Cloning And Functional Study Of Resistance-Associated Gene E77.43 Against Schistosma Japonicum Infection In Microtus Fortis

Schistosoma japonicum(S.japonicum) infection was not detected inan extensive investigation of the common lake rodent (Microtus fortis)that inhabits the endemic foci of marsh and lake regions of Hunanprovince in the Yangtze River valley of china. Subsequently, experimentalstudies suggest that Micotus fortis (M.fortis) might exhibit innateresistance to S.japonicum infection. And the quality of the innateresistance in M. fortis inherited steadily. It is likely that inheritance factorsin M. fortis may play an important role in regulating its innate immunityformation. Some recent advances have gained on immunological analysisin M. fortis, however, very little is known about the molecular mechanismof M. fortis genes involved in Schistosoma destruction and clearance ofinfection. In previous study, we gained a second subpool E77, thetransient expression product of E77 can inhibit schistosomula strongly invitro. Therefore, we subsequently performed the isolation and characteri-zation of a single gene that can inhibit schistosomula in vitro and in vivo. Part1: Cloning and tissue distribution of Resistance-associated GeneE77.43 against Schistosoma japonicum Infection in Microtus fortisWe have recently identified a second subpool E77, and theconditioned media of E77 significantly inhibited S.japonicum schistose-mula in vitro, were progressively subfractionated until the subpoolcontained single cDNA that inhibited S.japonicum schistosomula in vitro.The cDNA, initially named "E77.43", was then sequenced by standardtechniques. The full-length cDNA of E77.43 gene is 1494 bp in lengthand contains open reading frame of 333 bp. The predicated proteinsequence derived from the open reading frame produces a 111-amino acidpolypeptide, with a calculated molecular mass of 12.2 kD and theoreticalisoelectric point of 9.65. The nucleotide and protein sequences have nosignificant similarity to any sequence deposited in the GenBank data baseand protein data base, which showed that E77.43 is indeed novel. Inaddition, ScanProsite analysis revealed one N-glycosylation site, twoN-myristoylation sites, four protein kinase C phosphorylation sites. Ingeneral, analysis of the expression pattern of the novel gene is also animportant pathway for inference of its function. Firstly, distribution ofE77.43 mRNA in normal Microtus fortis tissues was characterized indetail by RT-PCR. We found that the expression of E77.43 was high inbone marrow, brain and testis, whereas its expression in liver, lung, spleen and kidney was weak. However, the expression of E77.43 was not detected in some tissues including stomach and muscle. The result ofexpression and distribution of E77.43 in Microtus fortis tissues should befurther study and analysis.Part 2: Prokaryotic expression,Polyclonal antibody preparation,Invitro-killing activity of recombinant protein on E77.43E77.43 is an ubiquitously expressed Microtus fortis gene. Thoughour result suggested E77.43 may play an important role in resistance toschistosoma infection, its function is still not completely understood.Analysis of protein is a preferable approach to obtain insight into thefunction of the novel gene. In this section, the coding region of E77.43cDNA was generated by PCR amplification and DNA fragment generatedby PCR was digested with BamH I and PstⅠand ligated into the pQE30vector. The transformants were isolated and confirmed by enzymedigestion and sequence analysis. The recombinant E77.43 fusion proteinwas expressed in prokaryotic expression system. New bands corres-ponding to the fusion protein of E77.43 was detected by SDS-PAGE. Inthe meantime, the recombinant E77.43 fusion protein was produced andpurified on His-Band resin and was dialyzed with PBS several times anddissolved in PBS. This purified recombinant E77.43 fusion protein wasused for the in vitro killing to schistosomula. Interestingly, the resultsindicated that recombinant E77.43 fusion protein has significant effect in killing schistosomula in vitro. Therefore, it is suggested that we havegained one resistance-associated gene E77.43 to Schistosoma infection inMierotus fortis.On the other hand, it is important to study if the relative amountsE77.43 mRNA correlate to the protein level. In this section, thepolyclonal anti-E77.43 antiserum was generated by immunizing rabbitswith purified E77.43 fusion protein. The specificity of anti-E77.43polyclonal antibody was identified by western-blot analysis. Subsequently, distribution of E77.43 in different tissues of Microtus fortis wasinvestigated by western blot. We extracted proteins from liver, lung, brain, spleen, muscle, kidney, bone marrow, testis and stomach and performedWestern blot analysis. We detected the highest amounts of E77.43 in brainand testis, whereas liver, lung, spleen, kidney expressed E77.43 onlyweakly. The expression of E77.43 was not detected in muscle. Theprotein expression of E77.43 corresponded to the results of mRNAexpression in RT-PCR. The preparation of recombinant protein andpolyclonal antibody can be lay foundation for the following functionalstudy of E77.43.Part3: Preparation of recombinant retrovirus and its protective effectin a mice model of the Schistosoma japonicum infectionBased on the resistance-associated genes E77.43 to Schistosoma japonicum infection in Microtus fortis, the aim of this work is to explorethe feasibility of the E77.43 gene therapy in schistosomiasis. Weconstructed a recombinant retroviral vector pRevTRE-E77.43/Tet-Off andpLXSN/E77.43 containing the E77.43 gene, after Packaging of retrovirusin vitro, we observed the expression of this gene and evaluated the viral(activity) titer in vitro, which may lay solid foundation on developingpRevTRE-E77.43/Tet-Off and pLXSN-mediated gene transfer as amethod of anti-schistosomiasis in vivo. The rRevTRE-E77.43/Tet-Offand rPLXSN/E77.43 was injected into tail of Schistoma japonicuminfected model mice. The recombinant virus injected mice couldefficiently express E77.43 and peaked at seven days after injection.Significant reduction in adult worms (31.0%) and high reduction (35%)in liver eggs was induced following gene therapy. Our findings suggestedthat pRevTRE-E77.43/Tet-Off gene therapy could be used for treatmentof schistosoma infection.Part4: Resistance to Schistosoma japonicum infection challenge inAAV2-E77.43 transferred miceTo observe the resistance of mice to Schistosoma infection bytransfection of E77.43 gene, and to identify the potentially use intreatment of Schistosomiasis. We constructed recombinant retroviralvector AAV2/E77.43 containing the gene, after packaging of AAV-virus in vitro, we inject the recombinant virus into mice. The mice werechallenged with Schistosoma cercariae and were sacrificed 41 days afterchallenge, worm burden and liver eggs were counted. The results showedthat the reduction in worm burden and liver eggs in AAV2-E77.43transferred mice were 27.3% (P＜0.001) and 26.2% (P＜0.001). Thisindicated that AAV2-E77.43 could significantly protect mice againstSchistosoma infection.In summary, here we report the isolation and characterization of onenovel gene E77.43 that resistant Schistosoma japonicum infection inMicrotus fortis. E77.43 distributes widely in most tissues (organ). Boththe results of in vitro and in vivo experiments show significant protectiveeffect against Schistosoma japonicum infection, it can be speculated thatE77.43 may participated in resistance mechanism in Microtus fortis.