| Objective: Polypeptide: N-acetylgalactosaminyl transferase is the initiation enzyme catalyzing the linkage of O-glucan chain. Recent study shows that O-glucosylation is closely related to molecular recognition, tumor formation, development and metastasis, as well as embryonic development. Due to the initial study on function of O-glucosylation in China, this thesis aims to obtain stably expressed pp-GalNAc-T2 gene clones for further study. At the same time, we can observe the distribution of pp-GalNAc-T2 at mRNA level in different human tissues, which gives useful clues for further research. Methods: By using Gateway technology, we have cloned the soluble part of pp-GalNAc-T2 with human brain cDNA library as templates; we also adopt RT-PCR to detect the expression of pp-GalNAc-T2 in six different human tissues. Results: 1. By using a pair of synthetic primers, with Mararhon-Ready?human brain cDNA library used as templates, we amplified a fragment of pp-GalNAc-T2 cDNA. Then we transformed it into prokaryotic pFASTBAC plasmids through pDONR?01 cloning vector, for cloning amplification. Two reconbinant plasmids are characterized through linearized length, or cloning PCR and sequence characterization. We also transformed the plasmids into DH5a engineeringbacterium. By the means of SDS-PAGE, the molecular weight of expression product is inaccordance with the reference report. 2. Among six different human tissues, mRNA of pp-GalNAc-T2 in gastric and intestinal tract are expressed at a high level, while they remain at a moderate level in brain and cardiac muscle, and at an even lower level in kidney and liver. Conclusion: We have obtained the prokaryotic reconbinant cloning and expression plasmid of pp-GalNAc-T2 successfully, which establish a foundation for further research. The expression of pp-GalNAc-T2 differs obviously in different tissues, showing the possible relationship between the expression of pp-GalNAc-T2 and the functions of tissues, which desires our further probing.
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