Lentivirus Mediated ShRNA Interference IRG1Gene Expression And The Effect In Brucella Infection

Brucellosis is caused by Brucella which is a kind of zoonotic infectious diseasesthat mainly damage the body lymphatic system and reproductive system. The diseaseis widely distributed around the world, the most damaging one of zoonosis.Brucellosis outbreak in the geographical distribution is tendency to spread gradually,the incidence rate is tendency to ascend in the world, which popularity spread not onlycause livestock production suffering serious losses, but also harm to human health isvery seriously. Therefore, prevention of brucellosis significance, not only in terms ofveterinary science, more prominent in the public health aspects.In terms of brucellosis prevention and control, research and development ofvaccine has been a hot of scientific research. However, the application of vaccinescurrently exist in varying degrees of defects which cause has not been widely usedand develop new vaccines slow progress. Therefore, this study intends to achievegene silencing on IRG1in mouse macrophage cell line RAW264.7gene expression byRNAi technology, by puromycin pressure screening to obtain IRG1gene silencingcell lines of stably integrated lentivirus gene, detect the role of regulation secretion ofIFN-γ and TNF-ɑ, verification the role of IRG1gene silencing cells outsideanti-Brucella infected. In order to screen more resistance-related genes andsubsequent gene screening and validation function to lay the foundation.Therefore, this study first obtain IRG1of about2000bp, after sequencing rightconnect to the dual luciferase expression vector. Then based on IRG1designed fourdifferent siRNA, make use of liposomes co-transfected293T cells, by dual luciferaseassay system detect siRNA1make Renilla luciferase expression levels significantlyreduce, thereby obtaining the best sequence for interference IRG1gene. The shRNA1successfully connected to the lentiviral vector, by restriction enzyme digestion and sequencing identified correctly, recombinant lentivirus transfected293T cells underthe fluorescence microscope shows a large number of red fluorescence; Lentivirusinfect293T cells resulting visible red fluorescence under a fluorescence microscoperevealed that the success of the lentivirus packaging. Experimentally measured bypuromycin kill cells the minimal lethal concentration of puromycin on293T cells is2.5μg/mL; After diluted enrichment virus infection293T cells use puromycin cultureand measurement the virus titer which is reached108TU/mL. Further study showedthat5.0×104TU/mL lentivirus weakest toxicity on RAW264.7cells, and optimumscreening concentration of puromycin is2.0μg/mL, This concentration lentivirusinfection RAW264.7cells, adding2.0μg/mL puromycin pressure screening to obtainIRG1gene silencing RAW264.7cell lines. Further analysis showed that: RAW264.7cells of stably integrated lentiviral gene under a fluorescence microscope shows alarge red fluorescence; IRG1gene silencing on RAW264.7cell growth, morphologyhad no significant effect.By Real-time PCR detect gene silencing effect of IRG1gene silencing cellsshowed that gene silencing cells gene inhibition rate of92.73%. Quantitative PCRresults showed that after M5and16M Brucella treatment gene silencing cells group,which were elevated mRNA levels of IRG18times and11.5times, compared with thecontrol significant difference. Brucella infection cell experiments show that,compared with the control, whether it is up-regulated IRG1genes induced by LPS orRNAi-induced inhibition of IRG1gene expression cells, MOI of Brucella were notsignificantly different.The above findings suggest that using dual luciferase detection system can screensiRNA that make mRNA levels of IRG1gene reduce significantly;lentivirus-mediated siRNA can package Lentiviral Particles that make IRG1genesilencing of RAW264.7cells; IRG1gene silencing can not have a significant impacton the Brucella infection (MOI);16M and M5Brucella infect gene silencing cellswhich have different impact on IRG1mRNA level，and compared to control havesignificant difference. This study lay the foundation for IRG1and related regulating genes resistant Brucella research.