Cloning And Expression Of Akabane Virus Glycoprotein G1 Gene And Establishment Of An Indirect Enzyme-Linked Immunosorbent Assay For Detection Of Akabane Virus Antibodies

Akabane disease is an pleomorphic infectious disease of cattle, sheep and goat, caused by Akabane virus, charactered by abortion, premature birth, stillbirth,and congenital deformities such as arthrogryposis-hydranencephaly (AH) syndrome.Virus genome encodes 4 structural proteins,which are two intramembrane proteins L,N and two extramembranous proteins G1,G2. Among them, glycoprotein G1 is the major protein presented on the envelope of the virus, which can induce strong specific immune response, and determine several important biological characters of the virus such as pathogenicity, neutralization, hemagglutination and cell conjugation. In this study, the gene G1 of AKAV OBE-1 strain was amplified from the AKAV genome by RT-PCR using specific primers designed according to M genome segment sequence in Genbank. The amplified product was cloned into pMD18-T vector and sequenced. Then the gene fragment was subcloned into pFastBacHT A donor plasmid of Bac-to-Bac baculovirus expression system as interest gene. Through the homologous recombination of donor plasmid with Bacmid DNA at the site of Tn7, the recombinant DNA containing the interest gene was obtained. The recombinant DNA was transfected into Sf9 insect cells by CELLFECTIN mediation, and then the recombinant virus BAC-G1P1 was generated. SDS-PAGE analysis indicated that Sf9 insect cells infected with BAC-G1P1 expressed interest proteins in solution form, which was approximately120kD in molecular weight as expected. Western blot and indirect immunofluorescence assay (IFA) proved that it had good immunological activity.Based on hydrophilicity, antigenity and surface probality analysis of the amino acids of Glycoprotein G1 of AKAV using bio-software DNAStar, the gene fragment G1-2 coding the high antigenic domain of AKAV G1 protein was selected, which was then amplified by PCR. The amplified product was cloned into pMD18-T vector and sequenced. Then the gene fragment was subcloned into pET-28a (+) vector directionally. The recombinant plasmid was transformed into E. coli BL21 (DE3). SDS-PAGE and Western-blot analysis indicated that the fusion protein was insoluble and approximately 44kD in molecular weight and had immunological activity. After purified by affinity chromatography, the concentration of purified protein was 2mg/ml and the purity was 92.6%. An indirect enzyme-linked immunosorbent assay (ELISA) was developed using the purified recombinant protein. The optimal reaction conditions of ELISA were determined: the coating concentration of the purified recombinant protein was 20μg/ml; the dilution fold of the serum samples was 1:200. There was no cross reaction with positive sera of other six infectious diseases. Bovine serum samples from Yun Nan (77) and Inner Mongolia (70) had been detected by serum neutralizing (SN) test before were detected by this ELISA. In accordance with SN test, the positive threshold of the ELISA was determined as 0.493 and 0.488 in two districts respectively. Comparing to SN test, the speciality of the ELISA was 73% and 86.9% respectively. The agreement ratio between the two methods was 80%(52/65) and 85.9%(55/64) respectively.