Expression, Purification And Characterization Of An LcrV Mutant Of Yersina Pestis

Plague is an acute infectious disease due to Yersinia pestis. In6th,14-15th and19th century,200million-persons succumb to plague pandemic, which caused large disaster to human; In recent years, under the circurstance of international anti-terrorism, the preventation and treatment of plague had been one of the international biomedical research hot point.LcrV(Low-Calcium Response V antigen), an important toxicity factor and key protective antigen of Yersinia pestis, which constitutes the needle tip of Type III secretion system injector, regulates secretion of Type III secretion system and directly be responsible for anti-inflammatory effect. LcrV plays the key role in infection and immunity of Yersinia pestis.LcrV has a molecular weight of37KD, and is composed of326amino acids residues. LcrV can form a homology dimer through the273Cysteine residues. The significance of LcrV in infection and immunity has not been reported in any documents.Vaccination had been proved to be an effective way to prevent plague. There were two-kinds of human plague vaccines in the world at present, one was whole-cell-killed vaccine,among-them, A formalin-killed Yersina pestis vaccine was licensed by FDA in USA; Another was attenuated live vaccine, which was mainly used in Russia and China. Although these two vaccines were safe, effective and used for long-term, there were some shortcomings such as highly safety control in manufacture, complicated immune process and difficult quality control, etc. In recent years, both domestic and international scientists are trying to develop a neotype plague vaccine. Researches showed that both rF1and rV antigen can protect experimental animals against Yersinia pestis’ attack, and solo was outstripped by combination. Because Fl antigen deficient Yersinia pestis still possesses virulence, the immunity effect of LcrV was extremely important to neotype plague subunit vaccine. Plague subunit vaccine had entered into clinical trials.Product quality need to be strictly controlled during new drug research and development. Because of Cys273, LcrV partly exists in the form of dimer. Although experiments showed both monomer and dimer of LcrV can induce protective immunity, it increased the quality control difficult in manufacturing. This research prepared LcrVm. compared its physic-chemical property and immunogenicity with LcrV, and appraisal its feasibility as candidate antigen of neotype subunit plague vaccine.Escherichia coli is one of the most popular host for producing recombinant proteins. In addition to its simplicity, safety, and well-known genetic properties, the major advantage of E.coli is its ability to produce proteins in large quantities and to grow very quickly compared with mammalian cells, which enables excellent space/time yields. This research, on the basis of our previous research,"that selected the E.coli system to high-level express the V antigen", considering the prokaryotic cell origin and the suitable molecular weight of V, we selected the E.coli system to express the Vm antigen. First, the code of Cys273in LcrV gene were mutated by using fusion PCR techniques. After this, LcrV mutant coding sequence was cloned into expression vector pMD-18T. The coding sequence of LcrVm was analyzed, and compared with LcrV with Vector NTI and then confirmed to obtain expected mutant gene; Then, the mutant gene was digested by Nde I/Hind III, extracted and subcloned into the corresponding restriction sites of the expression vector pET32a (+) to construct the mutant expression plasmid respectively. Finally, expression plasmid was transformed into the E.coli BL21(DE3) competent cells to obtain engineered strains by ampicillin selection on the LB plate. Randomly selected mutant strains were inoculated into LB culture medium. When the OD600reached about1.5,0.4mM of IPTG was added for induction for5h at37℃. The induced cells were analyzed by SDS-PAGE. The results showed that the mutant with expected size were high-level expressed successfully in a complete soluble form. The expression of the mutant is about46%of the total bacterial soluble proteins.LcrVm purification, at first, the mutant proteins were captured by Phenyl sepharose FF. Next, salt-ions were removed by Hiprep26/10Desalting, then the mutants were further purified by DEAE sepharose FF and Hiprep Superdex75to reduce host cell protein, nucleic acid and endotoxin. After the three step purification, the purity was more than95%. The N-terminal sequence analysis of rVm showed it was the same with natural LcrV. The purified protein can be detected by anti-LcrV with WB. Then Physico-chemical property of LcrVm was compared with LcrV.Furthermore, the immunogenicity of the various antigens formulation adsorbed to an aluminum hydroxide adjuvant was evaluated in mice models, immune serum antibody titer was detected by ELISA, humoral immune responses of the combined immunization were observed and compared with the separate immunization. Results showed antigenicity and immunogenicity of LcrV and LcrVm hadn’t evident discrepancy; after immunization, the antigen-specific high-titer IgG antibody was induced, synergism or interference action occurrence were less and stimulated cell immunity level was closed to each other and both were in low level. Analysis showed all physico-chemical properties of LcrV and LcrVm were similar except molecular weight, about200D discrepancy. Our results showed Vm antigen is a good candidate antigen of neotype subunit plague vaccine. In addition, the non-tagged mutant protein Vm could be helpful to study the structure and function of LcrV.