A Preliminary Study On Function Of Uncharacterized Felis Catus C11orf96 Gene

Most of the life activities in biological cells are performed and completed by proteins.Tens of thousands of proteins whose functions have been identified and described,but there are still many proteins and their functions that have not yet been discovered.It could be said that the process of human exploration of the mysteries of life is endless.In this paper,in the process of studying the pathogenic mechanism of animal calicivirus,such as Murine norovirus（MNV）,Rabbit hemorrhagic disease virus（RHDV）,Feline calicivirus（FCV）,a uncharacterized protein was discovered,and the expression level was significantly increased.According to the position of its coding frame,it was tentatively named as C11orf96 protein.Therefore,it is speculated that the C11orf96 protein may be involved in the infection process of many viruses.In order to explore the specific role and molecular mechanism of C11orf96 protein in viruses infection and replication,the results include the following four parts.1.Molecular cloning and bioinformatics analysis of C11orf96 gene.This study successfully cloned the CDS region of the cat,human,and mouse C11orf96 gene by RT-PCR.The full-length gene was 1 201 bp,but the encoded products were slightly different.The f C11orf96,m C11orf96,and r C11orf96 encoded 124 amino acids,h C11orf96encoded 123 amino acids.The average molecular weight of C11orf96 protein was about 17k Da.The results of bioinformatics analysis showed that the C11orf96 protein sequence of cat,mouse,rabbit and human were relatively conservative,with only a few amino acid differences in the N-terminal.It is predicted phosphorylation sites that there were 3 tyrosine（Tyr）and 15 serine（Ser）;it may interact with multiple transmembrane proteins,E3ubiquitin ligase sand zinc finger proteins.2.Preparation polyclonal antibody and establishment SYBR Green I RT-q PCR assay of f C11orf96.The recombinant plasmid p ET-32a（+）-f C11orf96-Fe was successfully constructed by seamless recombination.And the recombinant expression vector was transformed into BL21（DE3）,and the recombinant protein of f C11orf96-Fe was successfully expressed after IPTG induction,the recombinant protein of f C11orf96-Fe exists mainly in inclusion bodies,and the protein size was 49 k Da.Then,New Zealand white rabbit was immunized by the purified recombinant protein of f C11orf96-Fe,the f C11orf96polyclonal antibody was prepared,and has good specificity and immunogenicity.Using this antibody,it was found that f C11orf96 protein was mainly located in the cytoplasm by laser confocal technology.Furthermore,the cat different organs expression levels of f C11orf96protein was detected by immunofluorescence assay histochemistry（IFAH）,immunohistochemistry（IHC）,and western bloting（WB）,the study found that f C11orf96was distributed in all tissues and organs,with the highest expression level in the kidney.In order to study the antiviral infection mechanism of C11orf96 in the future,SYBR Green I absolute q PCR detection method was successfully established of C11orf96,m C11orf96,and r C11orf96.The transcriptional levels of C11orf96 in various organs of cat,mouse and rabbit were assessed by this method,with the highest expression levels in the cat kidney,the rabbit heart,and the mouse heart.3.The C11orf96 protein could inhibit the viral replication of RNA virus and participate in the regulation of host innate immune response.In order to study the relationship between C11orf96 and viral infection,this study knocked down or increased the expression levels of C11orf96 in cells,and then infected cells with virus.It was found that C11orf96 could inhibit the RNA viral replication of RHDV,FCV,MNV,and Vesicular stomatitis virus（VSV）.In order to further study the antiviral mechanism of C11orf96,it was chosen that FCV infected CRFK cells as the model to study the role of C11orf96 of FCV infection process.The results showed that C11orf96 could significantly promote the transcription levels and protein expression levels in the early of FCV infection.However,with the development of FCV infection,the high expression level of C11orf96 protein could inhibit the virus replication of FCV.Then,this study respectively constructed cell lines of stably expressing and knocking out expression,they were named CRFK-C11orf96-GFP and CRFK-C11orf96^-/--sg RNA.These cells were infected with FCV,and found that C11orf96could significantly inhibit the FCV replication.Furthermore,it was also found that C11orf96was related to many innate immune system molecules by Co-Immunoprecipitation（Co-IP）and high performance liquid chromatography-mass spectrometry（HPLC-MS）,and some molecules were detected by relative q PCR,such as:NF-κB2,RAC1,TBK1,and IRF7,was up-regulated by C11orf96.Therefore,these results suggested that the C11orf96 was involved in the regulation of host natural immune response.4.The C11orf96 protein inhibits viral replication by regulating NF-κB signaling pathway.To investigate the molecular mechanism of C11orf96 inhibits viral replication,it was found that C11orf96 could activate the activities of NF-κB promoter and IFN-βpromoter by the dual-luciferase reporter gene system.Furthermore,it was also found that C11orf96 could respectively interact with NF-κB pathway key molecules by Co-IP:TRAF6,P50 and p65.Interestingly,the relative q PCR and WB showed that the C11orf96 could cooperate with VSV to promote TRAF6 participate in mediated NF-κB signaling pathway,then promote the transcription levels of P50 and P65.Therefore,these results speculated that C11orf96 plays an antiviral role by promoting the secretion and expression of antiviral factors such as interferon and interleukin.In summary,this study found a uncharacterized host protein with broad-spectrum antiviral effect:C11orf96.In this study,the C11orf96 gene was cloned,polyclonal antibody was prepared,and SYBR Green I absolute q PCR was established.Moreover,it was found that the C11orf96 protein could inhibit the RNA viruses replication of FCV,MNV,and VSV,by positively regulating the NF-κB signaling pathway,promoting the secretion and expression of antiviral factors such as interferon and interleukin.This study not only discovered a new host protein,but also the effect of the C11orf96 protein on RNA viral replication was expounded from the perspective of host-pathogen interaction.It provides candidate targets for the design of broad-spectrum anti-RNA virus drugs.