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Iranian Journal of Microbiology، جلد ۸، شماره ۱، صفحات ۲۱-۲۸
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عنوان انگلیسی Expression of granulocyte colony stimulating factor (GCSF) in Hansenula polymorpha
چکیده انگلیسی مقاله Background and Objectives: During past decades Hansenula polymorpha has attracted global attention for the expression of recombinant proteins due to its high growth rate, minimal nutritional porequirements and use of methanol as a low cost inducer. Materials and Methods: The corresponding nucleotide sequences for the expression of heterologous genes in Hansenula poylmorpha were extracted and assembled in an E. coli vector. The constructed expression cassette included formate dehy- drogenase promoter (pFMD), a secretory signal sequence, a multiple cloning site (MCS) and methanol oxidase (MOX) ter- minator. Zeocin resistance gene fragment and complete cDNA encoding granulocyte colony stimulating factor (GCSF) were cloned downstream of the expression cassette in-frame with signal sequence. Restriction mapping and sequence analysis confirmed the correct cloning procedures. Final vector was transformed into Hansenula and recombinant host was induced for the expression of GCSF protein by adding methanol. SDS-PAGE and immuno-blotting were performed to confirm the identity of r-GCSF. Results: The expression cassette containing gcsf gene (615bp) and zeocin resistance marker ( sh-ble , 1200bp) was prepared and successfully transformed into competent Hansenula polymorpha cells via electroporation. Zeocin resistant colonies were selected and GCSF expression was induced in recombinant Hansenula transformants using 0.5% methanol and an approx- imately 19kDa protein was observed on SDS-PAGE. Western blot analysis using serum isolated from GCSF-treated rabbit confirmed the identity of the protein. Conclusions: Molecular studies confirmed the designed expression cassette containing gcsf gene along with pFMD and sig- nal sequence. The expressed 19kDa protein also confirmed the ability of designed vector in expressing heterologous genes in Hansenula cells.
کلیدواژه‌های انگلیسی مقاله Hansenula polymorpha,expression cassette,GCSF
نویسندگان مقاله یگانه طالب خان | yeganeh talebkhan
1biotechnology research center, pasteur institute of iran, tehran, iran.

سازمان اصلی تایید شده: انستیتو پاستور ایران (Pasteur institute of iran)

طناز صمدی | tannaz samadi
biotechnology research center, pasteur institute of iran, tehran, iran.

سازمان اصلی تایید شده: انستیتو پاستور ایران (Pasteur institute of iran)

آرمین سمیع | armin samie
biotechnology research center, pasteur institute of iran, tehran, iran and department of biology, azad university of damghan, iran

سازمان اصلی تایید شده: انستیتو پاستور ایران (Pasteur institute of iran)

فرزانه برخورداری | farzaneh barkhordari
biotechnology research center, pasteur institute of iran, tehran, iran.

سازمان اصلی تایید شده: انستیتو پاستور ایران (Pasteur institute of iran)

محمد عزیزی | mohammad azizi
biotechnology research center, pasteur institute of iran, tehran, iran.

سازمان اصلی تایید شده: انستیتو پاستور ایران (Pasteur institute of iran)

وحید خلج | vahid khalaj
biotechnology research center, pasteur institute of iran, tehran, iran.

سازمان اصلی تایید شده: انستیتو پاستور ایران (Pasteur institute of iran)

عصمت میراب زاده | esmat mirabzadeh
biotechnology research center, pasteur institute of iran, tehran, iran.

سازمان اصلی تایید شده: انستیتو پاستور ایران (Pasteur institute of iran)

نشانی اینترنتی http://ijm.tums.ac.ir/index.php/ijm/article/view/170
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