含人干细胞因子的pPIC9重组载体的构建
Construction of pPIC9 Recombinant Vector Containing Human Stem Cell Factor.
作者信息
Farhadi Behrooz, Shekari Khaniani Mahmoud, Mansoori Derakhshan Sima
机构信息
Department of Biochemistry, Faculty of Medicine, Tabriz University of Medical Sciences Tabriz, Iran.
出版信息
Adv Pharm Bull. 2013;3(2):303-8. doi: 10.5681/apb.2013.049. Epub 2013 Aug 20.
PURPOSE
Various cytokine regulates hematopoesis; they promote number of stages in stem cells biology such as proliferation, differentiation and endurance. Biological effects of SCF, as a hematopoietic cytokine; is triggered by binding to its ligand c-kit. Potential therapeutic applications of SCF include hematopoietic stem cell mobilization, exvivo stem/progenitor cell expansion, gene therapy, and immunotherapy. In this study we tried to construct of pPIC9 recombinant vector containing human SCF.
METHODS
hSCF cDNA was amplified by PCR and both hSCF cDNA and pPIC9 as yeast expression vector (shuttle vector) digested by EcoR I and Xho I restriction enzymes. Subsequent the digestion reaction, ligation reaction was carried out. In order to verifying of pPIC9 recombinant vector containing hSCF, PCR and sequence analysis was performed.
RESULTS
The construction of recombinant expression vector of pPIC9 containing hSCF cDNA was confirmed by sequencing method successfully.
CONCLUSION
rhSCF/pPIC9 vector can be transformed into the Picha pastoris yeast as a eukaryotic host in order to produce human SCF at industrial scale.
目的
多种细胞因子调节造血作用；它们促进干细胞生物学中的多个阶段，如增殖、分化和存活。干细胞因子（SCF）作为一种造血细胞因子，其生物学效应是通过与配体c-kit结合而触发的。SCF的潜在治疗应用包括造血干细胞动员、体外干细胞/祖细胞扩增、基因治疗和免疫治疗。在本研究中，我们试图构建含人SCF的pPIC9重组载体。
方法
通过PCR扩增人SCF cDNA，并用EcoR I和Xho I限制性内切酶分别消化人SCF cDNA和作为酵母表达载体（穿梭载体）的pPIC9。消化反应后，进行连接反应。为了验证含人SCF的pPIC9重组载体，进行了PCR和序列分析。
结果
通过测序方法成功证实了含人SCF cDNA的pPIC9重组表达载体的构建。
结论
rhSCF/pPIC9载体可转化到毕赤酵母中作为真核宿主，以便在工业规模上生产人SCF。
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