Preparation, Purification And Identification Of Polyclonal Antibody For Sodium-dependent Neutral Amino Acid Transporter2(SNAT2)

Objectives:Sodium-dependent neutral amino acid transporter2, transport of small neutral amino acids, is the second member of the SLC38family and widely expresses in mammals. SNAT2dysfunction can lead to many neurological diseases, such as Alzheimer disease and Parkinson’s disease. In the brain, SNAT2transports glutamine into cells mainly, and mediates glutamate/glutamine cycle, it has important physiological functions. In this study, the gene of SNAT2N-terminal75amino acids were cloned and expressed in E.coli. The recombinant protein was purified and anti-SNAT2polyclonal antibody was prepared. Affinity purification method was used to obtain specificity, high efficiency SNAT2polyclonal antibody. Successful preparation of SNAT2polyclonal antibody will be benefit to further studying structure and function of SNAT2Methods:In order to prepare SNAT2special antibody, the gene of75amino acids of the N terminal of SNAT2was amplied by PCR from pBK-CMV△-SNAT2-HA plasmid. The expression plasmid pET28a-SNAT2-75aa was constructed and transformed into BL21(DE3). SNAT2-75aa protein was induced to express by IPTG. After purification by Ni-column, the recombinant protein SNAT2-75aa was used to immunize New Zealand white rabbit in order to obtain SNAT2polyclonal antibody. In order to further purify the antibody, Thiopropyl sepharose6B column was used. The results of western blot show that the purified SNAT2polyclonal antibody has high specificity for SNAT2.Results:The titer of the SNAT2antibody was1:512000before purification, in order to further purify the antibody, Thiopropyl sepharose6B column was used and western blotting results showed that the purified SNAT2antibody has high specificity for recombinant protein SNAT2-75aa and SNAT2which was expressed in HEK293T. Conclusion:In this study we successfully expressed the SNAT2N-terminal75amino acids in E.coli and purified the recombinant protein by nickel column. The purifiedprotein was used to immunize rabbits and got SNAT2polyclonal antibody. Then the Thiopropyl sepharose6B column was used to purify the polyclonal antibody. The results of western blotting showed that the purified polyclonal antibody can recognize SNAT2expressed in HEK293T and SNAT2-75aa protein expressed in E.coli specifically, providing an efficient tool for further study of SNAT2.