The Prokaryotic Expression Of Matrix Protein In Fish Viral Hemorrhagic Septicemia Virus,and Preparation And Application Of Its Polyclonal Antibody

The Viral Hemorrhagic Septicemia Virus belongs to the Mononegavirales,Rhabdoviridae,Norarhabdovirus genus,is a species that can infect more than 80 kinds of fish such as rainbow trout,gums and turbot,causing the pathogens of necrosis of hematopoietic organs and the death of fish have brought enormous economic losses to the aquaculture industry at home and abroad.A structural protein-matrix protein encoded by the viral genome is a multifunctional protein that plays an important role in the process of virus infecting host cells.In order to study the function of M protein in detail,the M protein was expressed in prokaryotic expression and its polyclonal antibody was prepared.The main contents are as follows:1.Induced expression of M fusion protein and preparation of antibody serumThe full-length M gene of VHSV virus was amplified by RT-PCR.The target sequence was cloned into the prokaryotic expression vector pET-32a(+),transformed into BL21(DE3)competent cells,and then induced to express the fusion protein by IPTG.The results showed that the length of 606 bp M gene was amplified by RT-PCR.M fusion protein induced by IPTG mainly existed in the form of inclusion body,and the size was about36 kDa,which was slightly smaller than expected.The gel-purified M fusion protein was taken and injected into BALB/c mice by intraperitoneal injection to obtain the corresponding polyclonal antibody.2.Identification of polyclonal antibodiesThe antibody titer was detected by ELISA.The titer was greater than 1:102400.Western Blot showed that the polyclonal antibody can specifically recognize the M protein in the carp epithelial cells infected with VHSV,and can also identify the purified protein.M fusion protein;indirect immunofluorescence test results show that M protein is mainly located in the cytoplasm and cell membrane of EPC cells.The results suggest that the prepared polyclonal antibody can be used as an effective tool for studying the function of M protein and diagnosis of VHSV.