Prokaryotic Expression And Preliminary Application Of Influenza A Virus Nucleoprotein

Epidemdic influenza pathogens were influenza viruses, which were divided into A, B, C three types according to their nucleoprotein and matrix protein antigen. Influenza A virus belonged to Orthomyxoviruses Division, forming by eight segments of single stranded negative sense RNA chain, and encoding a total of ten polypeptides. The fifth paragraph of the RNA encoded nuclearprotein (Nucleoprotein, NP),which were highly conserved with the population and type of specificity in the influenza virus,were the basis of classification and diagnosis. Therefore, NP protein expressed in prokaryotic or eukaryotic expression system could be used for detecting of influenza A virus by enzyme immunoassay and seting up a method of explorating for the clinical diagnosis of influenza A virus to provide timely and accurate reference information.In this study, the synthetic NP DNA gene of the influenza A virus (A/duck/Zhejiang/11/2000 (H5N1)) were used as the PCR template.Then,the NP gene, which had a length of 1498 bp and encoded 498 aminoacids, were amplificated by PCR technique. And the recombinant plasmid were constructed and transformed into the expression vector. The relative molecular mass of the expressed influenza A virus NP protein were about 57 KD, and were used to immune the animals and prepare two types of anti-serum and purify the antibodies of the anti-serum, which would be used in the enzyme immunoassay for the detection of influenza A virus. In the end, the marked and packaged antibodies were used in the final double-antibody sandwich method for the preliminary exploration of detecting the influenza A virus.In this paper,the main contents:(1) In this study, the prokaryotic recombinant plasmid pET-30 Xa/LIC-NP was contracted by the template,which were the DNA gene sequences of influenza A virus H5N1 NP strain.And the nucleoprotein were induced in the prokaryotic recombinant plasmid,expressed and purificated in E. coli expression system;(2) Used the purified NP protein to immune the sheep and rabbit to get and purify the anti-NP charge anti-serum, and labeled the goat anti-NP polyclonal antibody and rabbit anti-NP polyclonal antibody with the enzyme of HRP; (3) The initial exploration of influenza A double-antibody sandwich detection method (ELIS A method), used of polyclonal antibodies can be prepared as a double-antibody sandwich of packaged and marked antibodies,which were used for analysising the feasibility of double-antibody sandwich detection method of influenza A virus.In this study, influenza NP protein antigen were expressed and purified in prokaryotic expression system,according to this method, which eliminated the natural bio-safety risks of the influenza virus.The initial exploration of double-antibody sandwich ELISA diagnosis method, providing support to the development of early diagnosis kits of influenza A virus.