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Construction of Leptospira Interrogans lipL32/1-ompL1/1 Fusion Gene Encoding Genus-specific Protein Antigens and Establishment of Its Prokaryotic and Eukaryotic Expression Systems

Author: ZuoShouFeng
Tutor: YanJie
School: Zhejiang University
Course: Pathogen Biology
Keywords: Leptospira lipL32 / 1 genotype ompL1 / 1 genotype Fusion gene / build The Prokaryotic Expression / eukaryotic expression Immune / Identification
CLC: R392
Type: Master's thesis
Year: 2004
Downloads: 77
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Abstract

Leptospira (Leptospira interrogans) infection caused by leptospirosis (Leptospirosis) is a worldwide popular natural foci Zoonosis also countries occurred when focused surveillance of infectious diseases in the flood. Leptospirosis serum mass, each group of cross-protection is weak or non-Leptospira serogroups popular in different regions of the advantages, type, it is both at home and abroad Leptospira serogroups local popular type to prepare multivalent Leptospira vaccine, but the infection protective effect of the vaccine does not contain other leptospiral serogroup extremely limited. In recent years, China in many areas the \In addition, leptospirosis is still commonly used whole-cell multivalent dead vaccine side effects, and can not easily be accepted and affect the promotion and application. Many areas of specific immunity of the population of the local epidemic of leptospirosis dominant flora decreased already exists the potential danger of leptospirosis outbreak of large-scale popular. Therefore, the search for leptospiral genus-specific protective antigen, thus developed genus-specific genetically engineered vaccine for the prevention and control of leptospirosis epidemic significance. It has been confirmed that 80 ℃ inactivated of the the saprophytic hyperbolic leptospirosis Sanbao the ridge group patoc type Patoc saline recovery I strain with all leptospirosis patients serum antibody agglutination, indicate the presence of Leptospira genus-specific antigen However, the nature of the antigen is unknown. Haake et al (1993) first identified group Leptospira genomic DNA from influenza typhoid a full-length 963 bp, the ompL1 gene encodes a 320 amino acid transmembrane proteins, and confirmed by Southern hybridization that the gene is present in the genomic DNA of the Multigroup Leptospira in. Haake et al (2000) reported that 4 Group 5 type Leptospira genomic DNA, encoding the major outer membrane protein (major outer membrane protein, MOMP) lipL32 gene exists, the size of the gene is 819 bp, encoding a 272 amino acid outer membrane lipid proteins. Zhejiang University, a master's degree thesis Zhao Shou wind is well known that different countries and regions popular advantage Leptospira serogroups often very different. Our findings confirm recent leptospiral serogroups in China. the mpLI genes can be divided into at least three genotypes. mPLI eight genotypes containing the fall group, influenza, typhoid and Siro group. mpLI few genotypes containing jaundice bleeding group dog group, Australia group, Bowmore crowd and 7 fever group. of mpLll3 genotype of leptospirosis are popular domestic non leptospirosis serogroups, but recombinant ompLI / l genotype expression yield (30%) higher than. mPLI left genotype (20%), and both rabbit antiserum cross microscopic agglutination test (microscoPieagglutination test, MAT) titers were Bu 16-128. Leptospiral serogroups in China, liPL32 genes can be divided into two genotypes, lipL32 / l genotype contains jaundice and bleeding group fall group, Bowmore crowd, influenza typhoid group, seven hot group Siro group 10 group of major epidemic of leptospirosis serogroups and liPL guilty the eight genotype expression yields (40%) was significantly higher than liPL32 / 2 genotype (10%). The genetically engineered vaccine has many advantages, but there are a single antigen rather less effective immune protection, higher production costs shortcomings. The liPL32 eight and using genetic engineering techniques. mpLlll two gene fusion construct liPL \prokaryotic expression system is simple to express the advantages of higher yield, but there is often a form of inclusion body the target protein denaturation and products susceptible to endotoxin contamination shortcomings. usually for expression in Pichia pastoris (P.pastoris) eukaryotic expression system of the host bacteria easily express soluble or secreted recombinant protein, endotoxin-free, but the operation is cumbersome and not easy to filter to the high production engineering strains in this study, we use the connection primer PCR constructed lipL32 of self-designed the eighty-one. mpLI / l fusion gene fusion genes lipL32 eighty-one ompLI eight prokaryotic expression system and Pichia pastoris eukaryotic expression system, W VII stem blot identification of prokaryotic expression product immunity, in order to further developed genus-specific leptospiral genetically engineered vaccine industry has laid the foundation materials and methods. Sheng genes build connection according to the llpL32 eight. mPLI / l genotype sequences of primers used PcR to build IIPL edge / I one that the I / B I fusion gene. order to be able to more convenient Ni-Ta affinity chromatography and collecting the purpose of the expression of recombinant proteins in eukaryotic expression systems using 6xHis sequence of the downstream primer was added with 1.5% agarose gel electrophoresis detection of amplified products aimed amplification products of the expected size for 1776bP (prokaryotic expression) and 1794bP (eukaryotic expression). Zhao Shou Feng Journal of Zhejiang University. nuclear the ordinary acid sequence Chau given by TA cloning method to build p [ Ic known T-liPL 2 / I a of the I B / I recombinant plasmid, the alkali lysis method to extract the plasmid of the positive clones were digested preliminary identification, the dideoxy chain termination method nucleotide acid sequence of the inserted fragment . original nuclear expression system's constructed using EcoRI and the public. 1 pairs digested pUCm a T-liPLJZ / the I a. namely B IH and expression plasmid pET-32a, the target fragment by recovered connection. the reorganization of the original expression vector pET- the ZaJ Imperial B 2 / I a I of B / I transformed E.eoli BLZIDE3 expression host bacteria build PET-Za a liPLJZ of / I that is Ll / I a E.coliB Arts ZIDE3 prokaryotic expression system alkaline denaturation method of extracting plasmid again sequenced eukaryotic expression system construction and screening using EcoRI and Notl digested pUCm a T-liPL 2 / I a the I / B I and plasmid corpse month heart eve of dogs purpose connection. fragments were recovered in the recombinant eukaryotic expression vector corpse corpse guilty of Xi said gamma B JZ / I a Yang B I of / I transformed in E.coliDHSQ alkaline denaturation method to extract the white colonies recombinant plasmid again sequencing. recombinant plasmid with Sall P. pastorisGSj \His Mut, His Muts colonies isolated MM and MD plates, and then G418 YPD plates to filter out high resistance to His Mut transformants constructed ppICgK lipL32 / l the one ompLI eighty-one Ppast

Construction of Leptospira Interrogans lipL32/1-ompL1/1 Fusion Gene Encoding Genus-specific Protein Antigens and Establishment of Its Prokaryotic and Eukaryotic Expression Systems

Abstract

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