Establishment And Identification Of The Transgenic Chicken Against Avian Influenza Virus H5N1

The avian influenza virus (AIVs), which is the type A influenza virus adapted to an avian host, has been an important pathogen for the poultry industry worldwide for many years. The highly pathogenic AI (HPAI) virus can cause disease and even mortality in human.Virus infection is not adequately controlled by vaccination or by biosecurity measures. A novel alternative strategy is to develop chickens that are genetically resistant to infection. The short incubation period and rapid onset of HPAIV lead us to generate transgenic chicken integrating with two or more anti-AIV candidate genes. In this thesis, the lentivirus vectors with anti-AIV H5N1specific siRNA NP604shRNA-expression fragment or single chain fragment variable (scFv) scFvl gene were microinjected into chicken embryo blood vessel for generating the transgenic chicken. The main results are as follows:In the previous work, we had screened out two kinds of antiviral genes specific to AIV subtype H5N1:siRNA NP604targeting the conservative regions of NP protein and chicken antibody single-chain variable fragments (scFvs:scFvl, scFv2and scFv3) specific to AIV HA protein. In this study, scFvl was selected for further research because of highly virus binding ability screened by ELISA. Chicken fibroblastic DF-1cells were transfected with scFvs in combination with siRNA NP604. Following infection with FJ13virus, copy numbers of the virus were significantly reduced from12h to at least60h post-infection compared to that achieved in cells transfected with scFv or siRNA NP604separately.siRNA NP604shRNA-expression fragment and scFvl fragment with a signal peptide gene were inserted into lentivirus vector GV115(EGFP fusionally expressed) and GV206respectively.293T cells were transfected with helper plasmid in combination with GV115or GV206. The supernatant containing a large number of lentivirus particles was collected and concentrated. Through fluorescence microscope observation and real time quantitative PCR determination, virus titers were2E+9TU/mL and2.00E+8TU/mL. The new constructed vector GV206-scFvl was confirmed to express scFvl protein by Western blotting..The lentivirus concentrate with siRNA NP604fragment or scFvl gene was microinjected into embryo peripheral vessel when chicken embryos developed to HH13-15. There were327chicken embryos injected and per embryo was injected with1uL virus concentrate.119embryos were injected with siRNA NP604lentivirus, while208embryos were injected with scFvl lentivirus.58and98chicks were hatched in2different groups. Through PCR detection,20siRNA NP604positive and19scFvl positive chimeric chicks were confirmed. The results of Western blot showed that positive chimeric chicks can express EGFP protein or scFvl protein in the blood. The green fluorescent protein can be observed in muscles, intestines, stomach, spleen, heart, liver, blood cells, trachea and gonads of siRNA NP604positive chimeric chicks under the luorescence microscope. Feeding the positive chimeric chicks to sexual maturity,2chimeric roosters with higher copies of exogenous gene in their semen were screened out from each group for the next chimeric chicks generation research.This study laid a solid foundation for breeding transgenic chicken that can express two specific anti-AIV H5N1molecules. If this goal is achieved, as transgenic siRNAs would be stably expressed in all body cells, they will be present to interfere with NP synthesis and limit viral replication upon viral invasion. Since scFvs are secreted into the blood after expression, they will bind to the HA of circulating virus, thereby preventing viral infection and spread. If they are efficiently expressed, the result might be significant inhibition of virus at the earliest stage of infection, before an adaptive immune response can be mounted.