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Iranian Journal of Public Health، جلد ۴۴، شماره ۱۲، صفحات ۱۶۳۲-۱۶۴۲
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عنوان انگلیسی Optimized Expression and Purification of Humbug in Pichia pas¬toris and Its Monoclonal Antibody Preparation
چکیده انگلیسی مقاله Background: The humbug gene is a truncated isoform of Aspartyl β-hydroxylase (ASPH) gene that is overexpressed in many human malignancies. In recent years, since humbug has received increasing attention, it is considered as a potential therapeutic molecular target. Therefore, it is necessary for preparing humbug protein and its monoclonal antibody to investigate its structure and function. Method: The optimized humbug gene, synthesized by Genscript in Nanjing, China on December 21st 2013, was expressed in Pichia pastoris cells that were cultured in a 10-L bioreactor. The recombinant protein was further obtained and purified by using ion exchange chromatography and Sephadex G75. The humbug protein was used to immunize Balb/c mice to generate the monoclonal antibodies. The specificity and sensitivity of the monoclonal antibodies were assessed by indirect enzyme-linked immunosorbent assay. Finally, the humbug monoclonal antibodies were used to detect the expression of humbug in several tumor cell lines via indirect immunofluorescence. Results: Firstly, the recombinant humbug was expressed in P. pastoris successfully and efficiently by using a gene-optimized strategy. Secondly, the purification process of humbug was established via multiple chromatography methods. In addition, four monoclonal antibodies against humbug were obtained from the immunized Balb/c mice, and the result of indirect immunofluorescence was indicated that the humbug monoclonal antibody showed the high affinity with humbug protein, which expressed in several tumor cell lines. Conclusion: The over-expression of recombinant humbug provides adequate sources for its structural study and the preparation of the humbug-specific monoclonal antibody can potentially be used in tumor initial diagnosis and immunotherapy.
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نویسندگان مقاله ting huyan | ting huyan


ruihua تنگ | ruihua tang


جینگ li | jing li


qi li | qi li


xiaoping xue | xiaoping xue


هیو یانگ | hui yang


نشانی اینترنتی http://ijph.tums.ac.ir/index.php/ijph/article/view/5559
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