Preparation and application of a novel monoclonal antibody specific for the heat shock protein 60 of Lawsonia intracellularis

Porcine proliferative enteropathy(PPE),an important infectious disease in pig production caused by an obligate intracellular bacterium Lawsonia intracellularis,is commonly associated with diarrhea and reduced weight gain in growing pigs widespread.An accurate method for detecting L.intracellularis is particularly important for preventing and controlling PPE.Heat shock protein 60(Hsp60)is an immunodominant bacterial antigen found in all eukaryotic and prokaryotic organisms.Thus,the purpose of the current investigation was to produce a novel L.intracellularis Hsp60 monoclonal antibody(mAb)useful for immunodiagnostics.Three hybridomas secreted anti-Hsp60 termed 3E5,4E2,and 9G6 were generated,and the titers of ascitic fluids of 3E5,4E2,9G6 were 1:1024000,1:2048000 and 1:2048000,respectively.The Western blotting analysis demonstrated that recombinant Hsp60(rHsp60)was recognized by mAbs 3E5,4E2 and 9G6.Subsequently,analyses of specificity showed all the mAbs were highly specific to L.intracellularis while could not significantly react with other enteric bacteria commonly found in the ileum of pigs,such as Escherichia coli,Salmonella Choleraesuis,Salmonella Typhimurium,and Brachyspira hyodysenteriae.Furthermore,the mAbs were useful for detecting L.intracellularis in the infected monolayer cells and histological sections of the ileum from PPE-affected pigs.Our research will provide a foundation for the development of immunological diagnostic tests.

Modification of tooth development by heat shock protein 60

several heat shock proteins have been investigated in relation to tooth development, no available information is available about the spatial and temporal expression pattern of heat shock protein 60 （Hsp 60）. To characterize Hsp 60 expression in the structures of the developing tooth germ, we used Western blotting, immunohistochemistry and in situ hybridization. Hsp 60 was present in high amounts in the inner and outer enamel epithelia, enamel knot （EK） and stratum intermedium （SI）. Hsp 60 also appeared in odontoblasts beginning in the bell stage. To obtain data on the possible effect of Hsp 60 on isolated lower incisors from mice, we performed in vitro culturing. To investigate the effect of exogenous Hsp 60 on the cell cycle during culturing, we used the 5-bromo-2- deoxyuridine （BrdU） incorporation test on dental cells. Exogenously administered Hsp 60 caused bluntness at the apical part of the 16.5-day-old tooth germs, but it did not influence the proliferation rate of dental cells. We identified the expression of Hsp 60 in the developing tooth germ, which was present in high concentrations in the inner and outer enamel epithelia, EK, SI and odontoblasts. High concentration of exogenous Hsp 60 can cause abnormal morphology of the tooth germ, but it did not influence the proliferation rate of the dental cells. Our results suggest that increased levels of Hsp 60 may cause abnormalities in the morphological development of the tooth germ and support the data on the significance of Hsp during the developmental processes.

Immunostimulatory role of rBmHSP60 from filarial parasite Brugia malayi

Objective:To evaluate the immunostimulatory potential of crossreactive molecule heat shock protein 60(HSP60)of filarial parasite Brugia malayi and Leishmania donovani.Methods:HSP60 of Brugia malayi(BmHSP60)was amplified using gene-specific primer,cloned in p Tri Ex4 vector,expressed in BL21-DE3 cells,and recombinant HSP60(rHSP60)of~65 k Da was purified by affinity chromatography using Ni-NTA column.The recombinant protein was desalted by the dialysis membrane,and the presence of endotoxin level was determined by Limulus amebocyte lysate assay.The recombinant protein was tested for cell proliferation,nitric oxide release,expression of Th1 and Th2 cytokines,and transcription factors(STATs)in vitro using murine macrophage cell line(J774 A.1).Results:Higher cell proliferation indicated that BmHSP60 had immunostimulatory potential.rBmHSP60 exposure upregulated the expression of iNOS,STAT1,STAT4,Th1 cytokines(IFN-γ,TNF-α,IL-12),and nitric oxide release.In addition,no remarkable change was observed in the expression of IL-6,IL-10,and STAT3 in macrophage cell line J774 A.1.The ELISA analysis showed the levels of IFN-γ,TNF-α,and IL-12 were upregulated while IL-10 level was downregulated,revealing that BmHSP60 triggered a Th1 immune response.Conclusions:Our study demonstrates that rBmHSP60 has immunogenic properties which effectively enhances the Th1 type immune responses,and can be used as an immunoprophylactic agent against leishmaniasis.Furthermore,in vivo studies are in progress to determine the protective role of rBmHSP60 against Leishmania donovani infection in a mouse model.

Relationship between Tumor Lesion of Kidney Tissue and Expression of HSP60 in Chickens Infected by MD

[ Objective] The paper was to investigate the relationship between pathological lesion and transcription, expression and distribution of heat shock protein 60 （HSP60） in kidney of chickens infected by Marek＇s disease virus （MDV）. [ Method] Tumor animal model was successfully established with chickens infected by MDV. Real-time RT-PCR, histopathological and immunohistochemistrical methods were used to detect the pathological lesion, transcription level, expression level and distribution of HSP60 in the kidney. [ Result] Obvious pathological changes appeared in kidney tissue of chicken after injection of MDV for 21 d. HSP60 was mainly distributed in the cytoplasm of tumor cell and interstitial macrophages. The expression of HSP60 in challenge group was always higher than blank control group and vaccine control group, and the difference reached extremely significant level from 28 to 86 day-ages （ P 〈 0.01 ） ; the expression of HSP60 in challenge group at 28 day-age was about 8.608 and 12.752 times of blank control group and vaccine control group, respectively. The mRNA transcription levels of HSP60 in challenge group showed a downward parabola during 7 to 42 day-ages; the mRNA transcription level of HSP60 in challenge group was rising in early stage （7 -21 d）, and then gradually dropped; it reached the peak value at 21 d, being 1.222 and 1.179 times of blank control group and vaccine control group, respectively. The mRNA transcription level of HSP60 was higher than two control groups throughout the entire experiment, and the difference reached extremely significant level from 14 to 28 day-agas （P 〈0.01 ）. [ Conclusion] HSP60 expression in kidney tissue and its mRNA transcription level can reflect MDV infection, tumor formation and development progress, with close relationship with pathogenesis of tumor diseases, which is expected to be used as one of the determination indicators for diagnosis and morbidity process of tumor diseases in chicken.