Studies On ELISA Kit For Detecting Avian Influenza Virus And Mucosal Immunization With Its Recombinant Nucleoprotein

Avian Influenza (AT) is one of fatal infectious disease caused by influenza A virus in avian. It first appeared in Italy more than 100 years ago(around 1878), and, so far, different strains were found and caused important economic losses in the avian industry throughout the world. At present, highly pathogenic avian influenza (HPAI) is listed in A infectious diseases by World Organization for Animal Health(OIE). Recently, AI has produced important economic losses and bad effect to society in our country, especially, events that AI infected human being happened in Hongkong(1997) and south-eastern Asian (2003-2004), which shocked mankind and caused our attention to commonality sanitation of AI. A common viewpoint is that prevention of AI is closely linked to persistent and steady development of avian industry and humankind's health. This research is aimed to develop ELISA for detection of AIV in order to provide technique to forepart diagnoses of AI. In addition, Because of negligible cross-protection against different AIV subtypes, it is very difficuly to prevent avian influenza from outbreaking. Therefore, we attempted to mix or fused expression product of Cholera toxin B subunit(CTB), a strong mucosal immunization adjuvant, with NP, a expression product of nucleoprotein(NP) of AIV, then chicken were immunized with mixture(CTB+NP) and fused product(CTB-NP) by nasal cavity in order to enhance secretory IgA(sIgA) titer of nasal cavity and trachea surface, which maybe control and prevent AI from happening. The results of research are summarized as following:1. Preparation of sera of chicken anti-AIV, rabbit anti-AIV, goat anti-rabbit IgG and goat anti-rabbit IgG-HRPAIV (H9N2) was inoculated into 9 to 10-day embryonating chicken eggs by the allantoic route, then allantoic fluid was harvested and centrifuged with 30,000r/m, and AIV deposit was redissolved by 0.01mol/L PBS (pH7.2)which is 1/30 volume of allantoic fluid. Chicken and rabbits were immunized with redissolved fluid of AIV emulsified with Frund's adjuvant, and sera of chicken anti-AIV, rabbit anti-AIV were acquired after three inoculations, and the titers of AGED reach 1:64. At the same time, purified serum of goat anti-rabbit IgG(AGID: 1:64) from goat immunized with purified rabbit IgG was labeled with horseradish peroxidase(HRP). The conjugate is characteristic of high activity of immunity and its work concentration reached 1:4000. This laid a foundation for the development of sandwich enzyme-linked immunosorbent assay (ELISA) for AIV.2. Development of enzyme-linked immunosorbent assay (ELISA) for AIV.A sandwich ELISA for detection of AIV antigen was developed by using purified chicken anti-ATV IgG as the first antibody coated on the ELISA plate and rabbit anti-AIV IgG regarded as the second antibody. The results showed that the optimum working concentration of chicken anti-AIV IgG and rabbits anti-AIV IgG were lug/mL and 5ug/mL, respectively. The dilution titers of positive AIV sample by ELISA was 16 times higher than that by hemagglutination test, and other AIV subtypes can also be detected by this method. No cross reaction was observed with Newcastle disease virus (NDV), infectious bronchitis virus (IBV), eggs drop syndrome virus (EDS^V), infectious laryngotracheitis virus (ILTV), infectious bursal disease virus (IBDV), avian pox virus (APV) and Marek's disease virus(MDV). 7 of 14 suspicious chicken groups showing respiratory tract syndrome or peritonitis, opthalmitis, egg drop were positive detected by this method.3. Development and Application of Sandwich Enzyme-linked Immunosorbent AssayTest Kit for Detecting Avian Influenza Virus AntigenA Sandwich Enzyme-linked immunosobent assay test kit based on previous work was developed for detection of avian influenza virus (ATV) antigen. The kit consists of eleven reagents and a piece of 16-well microtiter plate. There were highly specificity, sensitivity and repeatability in detecting AIV antigen with the kit. It was stable well when storing at below -lO'C for six months. It is very easy to manipulate and the result can be reported within 3 hours. So far, different subtype ATVs including H5 and H9 subtypes from chicken and H5 subtype from Landes Goose were isolated from some provinces such as Hubei, Anhui and Henan and so on detected by the kit.4. Gene fusion and expression of nucleoprotein (NP) of AIV and the Cholera toxin B subunit (CTB)Vector pGEX-KG-CTB expressing CTB gene was developed after CTB gene was cloned into expression vector pGEX-KG containing GST gene. Then NP gene was cloned into the downstream of CTB gene in pGEX-KG-CTB, and the expression vector pGEX-KGCN was constructed. After correct cloning sites and sequences were indentified, CTB and CTB-NP were expressed in Escherichia coli BL21 (codon plus) strain, The results of SDS-PAGE showed that molecular weight of GST-CTB and GST-CTB-NP were about 38.0kD and 94.0kD, respectively; which matches the expected molecular weight. Western blotting showed NP protein in GST-CTB-NP takes on strong biological activity.5. Mucosal immunization study on recombinant nucleoprotein of AIV45 chickens, 15 days old, were divided randomly into 5 groups at the start of experiments. Groups of 9 animals were immunized with NP, or NP+CTB, or CTB-NP, or CTB, at fhe same time, a group was control. All immunizations were given in nasal cavity for three times at 10-day intervals. Before every immunization and 10 days after the last immunization, sera, nasal wash, trachea wash and gut wash were collected for ELISA detection for corresponding immunoglobulin(IgG-NP, IgA-NP, slgA-NP). The results showed that high level IgG-NP and low level IgA-NP in sera happened. SlgA-NP antibody responses(day20 and day30) in nasal wash, trachea wash and gut wash were enhanced significantly by CTB(p<0.05). At day 30, ODs of slgA-NP in nasal wash and gut wash was significantly enhanced by CTB(CTB-NP group > CTB+NP group > NP group), and the difference were significant with each other(p<0.05). In all trachea wash, there was no difference(p>0.05) between CTB-NP group and CTB+NP group, but there was significantly difference(p>0.05)between the two groups and NP group; hi conclusion, when mixed with or fused expression product of recombinant NP, CTB was a candidate adjuvant for enhancing mucosal immunization of recombinant nucleoprotein of AIV.