| 摘要: | 研究期間：10108~10207;Central nervous system (CNS) infections continue to be a crucial factor for morbidity and mortality throughout the world. Bacterial meningitis is one of the most common and well known types that cause CNS infections, among all the microorganisms, only a small portion of them are capable to infect human brains. Escherichia coli K1 is a gram-negative organism causing meningitis; Many studies have focused on how this pathogen penetrating the blood brain barrier (BBB), which separates circulating blood from cerebrospinal fluid (CSF) in the CNS. BBB is formed mainly by human brain micro-vascular endothelial cells (HBMEC) and protects the brain from any toxins circulating in the blood. Thus, understanding the interactions between E. coli K1 and HBMEC is crucial for elucidating the mechanism of bacterial meningitis. Protein microarray has recently emerged as a useful and powerful tool for high-throughput protein screening studies, and has been applied to many kinds of investigations in biological and biochemistry research. One of the most unique points of proteome chips is the ability of systematic study. In our lab, we already have many kinds of protein microarrays, including yeast proteome microarrays, E. coli K12 proteome microarrays, human proteome microarrays and several types of antibody microarrays. Hence, we plan to probe HBMEC with E. coli K1 proteome chip to identify the K1 proteins that interact with HBMEC. We will base on the E. coli K12 library we already have and construct the E. coli K1 specific gene library compared to E. coli K12. The number of E. coli K1 specific genes is 480. We will use the library to fabricate the E. coli K1 proteome chip to study the interactions between the entire proteome of E. coli K1 and HBMEC. In the previous project, we have constructed 432 E. coli K1 specific gene clones by high-throughput infusion cloning method. Right now, we are constructing the rest E. coli K1 clones and transforming the vectors into the expression cells. In this proposal, we will optimize the protein expression, purification and protein chip fabrication in the first year. In the second year, we will optimize and build up the cell-chip probing model and the further validations and analysis such as bead binding assays, flow-cytomery, E. coli-HBMEC association assays (knockout and blocking) and invasion assays (knockout and blocking). In the third year, the identified E. coli K1 proteins will be probed with the human proteome chips to uncover the receptors in HBMEC that interact with the responsible E. coli K1 proteins for invasion. The identified candidate receptors will be further validated by flow-cytomery, association assays (knockout and blocking), invasion assays (knockout and blocking) and immuno-histochemical assays. |
