Cloning and Protein Expression of Human Influenza A Virus NS1 Gene and Its Application in Clinical Laboratory

• Main Authors: Shou-Tzu Li, 李紹慈
• Other Authors: chuan-Liang Kao
• Format: Others
• Language: zh-TW
• Published: 2008
• Online Access: http://ndltd.ncl.edu.tw/handle/55581543060986052110

碩士 === 國立臺灣大學 === 醫學檢驗暨生物技術學研究所 === 96 === Influenza A virus, a genus of Orthomyxoviridae, consists of 8 single-stranded negative sense RNA segments that encode 11 viral proteins. Influenza A virus causes seasonal outbreak during winter season in Taiwan. Laboratory diagnostic methods for influenza virus include virus culture, detection of antibody by serologic test, and detection of RNA by RT-PCR. Hemagglutination inhibition test (HI) is the major serologic method used for the detection of antibody to Hemagglutinine(HA) in Human serum. However vaccinated with inactivated influenza viruses in elder people and children have been started in Taiwan since few years ago, and for HI test, it is difficult to distinguish influenza-infected human and who immunized with inactivated vaccine. Segment 8 of Influenza A virus encodes non-structure protein 1(NS1), a 26kDa protein that is produced only in the influenza virus infected cells and usually is not included in the composition of inactivated influenza virus vaccines. Therefore NS1 antibody can be used as the marker to differentiate influenza virus nature infections from vaccination. In this thesis, first, we cloned the NS1 gene from influenza A virus (H3N2) isolated from National Taiwan University Hospital, and then used RT-PCR to amplify the NS1 gene. After the RT-PCR, full length NS1 gene was first cloned to TA vector, then subcloned into expression vector. The pET32 system was first used, but replaced later by pRSET. Due to low protein expression, Therefore, we have two full-length NS1 protein, NS1-p3 and NS1-pRB and N-terminal deletion mutant dNS1-NHB(C). Second, we used recombinant protein to establish an ELISA for NS1 antibody detection. Anti-NS1 antibodies were detected in unvaccinated healthy young adult serum, and we found there was no significant difference between pre-immune and post-immune serum. And for different age group, the anti-NS1 antibody of 6 month to 2 years-old was the lowest. These results indicated that the anti-NS1 antibody detection may be useful as an ideal method to differentiate the antibody of nature infection from the vaccinee.