發展分子診斷區分流行性感冒病毒基因型的方法與探討細胞中Disabled2蛋白參與流行性感冒病毒進行吞噬作用的可能機制; Development a new diagnosis method to separate serological subtype of influenza virus and possible roles of Dab-2 in the endocytosis pathway of influenza virus.

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Title:	發展分子診斷區分流行性感冒病毒基因型的方法與探討細胞中Disabled2蛋白參與流行性感冒病毒進行吞噬作用的可能機制;Development a new diagnosis method to separate serological subtype of influenza virus and possible roles of Dab-2 in the endocytosis pathway of influenza virus.
Authors:	陳嬿如;Yen-Ju Chen
Contributors:	中國醫藥大學：醫學檢驗生物技術學系碩士班
Keywords:	流行性感冒病毒;吞噬作用;血清亞型;HR-1;influenza virus;subtyping;endocytosis
Date:	2007-07-20
Issue Date:	2009-08-12 17:09:59 (UTC+8)
Abstract:	流行性感冒病毒藉由表面穿膜蛋白hemagglutinin (HA)和neuraminidase (NA)區分血清亞型，目前發現有16種HA蛋白及9種NA蛋白。本研究以Lightcycler Real time PCR 儀器複製病毒基因的特定高度保留區域，結合High Resolution I (HR-1)能夠敏銳的偵測聚合酶連鎖反應產物熔點曲線的特性，選取流行性感冒病毒基因中具高度保留性及演化性的M基因作為目標基因，發展一套能快速且準確偵測出病毒血清型的分子檢驗方式。實驗結果可區分H1、H3、H5、H7、H9五種不同血清亞型，且靈敏度可達到102個病毒；測試了21個經過細胞培養的臨床檢體，並且搭配定序分析序列，可正確區分出有10個H1檢體與11個H3檢體，並且可將單一核酸的突變偵測出來。約有65 %的流行性感冒病毒藉由clathrin組成的水泡進行吞噬作用進入細胞質當中，disabled-2也被證實會參與囊泡形成的過程，並且在囊泡與endosome融合之前，便脫離囊泡的結構。利用免疫螢光染色法與共軛焦顯微鏡觀察病毒感染A549細胞後，病毒在細胞中的位置，推測流行性感冒病毒感染細胞 4~6小時內進行吞噬作用。此外，於MDCK細胞大量表現Dab2蛋白時感染病毒，並以病毒斑分析法觀察，病毒斑數量沒有明顯的變化。Dab2蛋白是否影響流行性感冒病毒的吞噬過程，仍需要進一步研究的釐清。 Influenza A virus were subtype by hemagglutinin(HA) and neuraminidase (NA). Until now, sixteen kinds of HA and nine kinds of NA were found. In this study, a rapid and useful method to separate subtypes of influenza A virus by Lightcycler real time PCR combining with HR-1 analysis was developed. The primer set was designed on conserved MP gene, and the PCR products were cloned into plasmids. The results demonstrate that subtype H1, H3, H5, H7, H9 of influenza A viruses can be successfully separated by analyzing heteroduplex product, and the detection limitation of the method was 102 copies. Twenty-one samples were applied to the test, and 10 samples were correctly typed to H1, and the others were correctly typed to H3. About 65 % of influenza A viruses penetrate host cells by clathrin-dependent endocytosis pathway. Dab2 protein was thought to be one of the endocytic accessory proteins. Infection influenza virus to A549 cells and observed the viral protein sublocalization by confocal microscope observation combing immunofluorescence detection, we found that the viruses may process endocytosis before 4~6 hours post viral infection. Besides, there had no significant plaque formation changes in Dab2 over-expressed MDCK cells. The roles of Dab2 in endocytosis of influenza A virus infection is worthy to investigate further.
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