| Leptospira is a kind of special microbe in organic structure and genetic character which forms during the period of development and evolution.Leptospirosis,a kind of widespread zoonosis caused by leptospira,endangers the health of human as well as livestock. In order to control leptospirosis,it is urgent and important that clarity the pathogenic mechanism of Leptospira.Because of its particularity of structure and heredity , however most methods generally used in bacterial research are not suitable for study on leptospira.,Few reports about the virulence-associated genes appeared so far.The pathogenic mechanism of leptospira remain unclear.The research on differential gene between pathogenic and non-pathogenic microbe will contribute to the clarification of pathogenic mechanism.According to the differential DNA fragment AF 325818. between leptospira interrongans serovar Lai 017 strain and L.biflexa serovar patoc strain screened with method of substractive suppressive hybridization (SSH),we amplified the unknown region flanking the known region by cassette ligation and semi-nested PCR,and screened a differential geneSupported by China Natural Science Fundation (No. 30070670)OMPL17 only exists in 017 strain. After sequenced,it was registered in GenBank. Basing on prediction of the novel gene function with bioinformatics,we studied the role in the course of infection according to This research has theoretical significance and practical value to the pathogenic mechanism of leptospira serovar Lai 017 strain and the control of leptospirosis.For the purpose of defining the OMPL17 gene role precisely,we constructed the recombinant suicide vector by inserting Ampr in resistant gene at the Nhel site of ORF sequence and subcloned.We subcloned the OMPL17 gene into BamHI and EcoRI site of the suicide vector p2NIL,and inactived the gene by inserting Ampr in resistant gene at Nhel site of it's ORF.Then integrated the recombinant suicide vector into the chromosome of leptospira serovar lai 017 strain,constructed the double crossover mutant by electroporation and confirmed by PCR and DOT blotting analysis.We assessed the virulence of mutant strain by guinea pig with it.The test shows the OMPL17 gene contributed significantly to the overall virulence of 017 strain.In order to lay a foundation to guinea pig ,further study of the role of mutant strain 017 in other fields,we constructed the delection unmarked mutation by delecting OMPL17 gene with inverse PCR.The virulent test shows the same .results as the 017 strain mutant by inactived gene with insertion.At the same time,we proved that the differential novel gene encodes the outer membrane protein of leptospira interrogans serovar lai 017strain.The method is construction of the recombinant plasmid PGST-OMPL17 using the high-efficiency prokaryote expression vector PGEX1- A T,it can express 28KDa protein of OMPL17 induced by IPTG.This protein blot with anti-pan leptospira rabbit serum.The western-blotting which OMP of mutant 017stain blots with anti-OMPL17 protein rabbit serum shows that the OMPL17 protein is absent.We described the first report of screening the differential novel gene OMPL17 of leptospira interrogans serovar lai 017 strain,using two suicidal plasmid for the allelic exchange of disrupting gene with antibiotic resistance or unmarked delection mutation and proving the novel gene is the virulence factor of pathogenic strain.Our research provides significant information for the pathogenic methanism of leptospira lai and offers important laboratory materials for developing new drugs and gene-engineer vaccine.In addition to,it could help us to develop independently our unique gene resources and owe our own knowledge patent of gene as well as provide a new way to study the bacterial molecular evolution. Finally,the new technique of unmarked delection mutation will now allow the construction of potential vaccine strains without the inclusion of antibiotic resistance markers, the ability to make multiple defined mutations and the possibility of makin...
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