Colocalization Of Grass Carp Reovirus VP41 Protein And Inclusion Body And Its Role Study

Grass carp(Cyenopharyngodon idellus)is an economic fish in freshwater aquaculture in China.The viral pathogens of grass carp haemorrhagic diseases are reovirus in grass carp,which belongs to Reoviridae.Since the first grass carp reovirus was isolated in the 1980 s,more than 40 strains have been reported in the literature.The virus isolated from diseased fish can be divided into three genotypes according to its gene sequence : Type I,Type II,Type III.According to epidemiological surveys,type II is currently the most widely distributed,most prevalent and pathogenic virus type in China.The 11 gene segments of type II grass carp reovirus encode 11 proteins.It is found that the function of VP41 protein encoded by S8 segment is unknown,Homologous protein of VP41 can't be found in other genotypes of grass carp reovirus.As a specific protein in type II grass carp reovirus,VP41 may be associated with some of the unique biological characteristics of type II grass carp reovirus.Studying the function of VP41 is of great significance for elucidating the pathogenic mechanism of grass carp reovirus.In this paper,the grass carp reovirus Henan isolate GCRV-HN14 was used.In order to study the role of VP41 in viral replication,whether VP41 protein was localized to the virus inclusion body was studied.The virus inclusion body is a circular or elliptical structure formed by the virus in the cell,and is an important place for the virus components to assemble.Type I grass carp reovirus NS80 protein can form the basic framework of viral inclusion bodies,and can recruit other structural proteins and non-structural proteins of the virus into the virus inclusion body.In type II grass carp reovirus,the protein homologous to NS80 is the NS79 protein encoded by the S4 gene segment.This paper mainly studies the colocalization of VP41 and NS79 in cells,and the interaction of VP41 with other proteins to explain whether VP41 is involved in the assembly of virus.The main contents of this paper is as follows:1.Immunofluorescence detection of the interaction between VP41 and NS79 proteinThe VP41 and NS79 fragments were cloned from the genome of GCRV-HN14 virus,and the ORFs of VP41 and NS79 were inserted into pc DNA3.1(+)eukaryotic expression vector.The recombinant vector was successfully constructed by double enzyme digestion and sequencing.The constructed eukaryotic expression vector was transfected into CIK cells,and the cells were harvested 24 hours after transfection.The expression and localization of recombinant protein in the cells were detected by immunofluorescence.The results are as follows:(1)When transfected with pc DNA3.1-NS79 alone,the NS79 protein encoded by the S4 gene segment can form a circular agglomerate structure in the cytoplasm,similar to the function of the NS80 protein,and can also form basic structure of viral inclusion bodies.(2)When pc DNA3.1-VP41 was transfected alone,the VP41 protein encoded by S8 showed an irregular block distribution in the cytoplasm.(3)When the two vectors pc DNA3.1-NS79 and pc DNA3.1-VP41 were co-transfected into CIK cells,both VP41 protein and NS79 protein showed an irregular block distribution,and the two proteins colocalized,indicating that there are interactions.2.Preparation of Polyclonal Antibody against Grass Carp Reovirus VP41 and NS79 ProteinA partial fragment of VP41 and NS79 was amplified by using the genome of GCRV-HN14 virus as a template.The amplified fragment was inserted into the prokaryotic expression vector of p ET-32a(+)and transformed into E.coli DH5?.After screening positive bacteria,the sequencing results showed the construction were correct,the positive bacteria were expanded,and the recombinant plasmids VP41E-32 a and NS79E-32 a were extracted.The recombinant vector was transformed into E.coli BL21(DE3)and positive bacteria were screened.The positive bacteria were induced by IPTG,and the cells were sonicated,and protein electrophoresis showed that the recombinant protein was present in the form of inclusion bodies in the fragmented precipitate.The recombinant protein was purified by nickel column Ni-IDA,and the 100 ?g of the purified VP41 protein was injected into mouse,and 1 mg of the purified NS79 protein was injected into New Zealand white rabbits.After 4 times of immunization,blood was collected and the titer of polyclonal antibody was detected.The antibody titers were all higher than 1:64000.Western blot confirmed the specificity of the antibody and will be used to detect the interaction of VP41 and NS79 proteins in the virus-infected cells.3.Construction of eukaryotic expression vector of GCRV-HN14 other gene segmentsIn order to detect the interaction between other proteins,The ORFs of the gene segments S1,S2,S3,S5,S6,S7,S9,S10,and S11 were amplified,HA tag sequences were added to the carboxy terminus of the gene segment,and the ORFs of these fragments were inserted into eukaryotic expression vector pc DNA3.1(+),respectively.After double enzyme digestion and sequencing,the eukaryotic expression vectors pc DNA3.1-VP1,pc DNA3.1-VP2,pc DNA3.1-VP3,pc DNA3.1-VP5,pc DNA3.1-VP4,pc DNA3.1-VP56,pc DNA3.1-VP6,pc DNA3.1-NS38 and pc DNA3.1-VP35 were successfully constructed.