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Cloning and transformation human glucocerebrosidase gene in Agrobacterium tumefaciens LBA4404 strain

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Research Paper 01/04/2018
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Cloning and transformation human glucocerebrosidase gene in Agrobacterium tumefaciens LBA4404 strain

Mohamad Al-Hajaj, Zainab Al-Dallee
Int. J. Biosci.12( 4), 312-329, April 2018.
Certificate: IJB 2018 [Generate Certificate]
Key: Agrobacterium transformationGlucocerebrosidasePcambia1304

Abstract

Agrobacterium is broad used, after suitable modified, to be the utmost efficient vector for gene transport into plant cells. This study was carried out to produced Agrobacterium tumefaciens LBA4404 strain contains binary vector has human GBA  gene to became ready for  transforming any desired plant to expression glucocerebrosidase. Recombinant glucocerebrosidase is current treatment for gaucher disease which is lysosomal storage disease caused by mutations in the encode for glucocerebrosidase. In this study, Human whole blood was pivotal source of RNA by using GENEzol™ TriRNA Pure Kit, and the Hu-GBA gene was amplified by designed special primers,GBA gene was introduced inthe plant expression vector pCAMBIA1304 beneath the domination of the cauliflower mosaic virus 35S promoter and grated recombinant pcambia1304-GBA vector having GBA gene by restriction and ligation methods.Calcium chloride heat-shock transformation is used to introduce pcambia1304-GBA into E. coli DH5α and plantation on LB agar medium contains kanamycin 50 mg/l. The transformed colonies were proven by colony PCR and amplification GBA gene from isolated recombinant pcambia1304-GBA. The recombinant isolation plasmid was transferred to Agrobacterium tumefaciens LBA4404 using modified freezing-thaw method, transformation Agrobacterium colonies were enhanced by colony PCR. Results demonstrated that The1561bp-GBA gene was amplified from human total blood RNA which was confirmed by sequencing the PCR product which gives 100% identified withHomo sapiens glucosylceramidase beta (GBA), transcript variant 1, mRNA NM_000157.3 Gene Bank and colony PCR assured that Agrobacterium tumefaciens LBA4404 carried Hu-GBA gene.

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