SPC-A1細(xì)胞應(yīng)答呼吸道合胞病毒感染的敏感基因zlg10的克
發(fā)布時(shí)間：2018-12-31 11:04
【摘要】：呼吸道合胞病毒(respiratory syncytial virus,RSV)是引起嬰幼兒嚴(yán)重下呼吸道感染的重要病毒性病原體。該病毒流行廣泛,致病率高,多數(shù)兒童兩歲前都感染過RSV,在老年人和免疫力低下的人群中也會(huì)暴發(fā)流行。RSV屬副黏病毒科,肺炎病毒屬,病毒粒子直徑約150～300nm。RSV的基因組為單股負(fù)鏈RNA,長約15kb,編碼11種蛋白質(zhì),包括3種核衣殼蛋白(核殼蛋白N、磷酸蛋白P、大聚合酶蛋白L);3種跨膜蛋白(融合蛋白F、附著蛋白G、小疏水蛋白SH);2種基質(zhì)蛋白(M、M2)和2種非結(jié)構(gòu)蛋白(NS1、NS2)。RSV感染的臨床癥狀在不同年齡組往往不同,一般可致鼻炎、中耳炎、哮喘、支氣管炎、細(xì)支氣管炎和肺炎,尤其以后兩種疾病最為嚴(yán)重,甚至可導(dǎo)致死亡。目前能有效治療RSV感染的藥物很少,且療效不確定。世界衛(wèi)生組織(WHO)已將研制RSV疫苗列為全球疫苗計(jì)劃的優(yōu)先發(fā)展項(xiàng)目之一。但迄今為止,尚無安全、有效的RSV疫苗獲準(zhǔn)上市。 分子生物學(xué)研究已經(jīng)證實(shí)當(dāng)病毒感染宿主細(xì)胞時(shí),宿主細(xì)胞會(huì)發(fā)生變化,其中有一些特異性的序列表達(dá)量有所改變。mRNA差異顯示法(mRNA differential display,DDRT-PCR)是目前篩選差異表達(dá)基因最有效的方法之一。為了篩查到與病毒感染相關(guān)的宿主細(xì)胞敏感基因,本研究以呼吸道合胞病毒RSV感染的SPC-A1細(xì)胞與正常SPC-A1細(xì)胞為樣本,通過mRNA差異顯示技術(shù)獲得了SPC-A1細(xì)胞應(yīng)答RSV感染的mRNA差異表達(dá)譜,并且克隆到了50余個(gè)與呼吸道合胞病毒RSV致病相關(guān)的EST片段。隨后選擇在RSV感染后的SPC-A1細(xì)胞中高表達(dá)的EST片段g10-1為研究對象,經(jīng)克隆測序g10-1長264bp,采用生物信息學(xué)方法對g10-1進(jìn)行了鑒定后證實(shí)這是一個(gè)新基因片段,隨后電子克隆獲得的g10-1的延伸產(chǎn)物為761bp,含有一個(gè)編碼54個(gè)氨基酸的讀碼框,命名為zlg10基因。zlg10編碼的蛋白質(zhì)與己知蛋白質(zhì)均無顯著同源性,亞細(xì)胞定位實(shí)驗(yàn)證實(shí)是一個(gè)新的核蛋白質(zhì)。Northern Blot雜交分析驗(yàn)證后,設(shè)計(jì)了zlg10含完整閱讀框的特異性擴(kuò)增引物,通過RT—PCR方法克隆出了該基因。 為了了解新基因zlg10在RSV感染細(xì)胞過程中如何被調(diào)節(jié)以及在RSV感染過
[Abstract]:Respiratory syncytial virus (respiratory syncytial virus,RSV) is an important viral pathogen causing severe lower respiratory tract infection in infants. The virus is widespread and has a high pathogenicity. Most children have been infected with RSV, before the age of two years. RSV belongs to paramyxovirus family, pneumonia virus family, and can also break out in the elderly and people with low immunity. The genome of virus particles about 150~300nm.RSV is about 15kb long, encoding 11 proteins, including three nucleocapsid proteins (core-shell protein N, phosphate protein P, large polymerase protein L);). Three transmembrane proteins (fusion protein F, attachment protein G, small hydrophobic protein SH);) The clinical symptoms of two kinds of matrix protein (MM-2) and two kinds of non-structural proteins (NS1,NS2). RSV infection) are often different in different age groups. They can cause rhinitis, otitis media, asthma, bronchitis, bronchiolitis and pneumonia. In particular, the last two diseases are the most serious and can even lead to death. At present, there are few drugs that can effectively treat RSV infection, and the effect is uncertain. The World Health Organization (WHO) has made the development of RSV vaccine one of the priorities of the global vaccine program. But so far, no safe, effective RSV vaccine has been approved to market. Molecular biology has shown that when the virus infects the host cell, the host cell changes, and some of the specific sequence expressions change. MRNA differential display (mRNA differential display, DDRT-PCR is one of the most effective methods for screening differentially expressed genes. In order to screen host cell-sensitive genes associated with viral infection, we used SPC-A1 cells infected with respiratory syncytial virus (RSV) RSV and normal SPC-A1 cells as samples. The differentially expressed mRNA profiles of SPC-A1 cells in response to RSV infection were obtained by mRNA differential display technique, and more than 50 EST fragments related to the pathogenesis of respiratory syncytial virus RSV were cloned. Subsequently, the highly expressed EST fragment g10-1 in SPC-A1 cells infected with RSV was selected as the study object. The g10-1 was cloned and sequenced to 264bp. the g10-1 gene fragment was identified by bioinformatics method and proved to be a new gene fragment. The extended product of g10-1 was 761 BP, which contained a reading frame encoding 54 amino acids and was named zlg10 gene. The protein encoded by zlg10 had no significant homology with known proteins. The subcellular localization experiment confirmed that it was a new nuclear protein. Northern Blot hybridization analysis and verified, then designed a specific amplification primer containing a complete reading frame for zlg10, and cloned the gene by RT-PCR method. To understand how the new gene zlg10 is regulated in RSV infected cells and how it is infected with RSV
【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2005
【分類號(hào)】：R373
本文編號(hào)：2396484
[Abstract]:Respiratory syncytial virus (respiratory syncytial virus,RSV) is an important viral pathogen causing severe lower respiratory tract infection in infants. The virus is widespread and has a high pathogenicity. Most children have been infected with RSV, before the age of two years. RSV belongs to paramyxovirus family, pneumonia virus family, and can also break out in the elderly and people with low immunity. The genome of virus particles about 150~300nm.RSV is about 15kb long, encoding 11 proteins, including three nucleocapsid proteins (core-shell protein N, phosphate protein P, large polymerase protein L);). Three transmembrane proteins (fusion protein F, attachment protein G, small hydrophobic protein SH);) The clinical symptoms of two kinds of matrix protein (MM-2) and two kinds of non-structural proteins (NS1,NS2). RSV infection) are often different in different age groups. They can cause rhinitis, otitis media, asthma, bronchitis, bronchiolitis and pneumonia. In particular, the last two diseases are the most serious and can even lead to death. At present, there are few drugs that can effectively treat RSV infection, and the effect is uncertain. The World Health Organization (WHO) has made the development of RSV vaccine one of the priorities of the global vaccine program. But so far, no safe, effective RSV vaccine has been approved to market. Molecular biology has shown that when the virus infects the host cell, the host cell changes, and some of the specific sequence expressions change. MRNA differential display (mRNA differential display, DDRT-PCR is one of the most effective methods for screening differentially expressed genes. In order to screen host cell-sensitive genes associated with viral infection, we used SPC-A1 cells infected with respiratory syncytial virus (RSV) RSV and normal SPC-A1 cells as samples. The differentially expressed mRNA profiles of SPC-A1 cells in response to RSV infection were obtained by mRNA differential display technique, and more than 50 EST fragments related to the pathogenesis of respiratory syncytial virus RSV were cloned. Subsequently, the highly expressed EST fragment g10-1 in SPC-A1 cells infected with RSV was selected as the study object. The g10-1 was cloned and sequenced to 264bp. the g10-1 gene fragment was identified by bioinformatics method and proved to be a new gene fragment. The extended product of g10-1 was 761 BP, which contained a reading frame encoding 54 amino acids and was named zlg10 gene. The protein encoded by zlg10 had no significant homology with known proteins. The subcellular localization experiment confirmed that it was a new nuclear protein. Northern Blot hybridization analysis and verified, then designed a specific amplification primer containing a complete reading frame for zlg10, and cloned the gene by RT-PCR method. To understand how the new gene zlg10 is regulated in RSV infected cells and how it is infected with RSV
【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2005
【分類號(hào)】：R373
【參考文獻(xiàn)】
相關(guān)期刊論文 前6條
1 張艷敏,雷春蓮,楊玉琮,閻輝,成革勝;呼吸道合胞病毒下呼吸道感染T_H亞群功能狀態(tài)的研究[J];免疫學(xué)雜志;2002年02期
2 羅瑛,隋建麗,鐵軼,周平坤,孫志賢;生物信息學(xué)輔助定位及延伸輻射誘導(dǎo)未知表達(dá)序列標(biāo)簽[J];生物化學(xué)與生物物理進(jìn)展;2001年02期
3 韓為東,于力,樓方定,王全順,趙瑜,史子江,焦宏遠(yuǎn),周建軍;一個(gè)新的白血病相關(guān)基因LRP16全長cDNA的克隆、序列分析及表達(dá)特征[J];中國生物化學(xué)與分子生物學(xué)報(bào);2001年02期
4 郝純毅,趙敏,李勇,邢寶才,呂有勇,黃信孚;降低mRNA差異顯示技術(shù)假陽性率的一種方法[J];中國生物化學(xué)與分子生物學(xué)報(bào);2002年01期
5 池曉菲,舒慶堯;開放的差異基因表達(dá)技術(shù)研究進(jìn)展[J];中國生物化學(xué)與分子生物學(xué)報(bào);2002年03期
6 陳淑,姚明珠,俞麗萍;一次院內(nèi)新生兒呼吸道合胞病毒感染流行[J];實(shí)用兒科雜志;1992年02期
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