The Clonal Expression Of The Genes Specific For Penicillium Marneffei And The Preparation Of Polyclonal Antibodies

Backgrounds and Objectives: Penicillium marneffei (P.m) is a thermally dimorphic fungus that is endemic in Southeast Asia and southern China. It has been become one of the most common opportunistic infection pathogens in human immunodeficiency virus (HIV)-infected patients in that regions. It is an intracellular fungus and involves mainly the mononuclear phagocytie system of human. The golden standard of its diagnosis is through fungi culture. However, several factors restrict its usefulness in practice. Histologically, the closely resemblance to Pneumocystis carinii and Histoplasma capsulatum make troubles on differential diagnosis. HongKong University has developed a diagnosis kit based on specific recognizing MP1p antigen in P.m, but cross-reaction with Aspergillus still remains. Thus, we construct several cDNA librarys of P.m for finding new genes that can encode specific antigenic protein and preparing diagnostic antibodies to enhance the accurate diagnoses of P.m. Methods: Several cDNA librarys of P.m were constructed and sequenced for finding valuable genes. Bioinformatics analysis was conducted to predict the functions and location of 2 novel genes (PYA₀24₆0 and PYA₀12₉2). These 2 genes were expressed with glutathione S-transferase (GST) tags in E.coli expression system using pGEX-5X-1 plasmid and purify target proteins using Glutathione Sepharose 4B. Rabit-polyclonal antibodies were developed and purified by using antigen-based affinity chromatography. And then Western blot and Concanavalin A analysis were performed for identifying the functions and locations of them. Finally, the specificity of these 2 polyclonal antibodies was identified via immunohistochemical method. Results: More than 8,000 Express Sequence Tags (EST) were found in the cDNA library of P.m. There were 2 novel genes (PYA₀24₆0 and PYA₀12₉2) have been chosen for further research. They both encode putative signal peptide in N-terminal, N-glycosylation sites and threonine/serine-rich O-glycosylation sites in C-terminal, which provide strong evidences to their functions of involving cell wall synthesis. They can be easily removed from cell wall by glucanase digestion.The result of Concanavalin A analysis suggests that they likely to be glycoprotein. The Initial results of immunohistochemical study show that these two polyclonal antibodies are both positive to P.m while negative to other fungi. Conclutions: In this study, we construct a system looking for new genes and researching their functions in a pathogenic fungus of P.m. PYA₀24₆0 and PYA₀12₉2 are both encode highly specific antigens of P.m. This study highlights the necessity of further research on their future application.