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Cloning of Human Anion Exchanger2 (AE2) Gene and Construction of Mammalian Expression Recombinant pcDNA3.1-wt-AE2

Author: XiaoBin
Tutor: LaiZhongFang;HeMing
School: Nanchang University
Course: Pharmacology
Keywords: AE2 gene pcDNA3.1 clone transcription HUVECs
CLC: Q78
Type: Master's thesis
Year: 2008
Downloads: 6
Quote: 0
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Abstract


Objective: To clone human wild-type full-length AE2 cDNA and construct pcDNA3.1 eukaryotic expression vector . To identify the recombinant plasmid nucleic acid sequence by sequencing on the standard sequencer, and to evaluate the plasmid impact to the AE2 gene expression in transfected HUVECs cell line . These results lay a foundation for the further research on the function of AE2.Methods:1.Human AE2 cDNA was amplified by reverse transcription-polymerase chain reaction （RT-PCR） from total RNA isolated from cultured Hela cells maintained in DMEM+10%FBS.2.AE2 was inserted into the NheI and HindⅢsites of a mammalian expression plasmid pcDNA3.1, which contains Ampamycin resistance genes, to constructre combinant plasmid pcDNA3.1-wt-AE2.The recombinant pcDNA3.1-wt-AE2 was transformed into competent cell Escherichia coli TOP10 for positive screening and propagation followed by plasmid preparation and purification.3. The recombinant pcDNA3.1-wt-AE2 performed by the analysis of different restriction endonuclease digestioned automate sequencing.4.The confirmed recombinant pEGFP-N2, pcDNA3.1, pcDNA3.1-wt-AE2 was respectively transfected into HUVECs cell line by liposome method to observing the expression of GFP under immunofluorescence microscope, AE2 mRNA as well as protein levels were examined by RT-PCR and Westernblotting.Result:1. The AE2 cDNA was particularly amplified with primers we designed as expected.2. The recombinant of pcDNA3.1-wt-AE2 were successfully constructed.3. The recombinant of pcDNA3.1-wt-AE2 was structurally confirmed by analysis of restriction endonuclease digestion and sequencing. The inserted AE2 cDNA segment in the vector was sense orientation as a wild type AE2 gene and its sequence was structurally confirmed to be consistent with that of the published data （GenBank NM 003040）.4. The pEGFP-N2 vector was transfected into HUVECs cells by lipofectamine. We could find the green fluorescent in HUVECs cells under the fuorescent microscope.5. The results of RT-PCR indicated that pcDNA3.1-wt-AE2 could remarkably increase the expression levels of AE2 gene mRNA in transfected HUVECs cells （p<0.01）.6. The results of Western blot indicated that pcDNA3.1-wt-AE2 could remarkably increase the expression levels of AE2 protein in transfected HUVECs cells.Conclusion:1. The AE2 cDNA was particularly amplified with primers we designed as expected. The recombinant of pcDNA3.1-wt-AE2 were successfully constructed.2. The ecombinant of pcDNA3.1-wt-AE2 could significantly increase the expression of AE2 gene mRNA and protein level in transfected on HUVECs cells.

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