جدا سازي و كلون سازي ژن MAP30گياه Momordica charantiaدر باكتري اشريشيا كلي

Isolstion and cloning of MAP30 gene of Momordica charantia plant in Escherichia coli

در اين مطالعه استخراج DNA از گياه كارلا به روش دلاپورتا كه يك روش ساده و آسان و سريع مي باشد، صورت پذيرفت. كميت و كيفيت DNA استخراج شده به روش اسپكتروفتومتري و الكتروفورز ژل آگارز مورد بررسي قرار گرفت. پس از استخراج DNA براي ژن MAP30 بر اساس توالي هاي گزارش شده در سايت NCBI پرايمر هاي اختصاصي طراحي گرديد، و ژن به گونه اي تكثير شد كه امكان كلون آن در حامل هاي متعدد بوجود آيد. MAP30 در وكتور pTG19.T كلون و به باكتري اشريشيا كلي سويه DH5α منتقل گرديد. تاييد ترانسفورماسيون به كمك روش هاي انتخاب آنتي بيوتيكي، كلني هاي سفيد و آبي و آنزيم BamH 1 صورت پذيرفت. نتايج حاصل از توالي يابي و آزمون هاي تائيدي جداسازي و كلون نمودن صحيح ژن MAP30 را تائيد نمود.

The purpose of this study was to isolate and clone the MAP30 gene in E. coli which can increase the universal expression of the protein MAP30. The product of this gene is used as a anti-cancer and anti-viral molecule in numerous researches. In the study, Karela plant DNA was extracted accoding to delapota et al. (1983) which is a feasible and economic method. The quantity and quality of the extracted DNA was evaluated by spectrophotometry and agarose gel electrophoresis methods. DNA amplification of MAP30 gene was designed based on the deposited sequences in the NCBI data base and the gene was amplified in such a way that possessing the potential for colonization in several vectores. The MAP30 gene was cloned in pTG19-T vector and transferred to DH5α E. coli competent cell employing heat- shock method. The transformation was confirmed by applying a selective media containing antibiotics. Production of while colonies was the criterium for successful transformation while the blue colonies represent failure in the transformation process. The sequencing data and cloned DNA segments verified the appropriate and functionel insertion of MAP30 gene in to the E. coli. The obtained bacterial clones with MAP30 gene in their plasmids are preserved for further researches dealing with MAP30 protein function.