The Expression Of Pathogenesis-related Proteins Of Helicobacter Pylori And Exploration Of Crystallization Conditions

Helicobacter pylori (H.pylori) is a gram-negative, microaerophilic bacterium and its infection can lead to serious gastrointestinal diseases, such as peptic ulcer, gastritis and gastric cancer. The protein crystallography is one of most common methods for structure determination and has been widely used in protein function, pathogenic mechanism and drug development studies of H.pylori. Until now, about300protein structures of H.pylori have been determined using this method, whereas most proteins remain unanalyzed. Therefore, the aim of this study was to initiate structure analysis of proteins as well as statistical analysis on the centralized isolation results of H.pylori in a large number of clinical samples from multiple centers.The expression of functional proteins of H.pylori and screening of crystallization conditions were studied in the first part. In this study, we selected37proteins of H.pylori for expression and30of these proteins were expressed successfully. CagF, CagV, Cagδ, GyrB-N, GyrA-N, HP0900, HP0175and HP0170were identified as soluble forms. Combined with3proteins expressed in previous study, we selected9proteins for further purification and initial screening of crystallization conditions. There were19proteins in inclusion forms and the antigenicities were analyzed. The Western blot results indicated CagV, CagE, Cagβ, CagF, CagI, CagL, CagT, HP1299, FadA, GyrB-C, HP1111, HP0357, HP1476, HP1564and HP0549had good antigenicity and seemed to be potential drug targets and diagnostic biomarkers. The crystallization conditions of GyrA-N, GyrB-N, CagV, CagF, HP0900, HP0175, Hsp60and CbpA were screened successfully, whereas the crystals of CagV, HP0900, GyrB-N and CbpA were small and optimization was requested. There were single crystals founded for GyrA-N, CagF, HP0175and Hsp60, but the diffraction resolutions were less than5A. After optimization, the resolution of GyrA-N crystal reached4A.In this study, the crystallization conditions of8pathogenesis-related proteins of H. pylori were screened successfully. The results would provide basis for further structure determination as well as the function study.In the second part, we analyzed the culture positive rates of centralized isolation of H.pylori in a large number of clinical samples from multiple centers. From January2010to July2012, the gastric mucosal biopsies from66452patients were taken from the antrum for H.pylori culture. Furthermore,5736specimens from H. pylori positive patients were also cultured as control. These results were divided into three groups based on their transport time (5h,24h and delayed transport groups). The culture positive rates for different transport groups and transport temperatures were analyzed by chi-square test. The culture positive rate was31.66%of the66452specimens and71.72%of the H. pylori positive specimens. In the5h transportation group, the culture positive rate was30.99%while it was32.84%in the24h transportation group (P<0.001). In contrast, the rate declined significantly in the delayed transport group (26.25%, p<0.001). A decrease in culture positive rate also appeared when the average ambient temperature exceeded24℃, especially in the delayed transport group (23.17%). Our results indicated centralized culture of multicenter samples was feasible for individualized medical use.