with nature worm body antigen and the ES antigen.Meanwhile,we analyze the distribution of T3223-7 in Trichinella spiralis adult section,naked worm,also,the worm infected intestinal tissue by immunofluorescence.According to the results,the red fluorescence signal located in body skin,moreoverwe can also observe some red fluorescence signals around the worm body in the intestinal tissue.According to these results,we hypothesize that T3223-7 may be an ucleic acid enzyme protein expressed by skin cells,participating in the host immune escape or moulting process of the development in vitro.In conclusion,the DNase Ⅱ can degradate the DNA which may help the immune response or release from the worm body.It is beneficial for Trichinella spiralis to parasitism in the host.