The Mechanism Of Tim-3 Negetive Regulation Against Rna Virus Infection And Antibody Optimization

The invasion and spread of viruses can cause various diseases of the human body and seriously threaten human life and health.For example,influenza viruses such as H1N1 can cause large-scale influenza;Zika virus can cause microcephaly and has a high lethal rate;HIV virus can damage the body's immune system and lead to the acquired immunodeficiency syndrome;HSV virus can cause encephalitis,keratitis,herpes and other diseases;HAV,HBV,HCV,etc.can cause different types of viral hepatitis and in 2003 the SARS virus ravaged our country.Before the Spring Festival in 2020,a severe pneumonia epidemic caused by the novel coronavirus COVID-19 broke out and spread rapidly throughout the country and globally,posing a huge threat to people's lives and healthThe immune system is the guardian of the body against viruses,including two lines of defense,natural immunity and adaptive immunity.Natural immunity is the first line of defense against virus invasion,including immune structures such as skin and mucosa,and immune cells such as macrophages and neutrophils.T cell immunoglobulin and mucin-domain containing molecule-3(Tim-3)was first discovered to be expressed on CD4+Th1 cells and is an important member of the Tim family[1].It is encoded by the HAVCR2 gene and consists of 301 amino acids.The Tim family is comprised of an extracellular domain,including a N-terminal cysteine-rich immunoglobulin variable region(IgV)domain and a mucin-like region that mediates glycosylation,a transmembrane region,and an intracellular domain containing an organic phosphorylation site at the C-terminal[2].Since its discovery,research concerning Tim-3 has focused on the adaptive immune response,especially its role as an immune checkpoint molecule in the tumor microenvironment.In recent years,our laboratory has been able to detect its expression on macrophages,DC cells and NK cells,and found that it also plays an important regulatory role in the natural immunity[3].Nuclear factor 90(NF90)is a nuclear protein encoded by the interleukin enhanced binding factor-3 gene(ILF3)and mainly distributed in the nucleus of cells.Studies have found that there are multiple alternative splicing modes of the ILF3 gene,leading to the formation of multiple counterpart isomers of NF90 at the level of post-transcriptional translation,which ultimately makes NF90 a special protein family[4].With the continuous deepening of NF90 research,the important regulatory role of NF90 in viral infection has become more and more prominent.At the same time,it has attracted our greater interest in viral infection research as well.Literature research found that NF90 participates in the infection process of various RNA viruses such as H1N1,Ebola virus,and VSV,all of which are single-stranded RNA viruses,and NF90 plays an inhibitory role in the invasion of this type of viruses[5].At the same time,a study found that the downstream molecular signal that NF90 initiates to resist viral infection is not the classic interferon I pathway,but activates cytosolic protein aggregation and forms stress granules(SGs)and viral genes are encapsulated in the particles,inhibiting virus replication under the action of NF90 and several key proteins and thus exerting an antiviral effect[6].However,due to insufficient research on the molecular structure and function of NF90,an important molecular mechanism for regulating viral infection needs further investigation.After the virus successfully invades the first line of defense of the human body,chronic infection occurs in the body,and the adaptive immune regulation of T cells gradually appears,leading to the production of a variety of cytokines,such as interleukin-2(IL-2),interferon-?(IFN-?),etc.[7-8],resisting infection or inflammation and maintaining immune microenvironment.Among them,IL-2 is a key anti-inflammatory cytokine in the adaptive immune response.According to literature research,Tim-3 can affect the expression of IL-2,but the specific mechanism is not completely clear.The production of IL-2 is mainly regulated by two ways:the transcriptional activation of IL-2 by nuclear factor-?B(NF-?B),nuclear factor-AT(NF-AT),and transcription activation factor-1(AP-1)[9-10];and the activation of intracellular protein kinase C(PKC?1)signal can enhance the stability of mRNA,so as to achieve gene translation and post-translational modification[11].Therefore we aimed to investigate whether or not Tim-3 can inhibit the expression of IL-2 through regulation of the activation of the above signal pathwaysThe above research found that Tim-3 can affect different signaling molecules and play an important regulatory role in the innate immune and adaptive immune response,but at present there are no significant protein molecules that cross between the two signaling pathways.Therefore,the research and development of small molecule inhibitors and their use may not be prominent in the process of antiviral infection.We found that the use of Tim-3 blocking antibodies can significantly improve the immune system's ability to resist viral infections,and try to extend the application of Tim-3 antibodies to the field of antiviral infections.There are currently more than thirteen research projects on Tim-3 antibody.Based on the computer molecular structure simulation platform and the mammalian antibody library technology,we optimized the existing Tim-3 antibody in the laboratory to a certain extent,screened and identified the antibody after site mutation,and obtained the full-length Tim-3 antibody with high affinity and new sequence,contributing to the research and development of Tim-3 targeted drugs.This research aims to clarify the important regulatory role of Tim-3 in innate immunity and anti-viral infection,and explore the mode of action of Tim-3 in regulating NF90 in the process of anti-RNA virus infection,and provide a new perspective for the research of innate immunity and anti-viral infection.The common DNA viruses HSV and RNA viruses H1N1 and VSV were used to infect RAW264.7 cell lines,respectively,using QRT-PCR and Western blot techniques to explore the effects of RNA viruses on Tim-3 gene transcription and protein expression in macrophages;6-8 week-old Tim-3+/+ and Tim-3-/-model animals were injected VSV into the abdominal cavity,and set the PBS group as a control.After the infection,the peritoneal macrophages and spleen and lung tissues were taken,using QRT-PCR technology And methods such as life and death rate statistics to determine whether the loss of Tim-3 has the effect of reducing the degree of inflammation and improving the survival rate of the body.In addition,the tag plasmids of Tim-3,TRIM47 and NF90 mutant plasmids were constructed,and the immunoprecipitation technology was used to determine the specific mode of action and structural domains of the three co-regulating the virus infection process.Tim-3 can have a transient high expression of genes and protein levels during the invasion of RNA viruses(such ac H1N1,VSV,etc.);HE stained lung tissue sections and lethal dose infection model animals,all results show high expression the level of Tim-3 aggravates the virus infection process of the body;Tim-3 can promote the K48 ubiquitination modification of NF90-DZF-Lys297 through TRIM47,and mediate immune escapeIn order to further explore the signal pathway that Tim-3 regulates IL-2 expression in adaptive immune responses,this project uses JurkatTim-3-high and JurkatTim-3-low cell lines,activated by PMA/PHA,and use QRT-PCR method to measure the change of IL-2 gene transcription level,use ELISA and FACS methods to measure the change of IL-2 protein level,and use Western blot to explore the effect of Tim-3 on the phosphorylation pathway of IL-2.The protein expression level of IL-2 in JurkatTim-3-low cell line was significantly higher than that in JurkatTim-3-high cell line.The Tim-3 antibody was used to block the function of Tim-3 protein.The IL-2 gene and protein levels in JurkatTim-3-high cell line were significantly increased,and the phosphorylation levels of PKC?1 and P65 protein were significantly increasedThis research is based on the important role of Tim-3 target in the regulation of natural immunity and adaptive immune response.Aiming at the Tim-3 antibody commonly used in laboratories,the computer-assisted molecular model construction platform and mammalian cell antibody library technology,as well as Overlap PCR,Carrier digestion,enzyme ligation and transformation and other molecular biology techniques,through gene sequence comparison and ELISA method screening to obtain a certain degree of optimization of new sequence antibodies,successfully constructed a rich sequence diversity antibody molecular plasmid library.The use of Tim-3 antibody can enhance the ability of anti-viral infection in natural immunity and increase the expression of body cytokines in adaptive immune response.The optimization of antibodies based on Tim-3 targets is for future development of Tim-3 targeted drugs.Listing provides a certain referenceIn summary,this topic focuses on the Tim-3 target and conducts an in-depth study of its signaling pathways in natural immunity and adaptive immune response,and combines basic research with applied research.We must attach great importance to the diseases caused by viruses and their serious consequences,do more in-depth research on the body's immune regulation mechanism against viral infections,discover key molecules in basic research,and expand to applied research,striving for the future The treatment of clinical viral diseases and the spread of viral infectious diseases provide strong theoretical support and make more reasonable and effective treatment plans.