The Preparation And Identification Of Dugesia Japonica DjINX-11 Polyclonal Antibody

The freshwater planarian Dugesai japonica with astonishing regeneration capacity belongs to Platyhelminth Turbellaria is good mode for studying animal regeneration. Its generation was mediated by one somatic stem cell, named "neoblasts", which was the sole cells with the capacity of divding, proliferating and differentiating. It is very important how to identify the neoblasts for studying the charaters of neoblasts in order to uncover planarian regeneration mechanisms. In the early researches, neoblasts were identified by their morphological features—small and round/vial cells with a high nucleo-cytoplasmic ratio and heterogeneous distribution of chromatoid bodies, but this method required better equipments and some experience and the neoblasts could not be finely identified because of their accurate results; with the development of molecular biology, the molecular markers specific for cell division were used based on that neoblasts were the sole cells with the capacity of divding,proliferating and differentiating. These molecular markers were often used in the methods such as hybridization in situ or immunohistochemical analys mediated by antibodies. These molecular markers were all located in the cells, which leaded to tedious and time-consuming operation, and worse, the severe damages to cells. To simplify the process of marking neoblasts of Dugesia japonica and develop a simple, quick and specific marking method,connexin DjINX-11 was selected as the target marker for neoblasts. With the analysis of antigenicity, hydrophilicity and transmembrane domain using DNAStar lasergene and TMHMM server v. 2.0 (online toos), the parts (55-111 amino acids) of its first extracellular loop with strong antigenicity was targeted to construct the prokaryotic expression vector which was transformed into Escherichia coli BL21 ( DE3) and induced for 3 hours at 37?with IPTG (final concentrate 0.4 mM). The induced products were analyzed by SDS-PAGE and the target protein was purified to prepair the antigen for getting the polyclonal antibody(pAb). Cell fluorescence experiment and dual color fluorescence in situ hybridization (d-FISH)were used to detected the specificity of prepared pAb, Results showed that the pAb to DjINX-11 was able to label the cells with a high nucleus/cytoplasm ratio, the morphological features of neoblasts. Results from d-FISH showed that DjpiwiA-positive cells were completely overlapped with Djinx-11 -positive cells and the latters were only partially done with the former. Interestingly, Djinx-11-positive cells were all of the morphological features of neoblasts. In a word, the pAb to DjINX-11 laid the foundation for studying the functions of connexin INX-11 and the biological characteristics of DjINX-11-positive cells in Dugesia japonica.