Characterization Analysis And Expression Of VP2, VP3 And VP5 Genes Of MABV Y-6 In Insect Cells

MABV (marine birnavirus) belongs to the genus Aquabirnavirus of the family Birnaviridae which is characterized by two segments of double stranded RNA (dsRNA) in genome. The two segments of MABV genome include a segment A of 3.1 kbp and a segment B of 2.8 kbp. Segment A encodes a polyprotein (106 kDa) and a small protein VP5 (15-17 kDa). The polyprotein, whose protein order is NH2-VP2-NS-VP3-COOH, is cotranslationally processed and cleaved at the VP2-NS and NS-VP3 junctions by the NS (also named VP4)-associated protease activity. VP2 protein and VP3 protein are the major structural polypeptides. The small ORF encoding VP5 is partially overlaped with the 5'end of the polyprotein ORF, but as a different frame. The smaller segment B of the birnaviruses encodes the VP1 (90 kDa), which is an RNA-dependent RNA polymerase (RdRp). This paper presents the expression analysis of VP2e, VP3 and VP5 gene in the E.coli, insect cells or in the fish cells, and function analysis of VP2, VP3 and VP5 protein. The results are shown as follows. 1 , Expression and function analysis of VP5 geneVP5 gene was amplified by RT-PCR from MABV genome, and expressed in E. coli BL21 (DE3). Under the optimized expression conditions, at 28℃,0.1mM IPTG, the expression level of the fusion protein is 7.3% of the total bacterial proteins.The VP5 gene from MABV was fused to enhancing green fluorescence protein (eGFP) gene and inserted into the baculovirus genome under the control of polyhedrin gene promoter, and then was highly expressed in insect cells Tn-5Bl-4 (Trichoplusia ni cell line). The expressed VP5 protein was capable of enhancing insect cell viability, prevented membrane blebbing and delayed DN A internucleosomal cleavage when cells were infected with the recombinant bacmid-eGFP VP5 virus. The results suggested that the VP5 of MABV is a novel anti-apoptosis gene, which could regulate the cell apoptosis-off system. 2 Expression analysis of VP2e geneThe pGEX-6P-1-VP2e expression vector plasmids were constructed, and expressed in the E. coli (BL21). Under the optimized expression conditions, at 37℃,0.1mM IPTG, thequantity of expressed fusion protein GST-VP2e is as high as 28.6% of the total bacterial proteins. The expressed fusion protein GST-VP2e were purified, and used as an immunogen to raise GST-VP2e-specific polyclonal antibodies in male rabbits. The titer of polyclonal antibodies against GST-VP2e is up to 1: 512, 000.The VP2e gene was subsequently inserted into recombinant virus expression vectors through transposition, and transfected into insect cells of Tn-5Bl-4 for expression. The results show that VP2e gene was expressed efficiently in the insect cells, and the expressed proteins were accumulated to 15.8% of total insect cell proteins.The expression time course of VP2 protein in the MABV-infected fish cells was analyzed by western blot using the polyclonal antibody against GST-VP2e. Two specific immunoreactive bands of approximate 54 kDa were first observed at 24 h p.i. and remained detectable until 120 h p.i.. Fluorescent confocal laser scanning microscope examination shows that the VP2 protein is exclusively localized in the cytoplasm. 3, Expression analysis of VP3 geneThe pGEX-4T-2-VP3 expression vector plasmids were constructed, and expressed in the E. coli (BL21). Under the optimized expression conditions, at 30°C,0.8mM IPTG, the quantity of expressed fusion protein GST-VP3 is as high as 20.7% of the total bacterial proteins. The expressed fusion protein and GST-VP3 were purified, and used as an immunogen to raise GST-VP3-specific polyclonal antibodies in male rabbits. The titer of polyelonal antibodies against GST-VP3 is also up to 1: 512, 000.The VP3 gene was subsequently inserted into recombinant virus expression vectors through transposition, and transfected into insect cells of Tn-5Bl-4 for expression. The results show that VP3 gene was expressed efficiently in the insect cells, and the expressed proteins were accumulated to 8.2% of total insect cell proteins.The expression time course of VP3 protein in the MABV-infected fish cells was analyzed by western blot using the polyclonal antibody against GST-VP3. Only one specific immunoreactive bands of approximate 29 kDa was first observed at 24 h p.i. and remained detectable until 120 h p.i. Fluorescent confocal laser scanning microscope examination shows that the VP3 protein is also exclusively localized in the cytoplasm.4^ Fish anti-GST-VP2e antiserum and fish anti-GST-VP3 serum protect fish cells from MABV Y-6 infection.The fish anti-GST-VP2e antiserum and fish anti-GST-VP3 antiserum were raised by injection to fish with the purified GST-VP2e and GST-VP3, respectively. These antiserums were detected whether they play a protection role for MABV-infected fish cells by neutralization test and ELIS A. The titer offish antiserum against GST-VP2e is up to 1: 3, 200, while the titer offish antiserum against GST-VP3 is lower than that against GST-VP2e and is up to 1:800.