Dissertation
Dissertation > Medicine, health > Basic Medical > Medical Microbiology ( pathogenic bacteriology,pathogenic microbiology ) > Human Virology ( pathogenic virus)

Purification of Human Respiratory Syncytial Virus Fusion Protein with Immunomagnetic Microsphere Technology

Author FangZuo
Tutor HeJinSheng
School Anhui Medical University,
Course Immunology
Keywords immunomagnetic microspheres human respiratory syncytial virus F protein purification sandwich ELISA
CLC R373
Type Master's thesis
Year 2011
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Objective:Human respiratory syncytial virus (RSV) is a leading cause of severe respiratory infection in infants and young children worldwide. A safe and effective RSV vaccine is not available yet, so it is urgent to pursue potential preventive measures against RSV infection. The RSV fusion (F) glycoprotein is an attractive target for drug and vaccine development as it is highly conserved and major virus neutralization antigen, and essential for viral entry.? The purified F protein subunit vaccines (PFP-1, PFP-2 and PFP-3) demonstrate protective effects on the adults and children to some extent. Hence, PFP subunit vaccines are of great importance in the development of RSV vaccines. Purified RSV F protein is also valuable to establishe serologic diagnosis method for RSV. In this research, we use the immunomagnetic microspheres to purify F protein and develop a convenient, feasible method for purification of F protein.Methods:?RSV infected HEp-2 cells and medium were collected and centrifuged. The resulting supernatant was precipitated by PEG6000, and then the precipitate was dissolved and further purified through sucrose gradient ultracentrifugation. Purified RSV was used as antigen to immunize rabbits to prepare antiserum. The purified polyclonal IgG antibodies against RSV were used to coat the immonumagnetic beads. The optimal concentration and coating time of IgG for immonumagnetic beads were determined. We infect HEK293 cells with replication deficient first generation adenovirus vector encoding F protein, desiganted FGAd/F, and harvest the lysates. Then the resultant lysate was purified via immunomagnetic microspheres coated by rabbit anti-RSV polyclonal antibodies. Furthermore, through taking rabbit anti-RSV polyclonal antibodies as capture antibody and goat anti-RSV polyclonal antibodies as detection antibody, we established a sandwich ELISA method to analyze F protein quantitatively.Result:?After?we established optimal antibody concentration and coating time used to prepare immunomagenetic beads, we successfully purified F protein from the lysate of FGAd/F infected HEK293 cells. The purified F protein was confirmed as a functional homodimer of around 140-175 kD. Then we set up a method to quantitatively analyze concentration and purification of the RSV F protein by sandwich ELISA. The concentration of puried F protein is 116μg/mL, and we recovered 58μg purified F protein from 141μg total F protein and the recovery percentage was 41.1%. The purity of recoved F protein was around 90%.Conclusion: The F protein has be successfully purified from the lysate of FGAd/F infected HEK293 cells by the established immunomagnetic microspheres method, which is convenient and practical and offers a valuable alternative to purify F protein.

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