Preparation Of Rabbit Anti-wheat Calmodulin Polyclonal Antibody

As a Ca²⁺-binding protein, calmodulin (CaM) is considered to be a most important signal transduction molecule now. Ca²⁺/CaM complex has been stuied in one of the most in-depth and extensive way on plant cell signal transduction field. It is initially proved to be true that the Ca²⁺/CaM have involved in the hypersensitive reaction (HR) induced by wheat leaf rust fungus infection according to our preliminary work, which has been proved that the expression of wheat calmodulin (TaCaM) increased after the stimulation of the incompatible leaf rust race. In order to explore the role of CaM in the HR evoked by leaf rust further, the specific antibody preparation of TaCaM is necessary because it is the most effective and specific reagent in localization, quantitation and physiological functions research of CaM. An experiment was designed in this article to prepare rabbit anti-TaCaM antiserum, and purify it to obtain specific polyclonal antibodies by PVDF membrane binding method. This experiment have not only established the foundation, through which we could explore the spatial and temporal distribution features, and the effect of CaM in the interaction process of wheat and leaf rust, but also provided a feasibility test means to build various TaCaM isoforms recombinant, purify their respective specific polyclonal antibodies.The first step, CaM protein from wheat leaves was extracted and purified by heating, ammonium sulfate precipitation, isoelectric point precipitation, dialysis and other methods. And then, it was used as antigen to immune rabbits to prepare the anti-TaCaM antiserum. Its titer was tested in enzyme-linked immunosorbant assay (ELISA) by using the Arabidopsis CaM protein expressed in E. coli as antigen. The results indicated that the preimmune serum hardly had response to antigen, but the titer of the booster immune antiserum increased significantly. The titer of the rabbit anti-TaCaM antiserum after the last immune was 1:256 000. Next, the Arabidopsis CaM protein expressed in E. coli was used as antigen, the antiserum was purified by PVDF membrane binding method, and the purified polyclonal antibody was identified by the Western Blotting. The results showed that the purified polyclonal antibody can bind with purified TaCaM speciflcly. It is important that the CaM specific band also can be detected from the total protein of wheat, and the band become clearer than the band detected by rabbit anti-TaCaM antiserum. The results above demonstrated that the purified antibody was TaCaM specific polyclonal antibody purified before. In addition, the total RNA was extracted from wheat leaves, 3 pairs of primers were designed according to the reported gene sequences of 3 isoforms of camodulin which came from Zhongguochun, and then, the TaCaM cDNA was amplified by RT-PCR, all measurements above provided a platform for obtaining the varies recombinats of isoforms of camodulin.