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Development and Preliminary Application of Detection Strips for Newcastle Disease Virus Specific Antibody in Chicken

Author: JiaQing
Tutor: BiDingRen
School: Huazhong Agricultural University
Course: Preventive Veterinary Medicine
Keywords: Newcastle disease virus antibody HN protein Colloidal gold Immunochromatographic technology
CLC: S858.31
Type: Master's thesis
Year: 2010
Downloads: 150
Quote: 1
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Abstract


Newcastle disease (Newcastle disease, ND) the Paramyxoviridae mumps virus is a type of the paramyxovirus (Paramyxo virus) Newcastle disease virus (Newcastle disease virus, NDV) devastating disease caused by a highly infectious, the The disease seriously hindered the development of the poultry industry in the world. Immunity levels and evaluation of detection flocks of chickens immune status is of great significance for the control of the disease. At present, the antibodies against the virus detection technology is mainly confined to laboratory testing, there are some limitations in clinical application. In this study, the prokaryotic expression hemagglutinin neuraminidase protein, colloidal gold immunochromatographic technology, the establishment of the Newcastle disease virus antibody rapid detection method, and obtained the following results: 1. Newcastle Disease hemagglutinin neuraminidase protein prokaryotic NDV La Sota strains kept in our laboratory expression and purification through the chick embryo passage charge allantoic fluid extraction of viral RNA, two pairs of primers were designed with BamH Ⅰ and Hind Ⅲ digestion sites was amplified by RT-PCR full length and go signal peptide sequence of the transmembrane region of the HN gene fragment, two amplification products were cloned into the pGEX-KG vector was successfully constructed a recombinant plasmid pKG-HN and pKG-HNde-sp sequenced The two gene size of 1734 bp and 1593 bp, amplification La Sota and reported on the sequence in GenBank nucleotide sequence homology of 99%. Constructed pKG-HN and pKG-HNde-sp plasmid was transformed into E. coli BL21 (DE3) expression, induced by SDS-PAGE electrophoresis analysis, recombinant expression of the protein molecular weight of approximately 89 KD and 80 KD. After optimization of protein expression, the results show pKG-HNde-sp in temperature to 37 ° C, IPTG concentration of 1 ‰ and induced 5 h, the highest expression levels, Western-Blot analysis showed that the expression of the protein have a better immune school activity. The establishment of the Newcastle disease virus antibody rapid detection test strip prepared by 20 nm colloidal gold tri-sodium citrate reduction, mouse anti-chicken IgG Fc monoclonal antibody marker, mark the optimum pH was 8.0, best mark of 9.6 μL1.0mg/mL. To the gold standard mouse anti-chicken IgG Fc monoclonal antibody as a probe, rabbit anti-mouse IgG as a control line, as the test line capture reagents purified HN protein, initially established a detection of Newcastle disease antibodies colloidal gold rapid test strip amended. The preferred spraying membrane of the rabbit anti-mouse IgG concentration of 1.0mg/mL, HN expression of the best spraying membrane protein concentration of 1.1 mg / mL. The test strip and infectious bursal disease, infectious bronchitis, H9 subtypes of avian influenza subtype H5 avian influenza, Mycoplasma gallisepticum positive serum and specific pathogen free chickens negative serum reactions were negative; sensitivity and blood coagulation inhibition test results consistent. The method is simple, specificity, high sensitivity, and can be used for detection of Newcastle disease serum antibody levels. Rapid detection of Newcastle disease antibodies preliminary application of the test strip with the test preparation Newcastle disease antibody rapid detection kit for clinical application, testing 240 sera which detected 222 positive serum test strip, the detection rate was 92.5% ( 222/240); hemagglutination inhibition (HI) test detected 220 positive serum, the detection rate was 91.6% (220/240), both the rate of 99.1% (220/222), the results show that this The kit HI test results are basically the same, instead of the HI test in the clinical detection of Newcastle disease antibody titer.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Livestock, poultry, wildlife diseases > Poultry > Chicken
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