Production Of An Antibody Against Rat Recombinant Choline Acetyltransferase

Acetylcholine (ACh) is the first neurotransmitter to be discovered and plays a pivotal role in such fundamental brain processes as learning, memory, and sleep. Choline acetyltransferase (ChAT), the biosynthetic enzyme of ACh, is currently the most specific marker available for identifying cholinergic neurons. Thus, immunohisto-chemistry using antibodies directed against ChAT have provided the basis for more specifically delineating the anatomical distribution of cholinergic elements in the mammalian central nervous system. However, the purification of ChAT is fraught with difficulties because of its extremely low concentration in many tissues and its instability. It was very hard to get a high titer antibody against ChAT. The various anti-ChAT antibodies that have so far been reported were variable in their titer, specificity and their usefulness for immunohistochemical work. In the present study, a fusion protein expression system was introduced to obtain the rat ChAT protein and used it as an antigen for the production of an anti-ChAT antibody.Total RNA was extracted from the rat striatum and used for the fisrt strand cDNA synthesis by reverse-transcription. The ChAT gene fragment was then amplified using the reverse transcribed cDNA and a specific pair of primers of ChAT. In order to confirm the inditity of the amplified ChAT gene fragment, a recombinant palsmid was constructed by cloning the ChAT gene fragment into pGEM vector. The plasmid was subsequently transformed into E.coli strain JM109 competent cells and analyzed by coloney screening. The positive colonies were selected for mini-preparation of plasmid DNA and subjected to DNA sequencing. The ChAT-pGEM recombinant plasmid, with correct orientation and nucleotide sequences, along with the pMAL-c2 expression vector, were then digested by restriction enzymes Hind III and EcoR I. The cleaved ChAT gene was subcloned into pMAL-c2 vector to construct a fusion vector. The presence of the ChAT insert was detected by colony screening and the reading frame of the constructed expression plasmid was determined by DNA sequencing analysis. The ChAT cDNA was expressed in E.coli as a fusion protein with maltose-binding protein (MBP). The expressed fusion protein was purified by amylose-resin affinity chromatography and then...