Study On Expression Of APMV-1 F Gene And Development Of Multiplicity Indirect ELISA

Avian Paramyxovirus consisted of nine serotypes, which is APMV 1 to 9. APMV-1, which historically was called Newcastle disease virus (NDV), has previously been associated almost exclusively with disease in avian species. ND was found to be caused by a zoonotically transmitted Avian Paramyxovirus that had evolved the capability for cross-host transmission. It is well established that APMV-1 can cause an acute and rapidly clearing conjunctivitis, low fever and chills, were clearly documented as attributable to APMV-1 on the basis of virus isolation and serology. It is traditional ideal that waterfowls such as geese and ducks and mammal constantly were not considered to be infected by NDV, for the host restriction. Sine 1997, the infection spectrum of NDV has been expanding from chicken, pigeon, parrot and some rare birds to waterfowls. A recent report by a research team from New York state department of health, it is surprised that isolation of APMV-1 from a patient with a lethal case of pneumonia. Those cases show that the infection spectrum of NDV has been extending endlessly and bring some troubles to prevention and cure to NDV. According to previous research, there are lots of different about the antigenic variant of between NDV and Goose Paramyxovrus, so development of a new detection system based on coinstantaneous detect anti-NDV and GPMV serum have a important meaning to prevention and cure to NDV.According to the nucleotide sequence of NDV F₄₈E₉ strain in Genbank and the MCS of pET-22b (+), a pair of primer was designed and synthesized, and a complete F gene of NDV F₄₈E₉ strain was obtained by RT-PCR. Then F gene of NDV F₄₈E₉ strain was cloned into pMD18-T simple Simple vector, sequenced. The prokaryotic expression vector pET-F₄₈E₉-F was constructed by inserting the F gene into the pET-22b (+) vector. And the expression were induced by IPTG in E.coli BL21(DE3). SDS-PAGE and Western-blot analysis showed that F protein of NDV F₄₈E₉ strain amounted to 25.2% of the total protein of E.coli BL21(DE3)after induced by IPTG(1 mM)at 37℃for 6 hours. The molecular weight of expressed F protein was about 59 Ku, and was specifically recognized by polyclonal antibody against NDV F₄₈E₉ strain.It is well that Purified IgG of goose serum by caprylic acid-ammonium sulfate method, the result of SDS-PAGE show that there are two obviously lines, the one is heavy chain which is 66 Ku and the other is light chain which is 28 Ku respectively. PcAb against goose serum IgG was obtained by vaccination of rabbits with goose serum IgG. The vaccination procedure is the first time vaccinated with the mixture that is purified IgG and complete Freund's adjuvant, the second the aduvant is incomplete and the third is only purified IgG. After third vaccination, when the titer of PcAb was up to 1: 64, getting blood from heart, dissociating serum and the antiserum was last purified by caprylic acid-ammonium sulfate method. Then PcAb was successfully labeled with HRP. The labeling rate was up to 90.2%. And it could bind with Goose serum IgG. The ELISA experiment was operated by elementary procedure, with purified F protein of GPMV NA-1 strain as coated antigen, and rabbit HRP-labeled PcAb as a second antibody, was 0.21μg per well, dilution of HRP-labeled PcAb was 1: 1 500, confining liquid was 2% BSA and the OPD was chosen to be enzyme reaction substrate. Then the standard curve was obtained and regression equation was got by a regression analysis. It was y=0.2447x-0.2776,R2=0.9804. Based on the indirect ELISA for detection anti- GPMV serum, we development a new ELISA method that it can simultaneously detected the antibody from NDV and GPMV. According to the previous purified F proteins from NDV F₄₈E₉ strain and GPMV NA-1 strain, put them together with some percentage, then the multiplicity indirect ELISA was development. The ELISA system is the antigen was F protein of NDV F₄₈E₉ strain: F protein of GPMV NA-1 strain is 1:1, the total coated loading is 0.52μg per well. When it can detected NDV, the dilution of rabbit anti-chicken HRP-labeled PcAb was 1: 2 000 in 90 minutes, the other is the dilution of rabbit anti-goose HRP-labeled PcAb was 2 000 in 120 minutes. The well confining liquid was 2% BSA, time of reaction of OPD is 20 min. This ELISA has no cross reaction with other anti-virus serum from chicken viral diseases and goose viral diseases, such as MDV, IBDV, IB, GPV, Goose Adenovirus and so on.From the detection of many samples from clinic, we can see that the multiplicity indirect ELISA here established has higher sensitivity and lower minimum detect ability, so it laid the foundation for the clinical detection, preparation of immunoassay diagnostic kit and prevention and cure for APMV-1.