Prokaryotic Expression System Construction Of Leptospira Interrogans LipL32/1-lipL41/1 Fusion Gene And Detection Of LipL32 And LipL41 Gene Expressions In The Wild Strains And Specific Antibodies In The Patients' Sera

Leptospirosis, caused by infection of Leptospira interrogans, is a widespread zoonosis in the world, especially in paddy areas. These highly invasive spirochetal pathogens are capable of infecting a broad range of mammalian host, survive and propagate in kidney, contaminate soil and water. People infected through either direct contact with an infected animal or indirect contact with soil or water contaminated with urine from a chronically infected animal. So this disease is one of the infectious diseases especially monitored and controlled during periods of floods and water logging.Inoculating vaccine is the most effective measure to prevent and control Leptospirosis. But there are numerous serogroups of L. interrogans and there is a low or even no cross-protection among them. The epidemic dominant serogroups of the L. interrogans were found to be distinct in different areas. Therefore, the epidemically dominant serogroups of L interroans have been selected for preparing polyvalent vaccine production both domestically and aboard. However, this kind of vaccine offers little protection against other serogroups of the microbe that were not selected in the vaccine. In addition, there are serious side effects for the polyvalent vaccine made of several whole dead leptospira. A multivalence outer membrane vaccine, recently developed by scolars of our country, was the internationally first component vaccine with fine immunity and little side effects. However, this component vaccine still had no cross-protection among different serogroups of L. interroans. Thus, finding leptospiral genus-specific protective antigens is significantfor developing a universal vaccine to prevent and control Leptospirosis.Shang et al (1996) and Haake et al (2000) cloned HpL32 and lipL41genes with 819 bp in size encoding a lipoprotein with 272 amino acid residuals, 1068 bp in size encoding another lipoprotein with 355 amino acid residuals in the outer membrane. Simultaneously, they thought that lipL32 and lipL41genes exist in the DNAs of all pathogenic Leptospira but not in saprophytic one. Based on the results of our recent studies, all the 15 strains that represent the 15 serogroups of L. interrogans mostly epidemic in China were positive for lipL32 and lipL41 genes, but they can be divided into lipL32/1 and lipL32/2, lipL41/1 and lipL41/2 genotypes (GenBank No.: AY568680, AY568679, AY609321-AY609333, AY622673-AY622687).The lipL32/1 and lipL41/1 genotypes exist in the major epidemic L. interrogans serogroups such as Icterohaemorrhagiae, Canicola, Autumnalis, Pomona, Grippotyphosa, Hebdomadis and Sejroe.Certainly, genetic engineering vaccine possesses many advantages, but it has low immunoprotection probably due to single antigen and high cost for production. It is a possibility that multi-antigenic determinants and larger size of an antigenic molecule improve its antigenicity. Therfore, we chose the two genotypes UpL32/l and UpL41/l for constructing lipL32/1-lipL41/1 fusion gene and its prokaryotic expression system by using genetic engineering techniques, which would decrease steps during the producing procedure and cost of the vaccine, and probably improve the antigenicity of the target expression product through its larger molecular size. Generally, prokaryotic expression system using E. coli as expression host cell has the advantages of simple operation, easier cultivation, faster reproduction and higher expression.In the present study, PCR with a lingking primer was performed to construct lipL32/1-lipL41/1 fusion gene and its prokaryotic expression system using E.coli as the host cell. Western Blot was applied to determine immunogenicity of the target expression pruduct, which, hopefully, would lay a foundation for further development of genus-specific genetic engineering vaccine of L. interrogans. Theremore, we detected the carrying frequency of lipL32 and lipL41 genes in 97 wild strains of L. interrogans using PCR. Microscope agglutination test (MAT) was applied to measure the agglutinative titers of rLipL32/l, rLipL32/2 or rLipL41/l, rLipL4...