十字花科黑腐病菌對於胞外蛋白的分泌，主要是靠內、外膜分泌系統來完成。在外膜分泌系統的組成中，XpsG，-H，-I，-J是其中四個蛋白質。此四個蛋白質之前驅物NH2-端與prepilin之signal peptide有相當高的同源性，因此在外膜蛋白分泌系統中被認為很可能會形成pilus-like之構造，以幫助胞外蛋白的分泌。所以為了探討此pilus-like的構造是否形成，本研究擬構築XpsG-J四個重組表現質體，並表現蛋白引發其抗體，利用免疫與膠質過濾管柱層析法來探討在XC1701中各蛋白間是否有交互作用。目前本論文以pQE與pET表達系統已先後純化出XpsG與XpsI，並完成XpsG與XpsI之抗體。以分子篩濾層析分析，初步發現這二蛋白都在同一retenti-on time出現，因而認為此二蛋白有cofractionation的現象，至於兩者是否真的有直接關係，還有待以免疫層析或其他的實驗來做進一步瞭解。
XpsG,H,I and J,having homologous leader sequence of type IV pilin,arecytoplasmic membrane proteins that require for the secretion of extracellularproteins in Xanthomonas campestris pv.
campestris. The objective of this researchis to study the protein-protein interaction between XpsG,H,I and J and the
putativepilin-like structure. The specific aims include the construction of expression vector,the overexpression of
recombinant proteins,the induction of antibody and investigationof the protein-protein interaction in XC1701. In
this study,four expression vectors wereconstructed. Among them,the recombinant protein of XpsG and XpsI were expressed and purifiedby both pET32a and pQE9 expression vectors from E.coli BL21(DE3) and M15[pREP4] strain. Theantibodies against XpsG and XpsI were also obtained by immunizing rabbits with purified recombinantproteins. Gel filtration chromatography and Western blot analysis were then performed to analyze thecofraction of XpsD,XpsG and XpsI proteins. The preliminary data showed that XpsG and XpsI,which wereeluted in the same fraction,were not
cofractionation with XpsD. The protein-protein interaction betweenXpsG and XpsI in XC1701 still need other experiment such as immunoaffinity chromatography analysis to bedirect evidence for perfomed.
