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Developments And Preliminary Applications Of Three Detection Methods For Venezuelan Equine Encephalitis Virus

Posted on:2019-12-25Degree:MasterType:Thesis
Country:ChinaCandidate:C C JiaoFull Text:PDF
GTID:2370330542997333Subject:Prevention of Veterinary Medicine
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Venezuelan Equine Encephalitis?VEE?is a highly pathogenic disease for humans and other animals,and the pathogen is Venezuelan Equine Encephalitis Virus?VEEV?.Equines act as efficient amplifying hosts.Humans can be infected via mosquitoes.In humans,VEEV causes moderate flu-like clinical symptoms including fever,headache,myalgia,fatigue,nausea,and pharyngitis.VEEV was not isolated until 1938 from sick horse.VEEV has also been found to be highly infectious by aerosol and is considered to be potential biological weapons.As a result,VEEV has been classified as a Category B pathogen by both the Centers for Disease Control and Prevention and the National Institute of Allergy and Infectious Diseases.Currently,there are no licensed vaccines and effective antivirals for the treatment of VEEV infection in humans.Our coutry has yet to report VEE cases.The globalization of economic trade make it possible the introduction of VEE pathogen.In order to prevent the disease from being imported,China's entry-exit ports must strengthen the detection of VEEV.Therefore,it's particularly crucial that rapid and convenient assays be developed for detection of VEEVantibody and antigen.In order to provide technical support for the detection of VEEV and monitoring epidemic,based on VEEV TRD virulent strains this study established three methods named indirect ELISA for detecting VEEV antibody,double antibody sandwich ELISA for VEEV antigen and VEEV pseudovirus neutralization test for neutralizing antibody.1.Establishment and application of indirect ELISA for the detection of Venezuelan equine encephalitis virus antibodywe used E.coli expression system to express the extracellular domain of VEEV tE2glycoprotein.The conditions of induction were optimized and the target protein was purified and tested the purity.Establishing an indirect ELISA which can detect VEEV antibody,we employed the purified VEEV tE2 as coating antigen.After the optimization of the reaction conditions were optimized,the specificity,repeatability of the indirect ELISA was analyzed to evaluate practicality.The target gene of VEEV was successfully amplified by PCR to yield a product 1023 bp.PCR analysis and sequencing proved that the recombinant plasmid pET-30a?+?-VEEV-tE2 had been successfully constructed.The target protein was expressed in the form of inclusion body.The optimal conditions of protein expression were 37?,with the final concentration of0.6mM IPTG for 3h.The purity of target protein is 94%.The purified tE2 was used as coating antigen to establish indirect ELISA for detecting VEEV antibody.The optimal coating concentration of tE2 was 10?g/mL at 4?overninght;the optimal dilution of tested serum was 1:40 incubated at 37?for 1.5h;super blocking buffer as blocking agent at 37?for 1.5h;the optimal dilution of HRP-labeled goat anti-rabbit IgG was1:10000 at 37?for 1h.The indirect ELISA could specifically recognize the VEEV antibody.The intra and inter batch variation coefficients were less than 8%with good repeatability.This study successfully established the indirect ELISA for detecting VEEV antibody,which provided a simple and rapid method.2.Establishment and Application of Detection Method for Neutralizing Antibodies against Pseudoviruses of Venezuelan Equine Encephalitis Virus293T cells were co-transfected with the plasmid pNL4-3.Luc.RE containing the luciferase reporter gene and recombinant plasmid containing VEEV E123 gene.Supernatant was harvested after 48h,and was subjected by Western Blot and electron microscopy.The pseudovirus was selected for screening of susceptible cells and the infectivity of the pseudovirus.The neutralizing antibodies of some related samples were detected by VEEV pseudovirus.The recombinant plasmid pCAGG-VEEV-E123 was successfully constructed.Pseudovirus was identified by Western Blot,showing 42 kD VEEV E2 glycoprotein band and 24 kD HIV-1 P24 protein band.HIV-like pseudotype particles can be observed by electron microscopy in supernatant pVEEV-E123.Huh-7,BSR,BHK-21,Vero E6 and NA cells were infected with pVEEV-E123.The results showed that pseudovirus was the most susceptible to Huh-7 cells.Neutralizing antibody detection method was established by using pVEEV-E123.This method can specifically detect the neutralizing antibody titer of VEEV and has good repeatability.This method can be used for the detection of neutralizing antibodies in serum samples.The above results show that the successful establishment of the VEEV pseudovirus system and its application in the detection of VEEV neutralizing antibodies can effectively reduce the biosafety risk in the VEEV neutralization experiment.3.Establishment and application of double antibody sandwich ELISA for detection of Venezuelan equine encephalitis virus antigenRecombinant baculovirus rpFBD-2tE2 expressing VEEV tE2 was obtained using insect baculovirus expression system.Rabbit was immunized with the purification tE2to produce polyclonal antibody.Monoclonal antibody as the capture antibody and rabbit polyclonal antibody as the detection antibody were used to establish a double antibody sandwich ELISA method.This method can quantitatively detect the E2 content in the recombinant baculovirus rpFBD-2tE2 and VEEV V5?recombinant baculovirus expressing C-E3-E2-6K-E1?.The working conditions,specificity,repeatability and sensitivity were optimized and analyzed.The optimal concentration of capture antibody was 8?g/mL,the optimal dilution of detection antibody was 1:1600,The detection sensitivity of rpFBD-2t E2 was 8.619ng/mL.There was no cross reaction with other recombinant.Intra and inter assay coefficients of variation were less than 10%.The relative content of E2 protein in rpFBD-2tE2 measured by this method ranged from739.89 to 1025.17 ng/m L,while the relative content is 10.6838.90 ng/m L in VEEV-V5.The method can be used for detecting the E2 protein content in VEE vaccines and provides technical support for the evaluation and development of the vaccine.In summary,this study established three rapid and simple detection methods of VEEV antigen and antibody,which laid the foundation for VEE epidemic surveillance,antigen quantification of new vaccines,and evaluation of immune effects.
Keywords/Search Tags:Venezuelan equine encephalitis, indirect ELISA, double antibody sandwich ELISA, pseudovirus, neutralization assay
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