The Study Of The Polymeric Immunoglobulin Receptor Of Common Carp.

Polymeric Immunoglobulin Receptor (pIgR) is a kind of transmembrane glycoprotein which mediates the transportation of polymeric immunoglobulins including pIgA and pIgM across mucosal epithelial cells. This receptor interacts with pIgA/pIgM at the basolateral surface of epithelial cells resulting in transcytosis and subsequent cleavage of the pIgR at the apical surface, releasing a component of SIgA/SIgM or in a free form of secretory components(SC). PIgR and SC have innate anti-microbial properties, and can protect SIgA/SIgM form proteolytic degradation. The pIgR is very conservative in evolution, and has already been cloned and studied in several kinds of animals.In this paper, the extracellular part of carp. pIgR was cloned by using the methods of molecular cloning and recombinant technics, and connected to pET-28a(+) vector in BamHⅠand HindⅢsites. E.coli BL21(DE3) was transformed with the recombinant expression vector pET-28a(+)/pIgR and expressed the recombinant protein in adding of 1mM IPTG at 37℃. SDS-PAGE results showed that a protein with a molocular weight of 29.2kDa was expressed within the inclusion bodies. The protein was used as antigen to immune animals to gain the rabbit-anti-carp pIgR antiserum, which titer was 1:8 according to the double agar diffusion experiments.We studied the difference of expresstion of pIgR in several tissues of carp. and the change of expresstion of pIgR after the carps were stimulated. We use rabbit-anti-carp pIgR antiserum to study the distribution of pIgR in situ in several tissues of carp via immunohistochemical manipulations.In order to get the highly effective antiserum, we designed the DNA immunity experiment. Firstly, we designed the pIgR segment with kozak and ATG sequences and constructed the pcDNA3.1/pIgR vector. Then the highly purity recombinant vector was extrated and injected to mice to get the mice-anti-carp pIgR antiserum.For further research, recombinant plasmid pEGFP-N1/pIgR was transfected into 293T cell line and identified by green fluorescence. The whole cells'protein was collected and used to test the identity of mice-anti-carp pIgR antiserum by western-blot experiment.In conclusion, we studied the pIgR of common carp. from its gene and protein level, which is very important in fish innate immune.