Identification And Characterization Of Six Structural Proteins Encoded By Heliothis Virescens Ascovirus 3h

Heliothis virescens ascovirus 3h(HvAV-3h)is infectious to insect larvae of many worldwide serious pest species such as Helicoverpa armigera(Hübner),Spodoptera exigua(Hübner)and Spodoptera litura(Fabricius)(all Lepidoptera: Noctuidae).By causing stunted growth and loss of appetite in the larvae,it is considered a potential natural control agent.The HvAV-3h was first reported in 2012 from China,and then more related research has been carried out.However,the pathogenic mechanism of the virus causing acute infections and chronic death of the host remains unclear.It was hard to used as a biocontrol strategy because of the poor oral infectivity of ascoviruses.Until more data are collected,the structure of HvAV-3h virions,the functions of viral proteins,the mechanism of virus replication and assembly as well as release will remain unknown.Structural proteins play important roles in virus assembly,morphogenesis,and mediating viral infections and inducing protective immunity in the host.There are virion purification,prokaryotic expression,antibody preparation,RT-RCR,Western blot,immunohistochemistry,and bioinformatics analysis in the study.To identify and characterize the six proteins encoded by HvAV-3h.This study provides a foundation for further investigation of the structure of the HvAV-3h virus and mechanisms underlying ascovirus morphogenesis and infection.The contents and results of research are as follows:1.Virion purification and transmission electron microscopy analysis: Hemolymph samples from morbid larvae were layered onto a 25-55%(w/v)sucrose gradient and then centrifuged.There are four bands were collected: fat body,mixed layer,purely virion and dead body.The formvar-coated grid with the purified virions on it was negatively stained and checked by TEM.The analysis demonstrated that we collected purified HvAV-3h virions.2.Generation of anti-structural-protein antibodies and identification of structural proteins: Antibody of 3H-MCP(ORF 53)proteins have been prepared and stored in our laboratory before;The genes of orf 21,orf 107,orf 117,orf 122,orf 135 were ligated into the expression vector.Subsequently,the recombinant proteins were purified for preparation of polyclonal rabbit antiserum was used for the immunoassays.To determine if the six proteins are structural protein,Western blot analysis was carried out on purified HvAV-3h virions.The result suggesting that these proteins are constituent of HvAV-3h virions.The six structural proteins were named 3H-21,3H-MCP,3H-107,3H-117,3H-122,and 3H-135,respectively.3.By RT-RCR,Western blot,immunohistochemistry analysis of the six identified structural proteins,the results showed that six genes began to transcribe within 3-24 h;the protein was express within 12-72 h in vivo;A large number of structural proteins were observed in the fat body of H.armigera larvae in the late stage of infection.In summary,six structural proteins encoded by HvAV-3h were identified in the study.They are named 3H-21,3H-MCP,3H-107,3H-117,3H-122,and 3H-135 respectively.And then the corresponding antibodies were prepared.In vivo,genes which predicted encoding structural proteins begin to transcribe within 3-24 h;structural proteins begin to be expressed within 12-72 h;a large number of structural proteins were observed in the fat body of H.armigera larvae in the late stage of infection.