The Production And Purification Of D6 And Darc Chemokine Decoy Receptor Recombinant Proteins And Their Effects On Migration And Invasion In Mda-Mb-231 And Mcf-7 Cells

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dc.contributor.authorTan, Wee Yee
dc.date.accessioned2019-04-09T08:41:00Z
dc.date.available2019-04-09T08:41:00Z
dc.date.issued2018-07
dc.description.abstractD6 and DARC had been reported as a decoy chemokine receptor in cancer study. The involvement of D6 and DARC in breast cancer had been investigated and it was reported to negatively correlate with the progression and metastasis of breast cancer cells. This study aimed to construct recombinant clones of D6 and DARC, express, analyze and purify the proteins and then determine the effects of the recombinant proteins on breast cancer cell migration and invasion. D6 and DARC genes in MDA-MB-231 cell line were first amplified by one-step RT-PCR with gene specific primers and Pfu DNA polymerase. Besides, DARC was also amplified by PCR using Phusion DNA polymerase. For each of the clones, two pairs of specific primers were used to generate full length nucleotide sequences which were cloned into pPICZ and pPICZα expression vectors. The construction of TA clones and DNA sequencing analysis showed perfect match of TA-D6 and TA-D6α to reference sequence. However a base substitution was observed at 131 bp of TA-DARC and TA-DARCα. Site-directed mutagenesis was carried out to correct the mismatch. Generation of recombinant yeast expression vector was performed and DNA sequencing was carried out again to confirm in frame ligation of gene of interest to the N and C-terminal sequences of expression vectors. ClustalW analysis again showed a mismatch at 131 bp of pPICZ-DARC and pPICZ-DARCα, which was identical to the findings of TA-DARC and TA-DARCα.en_US
dc.identifier.urihttp://hdl.handle.net/123456789/7954
dc.language.isoenen_US
dc.publisherUniversiti Sains Malaysiaen_US
dc.subjectPurification of D6 and DARC chemokineen_US
dc.subjectdecoy receptor recombinant proteinsen_US
dc.titleThe Production And Purification Of D6 And Darc Chemokine Decoy Receptor Recombinant Proteins And Their Effects On Migration And Invasion In Mda-Mb-231 And Mcf-7 Cellsen_US
dc.typeThesisen_US
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