Role Of Receptor-interacting Protein,RIP1 In Anti-tuberculosis Immunity And Screening For Its Interaction Protein

Tuberculosis is a chronic infectious disease caused by M.tuberculosis?MTB?infection,which is co-infected with humans and animals and causes a serious threat to human health and animal husbandry.After macrophages phagocytose MTB in immune system,the triggered natural immune response is closely related to the anti-tuberculosis level.As the first member of the receptor-interacting proteins?RIPs?,RIP1 is thought to play a key role in innate immune response,but there is currently no reports about RIP1 in macrophage anti-tuberculosis immunity.In order to study the role of RIP1 in innate immune response after tuberculosis infection,we first established Rip1 gene stably overexpressing macrophage cell line?RAW-RIP1?by using lentiviral packaging infection,and using this cell line as a research material,observed RIP1 involved in anti-tuberculosis.The MTB infectious experiment showed that the proliferation of intracellular live bacteria was inhibited,and the inflammatory factors such as IL-12p40 and TNF-?were secreted.These results suggested that RIP1 may increase the anti-tuberculosis ability of macrophage by moderately activating inflammatory signaling pathways.Next,as the potential TB treatment target of RIP1,we screened the interaction proteins of RIP1 by yeast two-hybrid system,and hoped to study its mechanism of regulation of immune response from the perspective of protein interaction.First,we established RAW264.7 cDNA library in Y187 yeast strain:extracted the total RNA of mouse macrophage RAW264.7,synthesized the first strand cDNA by SMART^TM technology,and amplified the double strands by long distance PCR?LD-PCR?.Then,the ds cDNA was purified using CHOMA SPIN+TE-400 columns,and the purified ds cDNA was co-transformed into the Y187 yeast competent cells with the pGADT7-RecAB vector by LiAc method,and the transformant was plated at 150mm SD/-Leu for selection.The transformant was cultured at 30?for 3 days,and the transformants were harvested,resuspended in a frozen stock solution,and subjected to cell counting,and 1 ml of each tube was dispensed and stored at-80?.After identification,the number of independent clone reached 8.6×10⁶cfu,and the average insert cDNA length reached 1.45kb,which can be used for the screening of RIP1 interaction proteins.On this basis,we further inserted Rip1 into the pGBKT7 vector to construct a bait?Bati-RIP1?,transformed into Y2H yeast competent cells,and tested by toxicity and self-activation.The Bati-RIP1 were mixed with the library,hybridized at 30?,40 rpm/min for 20 h,the zygotes were collected by centrifugation,plated in 150mm DDO/X/A selection medium,cultured at 30?for 4 days,and the blue clones were selected for further screening.The QDO/X/A selection was processed for 5 days at30?,and the blue clones were selected to extract plasmids.Two RIP1 interaction proteins were identified by sequence,which were Isochorismatase domain protein 1?ISOC1?and PUM2,a member of RNA binding protein family?Pumilio?.Co-Immunoprecipitation confirmed that RIP1-ISOC1 and RIP1-PUM2 can indeed interact in macrophage.In summary,this study demonstrated that the receptor-interacting protein RIP1enhances the anti-tuberculosis ability of macrophage by moderately enhancing the inflammatory response after MTB infection,and is one of the potential therapeutic targets for tuberculosis.Through establish of RAW264.7 cDNA library,yeast two-hybrid screen were used to identify the interaction between RIP1-ISOC1 and RIP1-PUM2,which laid a foundation for further study on the mechanism of RIP1 regulating the immune response induced by MTB infection.