| Getah virus(GETV)is a member of the Togavirus family Alphavirus,and is also a typical arbovirus.Getah virus can infect a variety of animals,but it is more invasive to pigs,horses,and mouse.There are five structural proteins of Getah virus.In the process of alphavirus replication and budding,capsid protein(Cap)plays an important role,and in the process of virus exocytosis and entry,glycoprotein(E2)also plays an important role.GETV-Cap protein and GETV-E2 protein antibodies can not only be used to establish virus detection methods,but also provide convenience for the study of Getah virus.This study not only isolated Getah virus from the material of sick pigs,but also studied its biological characteristics and pathogenicity to suckling mice.The prokaryotic expression of Capsid and E2 proteins of Getah virus was used to immunize BALB/c mice for several times.Finally,polyclonal antibodies against GETV-Cap protein and GETV-E2 protein were successfully prepared.1.Separation and biological characteristics of pig-sourced getah virus of Getah virusThe RT-PCR method was used to detect the Capsid gene of the Getah virus in the piglet disease feed of a pig farm in Guangxi.Through the verification methods such as sequencing,it was preliminarily determined that the disease feed contained the Getah virus.After using Vero cells for multiple blind times and plaque purification,the porcine Getah virus was obtained and named GETV-GX.In order to explore its biological characteristics,its growth curve,temperature sensitivity and formaldehyde sensitivity and its infectivity to different cells were determined.After Vero cells were inoculated with viruses of different MOI(Multiplicity of infection),the cytopathic conditions at different time points were observed,and the titer of the cell supernatant at different time points of GETV-GX was determined to obtain its one-step growth curve.Using cell lines from different sources,we verified the susceptibility of GETV-GX to cells of different hosts and tissues,and found that it is not susceptible to Hela cells.The GETV-GX strain was placed in a water bath at different temperatures,and it was found that all the viruses were inactivated after it was incubated at 65°C for half an hour.2.The pathogenicity experiment of getah virus in miceAfter GETV-GX was intracranially inoculated into 3-day-old ICR suckling mice,the infected group showed 100%pathogenicity and lethality.In the infected group,the weight of the suckling mice ceased to increase after 24 hours.After 48 hours,some suckling mouse began to die with hunched back,tremor,and difficulty in eating.All the suckling mice in the infected group died 80 hours after inoculation.The p ET-E2 plasmid was constructed and used as a standard quality plasmid for q RT-PCR test,and finally a standard curve was obtained.After dissecting the sick and dead suckling mice,hemorrhages and lesions were found in the chest organs,brain,hind limbs of the suckling mice.Based on the standard curve of virus content,the q RT-PCR method was used to detect the viral RNA content of the various organs of suckling mice,and it was found that the brain,heart,lung,kidney,spleen,hind limbs,gastrointestinal and other organs have Getah virus.3.Preparation of GETV-Cap and GETV-E2 polyclonal antibodiesIn this study,the RNA of GETV-GX strain of pig origin was extracted,and GETV-GX capsid protein(Cap)and glycoprotein(E2)genes were amplified by RT-PCR method,and their prokaryotic expression vectors p ET-Cap and p ET-E2 were constructed.The p ET-Cap and p ET-E2 plasmids were transferred to BL21(DE3),and after inducing expression and purification,the prokaryotic expression proteins of GETV-Cap and GETV-E2 were obtained.After immunizing BALB/c mice with this for three times,the serum titer of the mice was tested and found that they all reached 10~5or more,and the polyclonal antibodies of GETV-Cap protein and GETV-E2 protein were prepared with this.After IFA and Western blot identification,it is found that they can specifically recognize GETV. |
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