The Screening Of Defensins Secreted By HT29 Cells Once Infected With EV71 And The Preliminary Identification Of Antiviral Activity Of HBD3 Against EV71

At present,studies on the antiviral effect of defensins are more,but mainly focusing on againsting enveloped viruses,and resisting the non-enveloped viruses less.Antimicrobial peptides synthesized in vivo have broad-spectrum and highly effective anti-microbial activity.Defensins with a strong killing effect on a variety of pathogenic microorganisms such as bacteria,fungi and viruses,etc,are a kind of antimicrobial peptides,widely distributed in animals,plants and insects.Antimicrobial peptides are important components of the innate immune system,involved in attacking the pathogen and regulating the body’s immune response.In this study,EV71 was used as a representation of the non-enveloped virus,and to screen the defensins secreted by HT29 cells once infected with EV71 virus.And then to detect whether HBD3(human β-defensin-3)having antiviral activity against EV71 preliminarily.Three aspects make up the main contents of this study:(1)to screen the defensins secreted by HT29 cells infected with EV71 Virus;(2)building the plasmid pcDNA3.1-smHBD3,and transfecting it into HT29 cells to ensure the expression of HBD3;(3)preliminary detection about the antiviral activity of HBD3 against EV71.1 To screen the defensins secreted by HT29 cells infected with EV71 VirusThe first step in this part was to develop an EV71 virus strain which can adapt to growing in HT29 cells.The second was to infect HT29 cells with this virus(0.01 MOI)and then detected the defensins mRNA expressed by the cells with RT-PCR methods.Finally,making further efforts to ascertain the mRNA of defensins screened out through gene cloning and sequencing.The results showed that,EV71 virus can stimulate HT29 cells expressing HBD3 mRNA.2 Building the plasmid pcDNA3.1-smHBD3,and transfecting it into HT29 cells to ensure the expression of HBD3Obtaining HBD3 cDNA(smHBD3)including its nucleotide sequence of signal peptide and mature peptide by PCR,and then,inserting it into the vector pcDNA3.1 to construct plasmid pcDNA3.1-smHBD3 by gene engineering.Finally,Once we confirmed the pcDNA3.1-smHBD3 right by restriction analysis with MluI、KpnI and EcoRI and sequencing,we got a lot of it.When the plasmid was transfected into HT29 cells,we identified the expression and distribution(intracellular or extracellular)of HBD3 protein within different time by ELISA.It was found that at 24h after transfection,the HBD3 protein could be detected,at 36h,there HBD3 can be found in extracellular and at 72h,the protein in the extracellular achieved the highest.3 Preliminary detection about the antiviral activity of HBD3 against EV71.In this chapter,we obtained the HBD3 protein by transfection,and then,to detect whether the protein have antiviral activity against EV71 through different ways.Firstly,the HT29 cells which were transfected 36h were infected with EV71(0.01 MOI)and at different time post-infection(0h,12h,24h and 36h),the viral titer of that virus was measured.Secondly,mixing EV71 virus(0.01 MOI)with the supernent of the culture medium conditioned by the 72h growth of HT29 cells transfected with the plasmid pcDNA3.1-smHBD3 at 37℃,after 4h,the HT29 cells were cultured with the mixture 48h and then the viral titer was measured.Finally,HT29 cells having 80%cell confluence were cultured with EV71 virus(0.01 MOI)and the supernent of the culture medium conditioned by the 72h growth of HT29 cells transfected with the plasmid pcDNA3.1-smHBD3 at 37℃、5%CO2.After 4h,the cells were cultured with cell maintenance medium 48h,and then the viral titer was measured.The results of three independent replication experiments showed that HBD3 has the antiviral activity against EV71,and this activity may be mainly exerted at the extracellular of HT29 cells.This research indicated that HT29 cells can express HBD3 mRNA once infected with EV71,and the HBD3 protein has antiviral activity against EV71,and this activity may be mainly exerted at the extracellular of HT29 cells.Besides,this paper gave another defensin associated with the non-enveloped virus,and paved the way in the future for studying the mechanism that HBD3 resistance EV71.