Preparation Of Monoclonal Antibody Against ORF25 Protein Of Cyprinid Herpesvirus 2 Virus And Cloning And Expression Analysis Of TRIM25 From Common Carp

Carp herpes virus type â…¡(Cyprinid herpesvirus CyHV-2), is a highly pathogenic virus, which could infect goldfish(Carassius auratus), crucian carp(Carassius auratus)and its variant. It was first reported in breeding goldfish in Japan;In 2011 and 2012,CyHV-2 caused severe death to allogynogenetic crucian carp(Carassius auratusgibelio).In 2013, CyHV-2 was identified in the cultured allogynogenetic crucian carp in Jiangsu, Beijing, Wuhan, Guangzhou and other places, confirming CyHV-2 has become more widely distributed in our country. Currently, there is no effective drug to treat the disease. To provide effective tool for the clinical diagnosis of the disease, it is urgent to establish a rapid, accurate and convenient detection method.CyHV-2ORF25 gene segment was amplified by PCR and cloned into the prokaryotic expression vector p GEX-KG, then the recombinant plasmid CyHV-2-ORF25-KG established.The results of SDS-PAGE is that the CyHV-2-ORF25-KG fusion protein(42KDa) was expressed in the insoluble composition with IPTG induction. One monoclonal antibody(Mc Ab 5C6)against ORF25 protein were generated by fusion of mouse myeloma cell line SP2/0 and spleen lymphocytes from immunized mice. The results of IFA(Indirect immunofluorescent assay) and Western Blot assay further demonstrated the characterizations of the Mc Ab 5C6.This study laid a foundation to the research for the pathogenesis and the detection method of CyHV-2.In order to clone and expression of common carp(cyprinus carpio)TRIM25, the total RNA was isolated from ovarian tissue of common carp. After the reverse transcription and amplification with PCR, sequence and structure analysis found that common carp TRIM25 similar as zebrafish(Danio rerio) and bitterling(Rhodeus uyekii), the evolutionary relation of phylogenetic tree also showed that these species were cluster at one branches. Further more, the coding sequence of TRIM25 was cloned into the vector p CDNA4.0. The recombinant plasmid p CDNA4-TRIM25 was identified by restriction enzyme digestion and gene sequencing; Then it was transfected into HEK293 T cells and FHM cells, and detected by indirect immunofluorescence assay and Western Blot. Theresults showed that TRIM25 protein was expressed in HEK293 T and FHM cells. This study shows that the recombinant plasmid p CDNA4-TRIM25 is constructed and express successfully, which will lay the foundation for research on the activity of TRIM25.