Construction Of Rabies Virus Pseudovirus And Its Application In The Detection Of Neutralizing Antibody

Rabies is a fatal zoonotic disease caused by rabies virus（RABV）.So far,there is no effective treatment and antiviral drugs.If the vaccine is injected before exposure or after exposure,the incidence rate of rabies will be greatly reduced,so as to achieve effective prevention.Rabies virus neutralizing antibody（RVNA）can be produced after rabies vaccination,and its titer is an important index to evaluate the immune effect of the vaccine.The traditional RVNA detection method has the disadvantages of long test cycle and the use of highly pathogenic live virus,which is not easy to be popularized on a large scale.In order to avoid the operation of live virus in the test and more safely and accurately monitor the RVNA level of RABV fixed virus a G strain for the production of human rabies vaccine in China,this study prepared a pseudovirus of the RABV a G strain based on the HIV lentiviral packaging system and applied to the detection of serum neutralizing antibody level of immune rabies vaccine.In this study,the recombinant eukaryotic expression plasmid p CMV-a G-G encoding the G protein of RABV a G strain was constructed.The G protein expression plasmid,pseudovirus packaging plasmid and green fluorescent protein（GFP）indicator plasmid were co-transfected into 293T cells for pseudovirus packaging.The RABV a G strain pseudovirus was identified in terms of protein expression level and cell infectivity.By optimizing the key factors such as the ratio of plasmid to transfection reagent and virus collection time in the process of pseudovirus packaging,the RABV a G strain pseudovirus with high titer was successfully prepared,and its virus titer can reach 10^5.5TCID₅₀/m L.In order to verify whether the prepared pseudovirus has the ability to combine with serum neutralizing antibody,a preliminary neutralization experiment was performed on the pseudovirus with positive and negative serum respectively.The positive serum had inhibitory effect on the infection of pseudovirus,while the negative serum had no inhibitory effect.There is a good linear relationship between the amount of pseudovirus inoculation and the number of fluorescent spots formed by infected cells.The titer of serum samples can be calculated by the corresponding serum dilution multiple when the number of pseudovirus fluorescent spots is reduced by 50%.Through the screening and optimization of the detection time of pseudovirus serum neutralization test and the inoculation amount of pseudovirus,the detection method of pseudovirus neutralizing antibody of RABV a G strain was preliminarily established and used to determine the neutralization titer of positive serum.In conclusion,the RABV pseudovirus carrying GFP reporter gene and G protein of a G strain was successfully prepared based on HIV lentivirus packaging system in this study.It has the ability to bind with neutralizing antibody in positive serum,and can be preliminarily applied to the determination of neutralizing titer level of immune serum of rabies vaccine,which provides the possibility to further improve the detection method of neutralizing antibody titer.