人類A型流行性感冒NS1基因的選殖與表現及其臨床實驗室應用

Abstract

A型流行性感冒病毒屬於正黏液病毒科，含有八條單股RNA，可以轉譯出11種病毒蛋白。台灣每年11月到3月是流行感冒流行的季節。目前實驗室診斷法有病毒培養、血清抗體檢測及核酸檢測等。病毒培養及核酸檢測為最常用之方法，但均有其缺點存在。而血清抗體檢測，主要以血球凝集抑制(Hemagglutination inhibition)試驗為主，但卻無法分辨所測出之抗體是自然感染或預防接種免疫產生的，因此就需要發展出可區別自然感染及預防接種所產生之血清抗體的檢測方法。 由於流行性感冒病毒非結構(NS1)蛋白質，只會在病毒感染複製之宿主細胞中產生，並不會出現在成熟病毒顆粒中，基本上不會出現在去活性病毒疫苗中，因此在預防接種者身上，就不會產生NS1相關的抗體。在流感病毒方面，用NS1抗體分辨自然感染或疫苗免疫的研究，在豬流感、禽流感及馬流感病毒近年來已有相關的文獻發表。 因此本篇論文的目的為利用大腸桿菌系統來表現人類A型流感病毒的NS1基因，純化細菌所表現出來的NS1重組蛋白質，並建立一套可用來檢測人類血清中NS1抗體的酵素連結免疫吸附試驗(ELISA)，來幫助流行性感冒病毒的臨床診斷並可藉此分辨自然感染及預防接種所產生的抗體。 首先，本實驗使用臺大醫院分離出來的H3N2流感病毒株 A / Taiwan / NTU6699 / 2006 (H3N2) 做為NS1基因來源，先用RT-PCR的方法增幅NS1基因，再用TA clone選殖出序列正確的片段，然後用限制脢切下送至蛋白質表現載體 pET32c，最後將構築好的帶有NS1基因的載體送入大腸桿菌中，表現帶有NS1 protein的重組蛋白質，並用Ni2+-column 進行純化。但在蛋白質表現的過程中，發現pET32C所表現的重組蛋白質量並不足夠，因此改用pRSET增加蛋白質的表現量並同時構築了一個不表現NS1蛋白質N端(RNA Binding domain)的重組蛋白質。之後純化上述的NS1重組蛋白質建立可偵測人類血清中抗NS1 抗體的酵素免疫吸附試驗(ELISA)，利用此系統分析未接受過疫苗注射的健康成年人檢體，發現可測得一定值以上的陽性反應，在疫苗前後血清的檢測上，由實驗結果發現，人類血清中存在的NS1抗體不會因為疫苗注射而使抗體價數上升，免疫前後的數值統計也沒有顯著差異，而不同年齡層的檢測結果，也發現在2歲以下年齡層抗NS1抗體的檢測結果較其他群組低。2到5歲逐漸升高，5歲以後稍微降低之後就趨於平緩。綜合以上實驗結果，顯示本研究所建立的抗NS1抗體的酵素免疫吸附試驗，在未來或許能幫助分辨人類血清中抗體的上升為流感病毒的自然感染或是疫苗免疫所造成的。

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.