Cloning, Expression And Purification Of HumanTMEM16A-specific Peptide/GST Fusion Protein And Preparation Of Antibody And Cloning Human TMEM16A/pcDNA3.1

Calcium-activated chloride channels (CaCCs) play fundamental roles in numerous physiological processes. Despite their physiological importance, the molecular identity of CaCCs has not been fully investigated until recently, transmembrane16A (TMEM16A) was demonstrated by three independent research groups to be a strong candidate for the CaCC molecular basis. Then the research about the TMEM16A gene can be divided into at least two directions:on the one hand, the researchers focused on the expression patterns, molecular structures of TMEM16A and its relationships with certain diseases; on the other hand, as the TMEM16A belongs to the TMEM16gene family which contains another10members and it has been reported that they may also be the components of ion transport channel, so there were many researchers who conducted various experiments to investigate the expression patterns and functions of the other TMEM16gene family members, and predicted their relationships with diseases. This review mainly summarizes the research progress of the CaCCs and the TMEM16A protein.Transmembrane protein16A (TMEM16A) has recently been identified as calcium-activated chloride channel protein. Human TMEM16A (hTMEM16A) is expressed in many cells and tissues, and is overexpressed in many cancers, but the function of hTMEM16A is still unclear. In order to study the structure and function of hTMEM16A protein, total RNA was extracted from the human colon cancer cells SW620to synthesize the first strand of cDNA by reverse transcriptase. The coding sequence of the hTMEM16A-specific peptides was amplified by PCR using this cDNA as template, and then cloned into the expression vector pGEX-2T. After comfirmation by restriction enzyme digestion and sequencing, the recombinant plasmid was transformed into expression cells E.coli BL-21. Soluble fusion protein was induced by0.1mM IPTG and purified by GST affinity chromatography and Superdex75gel filtration chromatography, and7mg/L high purity fusion protein was gained successfully. GST-tag of the fusion protein was confirmed by Western blotting. We used the purified GST fusion protein as antigen to immune rabbit and get the polyclonal antibody with the titer of1:100000by ELISA, By cell immune fluorescent find the protein express location in SW620cell line. At the same time, The coding sequence of the hTMEM16A peptides was amplified by PCR using this cDNA as template, and then cloned into the expression vector pcDNA3.1. To guarantee the foundation for further study the structure and function of hTMEM16A protein.