Studies On Preparation Of Virus-like Particles Containing H5 AIV HA Gene Fragment

H5 subtype avian influenza virus(H5 AIV) can infect almost all birds.It seriously endanger the development of poultry industry because of its high pathogenicity and significant mortality.H5 AIV can also break through species barriers and infect human beings directly,which is a serious threat to human life and health.It is important for us to detect rapidly H5 AIV.PCR-based methods have become a common means which detect H5 AIV rapidly.At present pure nucleic acids and inactivated viruses have been used as a quality control in the pcr-based detection,which is lack of good stability and bio-safety. So it is significantly important to creat a quality control which has good stability and bio-safety.Firstly,according to the principle of Armored RNA technology,the H5 AIV HA gene fragment was cloned into the expression vector which can express RNase-resistant virus-like particles,then the recombinant expressing virus-like particles was transformed into E.coli BL21(DE3) and was induced.The expression products was confirmed by the enzyme digestion assay.The virus -like particles RNA was extracted and was checked by RT-PCR.The expression products were purified by sucrose density gradient centrifugation and observed by transmission electron microscope.A series of experimental results indicate that virus-like particles containing H5 AIV HA gene fragment have been constructed successfully.That the prepared virus-like particles are resistant to RNase makes them have good stability and bio-safety.Secondly,the derivational and expressing conditions of the recombinant expressing virus-like particles was optimized by choosing different culture mediums,OD and limited times IPTG induced.The production of virus-like particles was enhanced by optimizing expression vector and contracting three expression vectors such as pS1,pS2,pS3 further according to the molecular regulation mechanisms of MS2 bacteriophage 130bp UTR sequence.At last,the virus-like particles containing H5 AIV HA gene fragment was used as a positive control,the real-time fluorescence PCR assay system is optimized by equality design method,and the standard curve of positive control in the real-time fluorescence PCR assay detecting H5 AIV was constructed.To sum up,the virus-like particle containing H5 AIV HA gene fragment provide a good basic for the fix quality detection of H5 AIV.