Construct Of Eukaryotic Expression Recombinant Plasmids PcDNA4/HisMax Of VP Gene Of Avian Encephalomyelitis Virus

Avian Encephalomyelitis Virus (AEV) is a picoranvirus with a predilection for the central nervous system and other parenchymous organs of chickens that is transmited by the oral-faecal route. The virus may be spread by the vertical and horizontal routes, and because of its great stability, contaminated areas may remain infectious for long periods. In young chickens AEV induces paralysis, ataxia and muscular dystrophy, while in older chickens, infection is usually subclinical, resulting in a decline in egg production and hatchability. Infectivity was shown to remain unaffected by chloroform, low PH, pepsin, trypsin and deoxyribonuclease. Magnesium cations were shown to stabilise preparations of the virus against heat inactivation. The buoyant density of virions is 1.31g/ml. The diameter of the virion was estimated to be 22 to 33nm. The AEV can be adapted to grow in chicken embryo. The inability of AEV grows effeciently in most cell cultures.The research consists of two parts. The first part was construction of cloned recombinant plasmids pUCm-T of VP gene of avian encephalomyelitis virus. AEV was inoculated in embryonated Specific Pathogen Free(SPF) chicken eggs in the infected-AEV group, and nothing was inoculated in the contrasted group, yet. A 1:5 dilution of isolate- NH937 of AEV and control group of PBS were inoculated to susceptible 6-day-old chickens embryos, respectively. After incubation for 10 days, the embryos brains and intestines of infected-AEV group and the contrasted group were collected on an aseptic condition, respectively. Making a comparison the size of the chickens embryos between the test group and the control group, the results showed that the size of the control group was bigger than that of the test group. Three pair of primer were designed according to avian encephalomyelitis virus isolate NH937. The structural protein VP1,VP3 and VP0 genes of AEV were amplified by RT-PCR. These genes were cloned into the plasmid pUCm-T, and the inserts were sequenced. The result of nucleotide sequencing showed VP1 gene consists of 810 nucleotides and it has the potential to encode 270 amino acids, VP3 and VP0 consists of 735,726 nucleotides and they encode 245,242 amino acids respectively. The VP1,VP3 and VP0 genes of AEV-NH937 strain shared respectively 90.62%,93.88%,95.45% homology with those from the Calnek vaccine strain of AEV, and that deduced amino acids sequences from the VP1,VP3,VP0 genes of AEV-NH937 strain shared respectively88.89%,97.96%,98.35% homology with those from the Calnek vaccine strain of AEV. The second part was construction of eukaryotic expression recombinant plasmids pcDNA4/HisMax of VP gene of avian encephalomyelitis virus. Cloned recombinant plasmids pUCm-T of VP gene of avian encephalomyelitis virus were digested with EcoRⅠand XhoⅠ,and inserting VP1,VP3,and VP0 genes were purified by DNA Gel Extraction Kit. The expression recombinant plasmids were constructed in vitro by inserting VP1,VP3,and VP0 genes into vector pcDNA4/HisMax, and then transformed into E.coli DH5α. The result of nucleotide sequencing showed pcDNA4/HisMax-VP1,pcDNA4/HisMax-VP3 and pcDNA4/HisMax-VP0 expression recombinant plasmids were constructed successfully. The research is a primary foundation in order to develop AEV nucleic acid vaccines.