Preparation And Identification Of Polyclonal Antibodies Against Chicken Protocadherin1,8,17

BackgroundProtocadherins (Pcdhs), predominantly expressed in the central nervous system, are a group of transmembrane proteins with homophilic binding activity. Some studies had shown that Pcdhs, which can not only strengthen the connection of neuron synapses as adhesion molecules but also play a certain role in the signal transduction process as receptors, have direct connection with the development of neuron synapses. At present, some mutations of pcdh genes have been proved to have connections with human disease. Although, the function of some Pcdhs had been proved, the mechanisms are still unclear. Because there are no Pcdhs antibodies in sale, the further study on Pcdhs is limited. Therefore, this study aims to establish a fast preparation method for polyclonal antibodies against chicken Pcdhs, to develop assay methods helping to understand the mechanisms of Pcdhs, and to provide new approaches on the diagnosis and therapy of related diseases in clinical.ObjectiveTo establish a preparation method of Pcdhs polyclonal antibodies and prepare the polyclonal antibodies of Pcdh1,8,17.Methods1. The amplification of target fragments(1) Analyzing transmembrane domains of Pcdh1,8,17through the Globplot2.3software. Primers were designed with DNAStar software, including intra-membrane primers from cytoplasmic domain and extra-membrane primers from extracellular domain.(2) Total RNA was extracted from the7days of incubation of brain tissue of chicken embryo (E7) and cDNAs were synthesized through RT (reverse transcription). The cDNAs were used as templates and the target fragments were amplified from the cytoplasmic and extracellular domains of pcdhl,8,17(the cloned fragments were named as101,102,801,802,1701and1702seperstely, and the cytoplasmic domain and extracellular domain were parallel tested. In this experiment,pcdh101,802,1701and1702were studied.). 2. The construction of prokaryotic expression vectors101,802,1701and1702were cloned into pGEX-2TK and pET-32a vectors seperately, and the recombinant plasmids of pGEX-2TK-101, pGEX-2TK-802, pGEX-2TK-1701and pET-32a-1702were constructed, identified by restriction enzyme digestion and sequencing.3. The inducation and purification of target proteins(1) The recombinant plasmids were transformed into E.coli BL21and the expressions of target proteins of GST-101、GST-802、GST-1701and (His)6-1702were induced by IPTG.(2) The recombinant oligopeptides fused with the carrier protein glutathione-S-transferase and six histidines ((His)6) separately were purified with Glutathione-sepharose4B and Ni-NTA Sefinose Resin, respectively.4. Immunizing ratsRats were immunized by injecting the emulsified antigen subcutaneously into the hind footpads and the antiserums were collected.5. The identification of polyclonal antibodiesThe titer and specificities of polyclonal antibodies were examined by indirect ELISA and Western blotting.6. Detecting the expression levels of pcdh1,8,17in vivoThe expression levels of pcdhl,8,17were detected by PCR under the condition of GAPDH as the internal reference.Results1. The target fragmens of the intra-membrane of pcdh1,17(pcdh101,1701) and the extra-membrane of pcdh8,17(pcdh802,1702) were cloned seperately.2. The recombinant plasmids of pGEX-2TK-101, pGEX-2TK-802, pGEX-2TK-1701and pET-32a-1702were constructed.3. In E.coli BL21, the fusion proteins GST-101, GST-802, GST-1701and (His)6-1702were expressed by inducation with IPTG and t purified.4. Obtaining the polyclonal antibodies of anti-GST-101,-802,-1701and anti-(His)6--1702after immunizing rats.5. The results of ELISA showed that the best dilution ratio of anti-GST-101,-802,-1701and anti-(His)6-1702were1:3000,1:1600,1:800and1:3200respectively. 6. The results of western blotting showed that the antibodies could not only combine with the antigens used to immunize rats but also react with the full proteins of Pcdhl,8,17expressed in brain tissue of chicken embryo.7. The mRNA expression levels of pcdhl,8,17in some periods of chicken embro were detected by RT-PCR.ConclusionsThe preparation method of chicken Pcdhs polyclonal antibodies was established and the polyclonal antibodies of Pcdhl,8,17were prepared successfully. These antibodies can be used for ELISA and Western blotting. The results of Western blotting showed that the antibodies could react with the full length proteins of Pcdh1,8,17expressed in brain tissue and spinal cord of chicken embryo which can contribute to develop assay methods and helping to further understand the mechanisms of Pcdhs.