Establishment And Identification Of P815Cell Line Stably Expressing Chicken Ii Gene

Invariant chain(Ii) is a chaperonin of MHC II molecule,it assists the submission ofexogenous antigen mediated by MHC II molecule.Recent studies indicated that Ii mediatesinflammation，tumour etc. as a receptor of MIF. Ii of human and mammal have been deeplyresearched,but the research of poultry Ii was started recently.For deeper research onchicken-Ii,we planed to establish P815cell line stably expressing chicken-Ii.First, on the basis of recombinant plasmid PMD18-T/Ii and Ii gene sequence in it,wedesigned PCR primer and acquired chicken-Ii gene sequence by PCR.Then we constructedeukaryotic fluorescence express recombinant plasmids:pDsRed2-N1/Ii，pEGFP-C1/Ii.Afterantibiotics resistant gene screening, double digestion,and sequencing appraisal,weconfirmed the recombinant plasmids.Second, we transfected the recombinant plasmids to COS7and observed theexpression of GFP-Ii and RFP-Ii，we found that fluorescence was mainly distributed oncell endomembrane system.Then,we chose Ii gene sequences of human,cattle,chicken,parrot and made some bioinformatics analysis including sequence alignment, evolutionarytree rendering, protein structure prediction and location in cells prediction.We found thatthe homology of them achieved61.69%, among them,human and cattle belong to mammal,their homology achieved81.3%,chicken and parrot belong to poultry,their homologyachieved85.6%.Cladogram displayed the same pattern.After Protein structuresimulation,we found that their structure were very similar,so we inferred the functions ofthem are similar,and the location in cells prediction confirmed our conjecture.At last,we transfected pEGFP-C1/Ii to P815cell line, acquired cell line could keepalive in3mg/ml G418environment,RT-PCR proved that the cell line could stably expressGFP-Ii.Under fluorescence microscope the fluorescent light of P815-GFP-Ii distributed oncell endomembrane system.On the basis of recent reaserch and location in cellsprediction,we inferred if Ii distributes in nucleus,it mainly locates on caryotheca.This research has successfully constructed recombinant plasmids pDsRed2-N1/Ii，pEGFP-C1/Ii, and acquired P815cell line stably expressing chicken-Ii.It has builded tumorcell model for testing and evaluating the efficacy of antitumor immunology drugs aimmedat Ii,and laid foundation for deeper research on Ii.