title: Molecular characterization and polyclonal antibody generation against core component CagX protein of Helicobacter pylori type IV secretion system.
abstract: Gram-negative bacteria Helicobacter pylori cause gastric ulcer, duodenal cancer, and found in almost half of the world's residents. The protein responsible for this disease is secreted through type IV secretion system (TFSS) of H. pylori. TFSS is encoded by 40-kb region of chromosomal DNA known as cag-pathogenicity island (PAI). TFSS comprises of three major components: cytoplasmic/inner membrane ATPase, transmembrane core-complex and outer membranous pilli, and associated subunits. Core complex consists of CagX, CagT, CagM, and Cag3(δ) proteins as per existing knowledge. In this study, we have characterized one of the important component of core-complex forming sub-unit protein, i.e., CagX. Complete ORF of CagX except signal peptide coding region was cloned and expressed in pET28a vector. Purification of CagX protein was performed, and polyclonal anti-sera against full-length recombinant CagX were raised in rabbit model. We obtained a very specific and high titer, CagX anti-sera that were utilized to characterize endogenous CagX. Surface localization of CagX was also seen by immunofluorescence microscopy. In short for the first time a full-length CagX was characterized, and we showed that CagX is the part of high molecular weight core complex, which is important for assembly and function of H. pylori TFSS.
authors.affiliation: Special Centre for Molecular Medicine; Jawaharlal Nehru University; New Delhi, India; Department of Biochemistry; Faculty of Science; M.S. University of Baroda; Vadodara, Gujarat India.
authors.forename: Gopal Jee
authors.initials: GJ
authors.lastname: Gopal
authors.affiliation: Department of Biotechnology; National Institute of Technology; Raipur, Chhattisgarh India.
authors.forename: Awanish
authors.initials: A
authors.lastname: Kumar
authors.affiliation: Special Centre for Molecular Medicine; Jawaharlal Nehru University; New Delhi, India; Department of Medical Oncology; Dana Farber Cancer Institute; Boston, MA USA.
authors.forename: Jagannath
authors.initials: J
authors.lastname: Pal
authors.affiliation: Special Centre for Molecular Medicine; Jawaharlal Nehru University; New Delhi, India.
authors.forename: Gauranga
authors.initials: G
authors.lastname: Mukhopadhyay
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D000942:Antigens, Bacterial / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D058947:Bacterial Secretion Systems / Q000235:genetics / Q000276:immunology*; D001665:Binding Sites; D015195:Drug Design; D016480:Helicobacter pylori / Q000276:immunology*; D008969:Molecular Sequence Data; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods*; D011817:Rabbits
chemical_list: D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial; D058947:Bacterial Secretion Systems
keywords: Cag-PAI; CagX; H. pylori; molecular characterization; type IV secretion system