The Expression Of Recombinant Candida Albicans Hsp60 Fusion Protein And Study Of Immune Responses

In recent years, various forms of candidiasis have become increasingly prevalent and a major infectious threat that demands increasing medical attention. They are mainly caused by opportunistic fungal agents in immunocompromised hosts. In particular, candidiasis and aspergillosis are common in hospitalized patients and carry a high mortality toll even in the presence of effective therapy. Early and accurate diagnosis of these systemic infections is often difficult because of the nonspecificity of clinical symptoms and lack of standardized diagnostic tools. Moreover, antifungal therapy may be frustrated by toxicity and emergence of resistance. Thus A prophylactic and/or therapeutic vaccine would be the safest and most effective means to meet this pressing medical need.Heat shock proteins (hsps) are the most highly conserved proteins in prokaryotes and eukaryotes.In normal physiological condition hsps show less expression, however hsps were regarded as stress-induced proteins that showed higher expression after stimulation with ultraviolet radiation, heat shock, or viral or bacterial infection.Hsp60 is the protein inside the cell, which has close relation to cellular metabolism. In the cell, as molecular chaperones hsp60 performs vital functions in protein synthesis, folding, and translocation, besides hsp60 participates signal transmission in the cell.Hsp60 shows higher expression after stimulation and can be rcognized by cell surface receptors, transport target antigenic peptides out of the defective cell, present them to the immune system and stimulate the effective anti-specific defence. Respecting above hsp60 shows satisfactory immunogenicity in many anti-infection studies. But the mechanism of hsp60, especially immunology mechanism, should be top and difficult question in the future study. And so far, there is any report about the study of hsp60 gene in anti-infection. Therefore our group adopts hsp60 as study object, primarily studies the immunological activity of fungi hsp60 fusion protein. and establishes the experiment and theoretical basis for further developing anti-candida vaccine in the future.Our group aims to estimate immune protection effect of hsp60 protein coming from gene engineering by animal experiments and primarily approach its mechanism of action, which will settle experimental fundament for developing anti-candida vaccine.We designed primer according to hsp60 gene sequence of Candida albicans in the Genebank, and obtained hsp60 target fragment by RT-PCR amplifying. And then purified gene fragment of candida hsp60 was inserted into pMD18-T expression vector. We screen the clonies by white-blue screening and chose the white colony to extract plasmid. And then we successfully obtained the target fragment by cutting it with SacⅠand HindⅢ.Further we identified target fragment by PCR and gene sequencing and then confirmed that there were 1701bp fragment in the plasmid of obtained pMD18-hsp60 and the homogeneity is up to 99% compared with hsp60 gene sequence of Candida albicans in the Genebank.The plasmid of pMD18-hsp60 and pET28 (b) were cutting by SacⅠand HindⅢand then were retrieved by repeating freeze thawing. And so we successfully constructed recombinant pET28 -hsp60; hsp60 protein was expressed by Ecoli BL21 transformed by the recombinant pET28-b-hsp60 and induced by 1.0 mM IPTG; and then were analyzed by SDS-PAGE and Western blot. Then we got a conclusion that its molecule weight is 65KD and that was identical to specific binding of 6 X his mAb, which demonstrated the espress protein was correct. Through a great quantity induce expression, the pET28-b-hsp60 recombinant protein were purified by Ni<2+>-Saphrose-4 metal chelate affinity chromatography.The antiserum from mouse immunized by purified fusion protein hsp60 as antigen 3 times, and once in two weeks. Two weeks later after the last immune, through the below index to detect the immunologic competence of fusion protein hsp60.1. To detect the titer of antibody in mice blood serum by ELISA;2. Lymphocyte transformation test: after two weeks about the last immune, to isolate immunized mice splenic lymphocyte and by ConA stimulation to use MTT to detect lymphocyte cell multiplication, and then to detect whether the hsp60 fusion protein can activate cellullar immunologic response;3. We used lethal dose (2.0×107/ CFU 0.1ml) Candida albicans standard strains to attack the immunized mice and the control group at the same time by infecting to mice in vein. Three days later we counted the number of colonies by getting left kidney, and compared the immunized mice and the control group;4. Immunoprotection test: we used sublethal dose (1.0×106/CFU0.1ml) Candida albicans clinical strains to attack the immunized mice and observed the survival rate in a short time to estimate the protection effect.We successfully constructed pET28b-hsp60 expression vector, and also constructed and purified recombinant hsp60 protein. We used the immunized mice to detect the immunologic competence of hsp60 fusion protein. Consequence displayed:1. The titer of antibody to the immunized mice was significantly high compared with the control group. It suggested that hsp60 protein activated mouse humoral immune response.2. The hsp60 protein immunized mice can increase the SI of Tlymphocyte cell significantly compared with the control group (separate as 2.468±0.356; 1.125±0.312, P<0.001). It suggested that hsp60 protein activated mouse cellular immune response.3. The number of colonies to immunized mice was apparently less than the control group (1.70×105±0.20×105 CFU比4.26×105±0.15×105 CFU, P<0.001). It showed that hsp60 protein immunized mice can antagonize fungi infection.4. By 2 months observation showed that the survival rate of hsp60 protein immunized mice was 80% that was higher than the control group which was 16%.It suggested hsp60 protein immunized mice can antagonize fungi infection. Our group primarily approached immunological competence of recombinant Candida albicans hsp60 fusion protein and got a conclusion that it not only induced humoral immune response, but also induced cellular immune response. It showed that it can function well in antagonizing fungi infection, which lay an experiment and theoretical basis for anti-candida vaccine in future, and suggestede that the study of hsp60 vaccines is feasible and demonstrated a new way for future in developing vaccine against fungi infection.