Eukaryotic Expression Of Nucleoprotein Of Avain Influenza Virus And Preliminary Development Of Protein Chip

Avian influenza is a contagious disease of animals caused by viruses that normally infect only birds and, less commonly, pigs. Avian influenza viruses are highly species-specific, but have, on rare occasions, crossed the species barrier to infect humans. With the rapid development of poultry industry many countries continued to report outbreaks of avian influenza, so it is very important to rapidly diagnose influenza virus.Based on serological diagnosis of avian influenza, this study provided a new type of protein microarrays, which used to quickly determine poultry influenza antibodies, and can also distinguish between influenza virus infection and immune.Protein microarray technology is a kind of biochips, and it is fast and efficient, parallel, high throughput in protein detection technology, can be used to examine the multiple disease to realize the accuracy and speed in diagnosis. With the protein microarray technology applied to animal viral disease diagnosis is one of the important contents of promotion of this technology. This technology platform construction involves protein samples preparation, the choice of sealed liquid, the conditions of printing, concentration and hybrid time of antibody and data processing, etc.The NP of avian influenza virus was expressed in baculovirus expression system In this process the gene of NP was firstly amplified in RT-PCR, then NP baculovirus expression plasmid and transfer plasmid was construced in turn, then NP was expressed in insect cells, then with Western-blot analyzing, selected active protein to construct protein microarray. Then select the eukaryotic expression of NP as antigens, dilute to the appropriate concentration printed on the slides wrapped with aldehyde group, fixed, then this protein microarray hybrided successively with avian influenza virus positive serum and fluorescent marked second antibody, obtain result with the micro PE scanning,preliminarily established protein microarray diagnostic avian influenza work platform. After that further to analized the relation between NP protein concentration, antibody concentration and function of time, fixed time, closed buffer and chip specificity, to optimize detection conditions. Then combine the property of nonstructural proteins with influenza virus, to construct a differential diagnosis chip to distinguish infection serum and immune serum, and analyzes its differential diagnosis effect.Results showed that the NP gene of influenza virus was successfully expressed in insect cell, the NP product has good biological activity,and the NP can be constructed the diagnosis protein chip;When NP solution concentrationwas 2000μg/mL,fixed with 24h, blocked by 1% BSA for 2h, then reacted with diluted 400 degrees avain serum, dilute in 2000 times of second serum, protein microarray present strong fluorescence signal, and without nonspecific signal;Using influenza virus NP and NS constructed differential diagnosis of chips, the result shows that the fluorescent signals of NP and NS are strong with infected serum, the fluorescent signal was close to positive serum,in contrast NS appeared no fluorescent signal, NP fluorescent signals is presented to the strong fluorescent signals with immunization serum, this explains the differential diagnosis chip can differentiate infectious serum and immune serum.This experiment constructed protein chip can be applied to diagnose avian influenza and differentiate infection serum and immune serum,this protein chip is an efficient, fast and accurate antibody test, and laid the foundation for the next high-density chips, plays an important role in the diagnosis A influenza virus.