Development And Preliminary Application Of Detection Strips For Newcastle Disease Virus Specific Antibody In Chicken

Newcastle disease (ND) is a highly contagious and devastating disease of pourlty all over the world, caused by Newcastle disease virus (NDV) which belongs to the Paramyxoviridae family. It's a serious blow to the world poultry industry.It is important significance to control the NDV by checking and evaluating the level of immunization. Currently, some methods used in laboratory testing are not acceptable for real time, rapid and spot application. This article focuses on building and evaluating the method for rapid detection of antibodies against NDV which based on colloidal gold immunochromatographic technique and the Hemagglutinin-neuraminidase (HN) protein prokaryotic expression. The results were detailed as following:1. Expression and purification of Hemagglutinin-neuraminidase (HN) of NDV.Chick embryo passage preserved in the laboratory with NDV La Sota strains, collected allantoic fluid and then got virus by ultracentrifugation, used Trizol to get virus RNA. According to published HN gene sequence of NDV La Sota strains in NCBI, primers with BamHⅠand HindⅢrestriction sites were designed and synthesized.1734 bp and 1593 bp fragment were amplified which corresponded HN gene full length and the lack of signal peptide and transmembrane region, transmembrance region and cytoplasmic fragment by RT-PCR and cloned into the pGEX-KG vector. The recombinant plasmids were proved by PCR and enzyme analysised, and named pKG-HN and pKG-HNde-sp. The sequence analysis showed that genes consisted of 1734 bp and 1593 bp respectively, which corresponded to the length of published HN genes, and their homology were up to 99%. The constructed plasmids was transformed into E.coli BL21 (DE3) was induced by IPTG, the expressed proteins was analysised by SDS-PAGE, the results showed that the recombinant proteins'molecular weight respectively were 89 KD and 80 KD. Through optimizing the expression conditions of protein, the best result was recombinant pKG-HNde-sp expressed most protein in 37℃, 1‰IPTG, induced 5 h. The immune reactivity of the protein was tested with Western blotting, and it had immunologic reactivity.2. Construction of an immunochromatographic assay for rapid detecting antibodies against NDV in chicken.Using trisodium citrate reduction method to prepare 20 nm colloidal gold particles and then using mouse against chicken IgG Fc monoclonal antibody (McAb) tag it, the optimal label condition was established in which the pH for labeling McAb is 8.0, and the mount of McAb is 1.0 mg/mL 9.6μL. Colloidal gold particles labeled with McAb IgG Fc were used as the detector reagent, the recombinant HN protein which was purified and rabbit IgG against mouse were immobilized on test line and control line of the NC membrane, respectively, and an immunochromatographic assay for rapid detection NDV antibody was constructed. The recombinant HN protein and rabbit IgG against mouse respectively coated in test line and control line were about 1.0 mg/mL and 1.1 mg/mL The experiment results indicated that the strips had no cross-reactivity with the positive serum of H5AIV/H9AIV/IBDV/IBV/MG and negative serum from SPF chicken which demonstrated the specificity of the strip was well. The sensitivity of the strip was coincidence with the standard HI test and the repeatability was also well. All results showed that the strip had features of good specificity, good repeatability, highly sensitivity and rapid. The immunochromatographic strip was successfully established and can be applied to rapid detection of NDV antibody in serum of chicken.3. The preliminary application of an immunochromatographic assay for rapid detectingantibodies against NDV in chicken.240 serum samples were tested by the strip and the standard hemagglutinin inhibition (HI) test, respectively, there were 222 and 220 positive samples test and the detectin rate was 92.5%(222/240) and 91.6%(220/240) by the strip and the standard HI. The two methods show good correlation about 99.1%(220/222).