Isolation of a novel member of small G protein superfamily and its expression in colon cancer

来源 :World Journal of Gastroenterology | 被引量 : 0次 | 上传用户:dr_rush
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AIM:APMCF1 is a novel human gene whose transcriptsare up-regulated in apoptotic MCF-7 cells.In order to learnmore about this gene’s function in other tumors,we clonedits full length cDNA and prepared its polyclonal antibodyto investigate its expression in colon cancers withimmunohistochemistry.METHODS:With the method of 5’ rapid amplification ofcDNA end (RACE) and EST assembled in GenBank,weextended the length of APMCF1 at 5’ end.Then the sequenceencoding the APMCF1 protein was amplified by RT-PCR fromthe total RNA of apoptotic MCF-7 cells and cloned into theprokaryotic expression vector pGEX-KG to constructrecombinant expression vector pGEX-APMCF1.The GST-APMCF1 fusion protein was expressed in E.coli and used toimmunize rabbits to get the rabbit anti-APMCF1 serum.Thespecificity of polyclonal anti-APMCF1 antibody wasdetermined by Western blot.Then we investigated theexpression of Apmcfl in colon cancers and normal colonicmucosa with immunohistochemistry.RESULTS:A cDNA fragment with a length of 1 745 bpwas obtained.APMCF1 was mapped to chromosome 3q22.2and spanned at least 14.8 kb of genomic DNA with sevenexons and six introns contained.Bioinformatic analysisshowed the protein encoded by APMCF1 contained a smallGTP-binding protein (G proteins) domain and washomologous to mouse signal recognition particle receptorβ(SRβ).A coding region covering 816 bp was cloned andpolyclonal anti-APMCF1 antibody was prepared successfully.The immunohistochemistry study showed that APMCF1 hada strong expression in colon cancer.CONCLUSION: APMCF1 may be the gene coding human signal recognition particle receptor p and belongs to the small-G protein superfamily. Its strong expression pattern in colon cancer suggests it may play a role in colon cancer development. AIM: APMCF1 is a novel human gene whose transcripts up-regulated in apoptotic MCF-7 cells. In order to learnmore about this gene’s function in other tumors, we clonedits full length cDNA and prepared its polyclonal antibody to investigate its expression in colon cancers withimmunohistochemistry. METHODS: With the method of 5 ’rapid amplification of cDNA ends (RACE) and EST assembled in GenBank, we extended the length of APMCF1 at 5’ end. The sequence encoding the APMCF1 protein was amplified by RT-PCR from the total RNA of apoptotic MCF- 7 cells and cloned into the prokaryotic expression vector pGEX-KG to construct recombinant vector pGEX-APMCF1. The GST-APMCF1 fusion protein was expressed in E. coli and used to immunize rabbits to get the rabbit anti-APMCF1 serum. Specificity of polyclonal anti-APMCF1 antibody wasdetermined by Western blot. The we were the expression of Apmcfl in colon cancers and normal colonic mucosa with immunohistochemistry .RESULTS: A cDNA fragment with al ength of 1 745 bp was obtained. APMCF1 was mapped to chromosome 3q22.2 and spanned at least 14.8 kb of genomic DNA with sevenexons and six introns contained. Bioinformatic analysis of the protein encoded by APMCF1 contained a small aGTP-binding protein (G proteins) domain and washomologous to mouse signal recognition particle receptor β (SRβ). A coding region covering 816 bp was cloned and polyclonal anti-APMCF1 antibody was prepared successfully. The immunohistochemistry study showed that APMCF1 hada strong expression in colon cancer. CONCLUSION: APMCF1 may be the gene coding human signal recognition particle receptor p and belongs to the small-G protein superfamily. Its strong expression pattern in colon cancer suggests it may play a role in colon cancer development.
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