Construction And Identification Of Chick Embryo Fibroblast Cell Lines Stable Expression RIG-I Of Goose

Retinoic acid inducible gene-I (RIG-I) is a kind of pattern recognition receptor intracellularfound in recent years, which is an important component of innate immunity.RIG-I canspecifically recognized and combined with RNA produced in the process of viral infection inbody, replication and proliferation in cells, then change itself conformation, bind to adaptermolecule downstream, turn on signal path, so that the body play the role of innate immunityeventually.RIG-I is conservative in the evolution, there is no RIG-I gene within the chicken genomebut in duck and goose. Chickens have massive infection when every outbreak of Newcastledisease and avian influenza, RIG-I can recognize and bind to RNA generated when these twokind of viruses replicate in cells, whether there is a connection between them aroused theinterest of many researchers. The experiments build chicken embryo fibroblast (CEF) linesstable expression RIG-I gene of geeseof by using PiggyBac system, lay a foundation forsubsequent experimental study. The main contents and results in the study are as follows:1. The construction of chicken embryo fibroblast(CEF) stable expression RIG-I gene ofgooseAccording to the RIG-I nucleic acid sequences on the GenBank and combined withPiggyBac-513B-1carrier sequence, the specificity primers were designed. The plasmidFlag-RIG-I as template for PCR amplification, RIG-I was connected with carrierPiggyBac-513B-1after enzyme digestion, and filtered using puro after transfecting into cells.Single cell lines obtained through twice sub-clone continued passage, and the samples werecollected to verify the stability.2. Preparation of Polyclonal antiserum RIG-I gene of goose CARD domainAccording to the RIG-I nucleic acid sequences on the GenBank and combined withpET-32a vector sequence, the specificity primers were designed. The plasmid Flag-RIG-I astemplate for PCR amplification, RIG-I was connected with pET-32a vector after enzymedigestion, transformed into BL21(DE3) competent cells. Proteins were induced to express andthen purified，immuned SPF Balb/c mice to obtain polyclonal antiserum which titer was1:200million or more. 3. Identification of cell lines stable expressionSingle cell lines obtained through twice sub-clone continued passage, and the samples werecollected to verify the stability. Samples were collected in1st generation、5th generation,10thgeneration,15generations, the20th generation and30generations，the verification mRNA andprotein expression levels of RIG-I gene by RT-PCR and Western Blot，assisted verification byimmunofluorescence. The results showed that the constructed cells were stable and can be usedfor subsequent study.