發(fā)布時(shí)間：2018-06-02 12:30

  本文選題：蛋白質(zhì)組學(xué) + 雙向電泳��； 參考：《第三軍醫(yī)大學(xué)》2007年碩士論文

【摘要】： 輪狀病毒感染是全世界范圍內(nèi)嬰幼兒腹瀉的主要病因。MA104細(xì)胞是研究輪狀病毒感染生物學(xué)的理想宿主細(xì)胞。對輪狀病毒感染后宿主細(xì)胞基因表達(dá)的變化,通過RT-PCR、Northern blotting及cDNA微陣列的方法進(jìn)行了很多研究,但是蛋白質(zhì)才是生命活動(dòng)的最終體現(xiàn)者。為了揭示輪狀病毒的致病機(jī)理及宿主細(xì)胞對輪狀病毒感染的反應(yīng)特點(diǎn),我們用蛋白組技術(shù)對輪狀病毒感染前后胞內(nèi)蛋白表達(dá)譜的變化情況進(jìn)行研究。首先建立輪狀病毒感染MA104細(xì)胞模型。我們代表性的選用非唾液酸(SA)依賴的人源輪狀病毒W(wǎng)a株和唾液酸依賴的猿猴輪狀病毒SA11株,從對MA104細(xì)胞感染能力、胞內(nèi)轉(zhuǎn)錄復(fù)制能力、誘導(dǎo)細(xì)胞凋亡壞死能力等方面進(jìn)行觀察,確定了輪狀病毒感染MA104細(xì)胞的合適條件:輪狀病毒W(wǎng)a株以MOI=2,SA11株以MOI=8感染MA104細(xì)胞12h,既能保證較高的感染率,又使宿主細(xì)胞病變不過于嚴(yán)重,細(xì)胞結(jié)構(gòu)保持完整,為進(jìn)行感染后細(xì)胞蛋白表達(dá)譜研究的理想選擇。 為了分析感染病毒后宿主細(xì)胞蛋白表達(dá)譜的變化,用13cm pH3-11NL的IPG預(yù)制膠條做IEF,雙向電泳分析感染了輪狀病毒及模擬感染的MA104細(xì)胞蛋白譜表達(dá)情況。Wa株感染組分別可檢測出772和798個(gè)蛋白點(diǎn),SA11感染組分別可檢測出1230和1312個(gè)蛋白點(diǎn)。通過圖像分析及膠與圖像的比對,重復(fù)3次,發(fā)現(xiàn)Wa株感染組,感染12h后,有14個(gè)蛋白點(diǎn)表達(dá)上調(diào)2倍以上,37個(gè)蛋白點(diǎn)表達(dá)下調(diào)50%以上;SA11株感染組,感染12h后,有24個(gè)蛋白點(diǎn)表達(dá)上調(diào)2倍以上,29個(gè)蛋白點(diǎn)表達(dá)下調(diào)50%以上。而在這些上調(diào)表達(dá)的蛋白點(diǎn)中,Wa株感染組與SA11感染組僅發(fā)現(xiàn)2個(gè)在等電點(diǎn)及分子量接近,提示可能為相同的蛋白。鑒于輪狀病毒感染后會(huì)引起細(xì)胞蛋白表達(dá)的普遍下降,那些感染后表達(dá)反而上調(diào)的蛋白在輪狀病毒于宿主細(xì)胞相互作用中似更有意義,所以我們選擇在感染后表達(dá)上調(diào)的蛋白點(diǎn)用于蛋白鑒定。 為了鑒定輪狀病毒感染后宿主細(xì)胞內(nèi)上調(diào)表達(dá)的蛋白質(zhì),我們采用MALDI-TOF-MS技術(shù),并通過mascot用NCBInr數(shù)據(jù)庫進(jìn)行數(shù)據(jù)庫檢索。由于考慮到MA104細(xì)胞為猿猴來源,同時(shí)選取的蛋白中可能也會(huì)有病毒表達(dá)的蛋白質(zhì),我們對每個(gè)點(diǎn)同時(shí)搜索了靈長類和病毒兩個(gè)庫。輪狀病毒wa株感染組,送檢11蛋白點(diǎn),檢出陽性結(jié)果7個(gè),排除蛋白質(zhì)的種屬特性,其中兩個(gè)為同一蛋白HSP27,其它分別為與zeta晶體蛋白高度同源的未命名的蛋白產(chǎn)物(gi|90085272),理論上蛋白質(zhì)(gi|114583139),環(huán)戊烷介導(dǎo)的運(yùn)動(dòng)受體(hyaluronan-mediated motility receptor (RHAMM)),輪狀病毒非結(jié)構(gòu)蛋白NSP3,角蛋白8。輪狀病毒SA11株感染組,送檢16個(gè)蛋白點(diǎn),陽性點(diǎn)4個(gè),除去種屬差異性,分別為輪狀病毒非結(jié)構(gòu)蛋白NSP3,plectin 1 isoform 6, cortactin-binding protein 2與leucine-zipper-like transcription regulator, 1 isoform 1的復(fù)合物,KRT8 protein與reticulocalbin 1的復(fù)合物。這些蛋白可能與抑制宿主蛋白表達(dá)、胞內(nèi)信號傳遞、胞內(nèi)代謝、病毒復(fù)制與運(yùn)輸及宿主細(xì)胞發(fā)育分化相關(guān),顯示出輪狀病毒感染后對宿主細(xì)胞的改造,使之適合于自身在胞內(nèi)生存。 為了對蛋白質(zhì)譜鑒定的結(jié)果進(jìn)一步確認(rèn),我們選取HSP27進(jìn)行驗(yàn)證。Western blot顯示,在被輪狀病毒W(wǎng)a株感染12后,MA104細(xì)胞HSP27表達(dá)顯著上升。用SA11感染MA104,同樣可發(fā)現(xiàn)HSP27表達(dá)明顯上調(diào),這可能是由于質(zhì)譜鑒定的原因未能將該點(diǎn)鑒定出來,因?yàn)镾A11感染組選取的蛋白點(diǎn)中,有兩個(gè)點(diǎn)與Wa株感染組中兩個(gè)鑒定為HSP27的點(diǎn)的位置相對應(yīng)。換用HT-29細(xì)胞作為這兩種毒株的感染宿主細(xì)胞時(shí),發(fā)現(xiàn)HT-29細(xì)胞HSP27表達(dá)量顯著低于MA104細(xì)胞,且輪狀病毒感染不能誘導(dǎo)其增加表達(dá),說明HSP27在輪狀病毒感染中的上調(diào)表達(dá)可能是宿主細(xì)胞特異性的,提示其在輪狀病毒感染中可能存在重要作用。其它的蛋白點(diǎn)則有待于進(jìn)一步確證。
[Abstract]:Rotavirus infection is the main cause of infantile diarrhea worldwide,.MA104 cells are ideal host cells for studying the biology of rotavirus infection. Changes in gene expression of host cells after rotavirus infection have been studied by means of RT-PCR, Northern blotting and cDNA microarrays. In order to reveal the pathogenic mechanism of rotavirus and the response characteristics of the host cell to rotavirus infection, we studied the changes in the intracellular protein expression profiles before and after the rotavirus infection. First, we established the MA104 cell model of rotavirus infection. Our representative selection is not spittle. Liquid acid (SA) dependent human rotavirus Wa strain and sialic acid dependent monkey rotavirus SA11 strain were observed from the ability to infect MA104 cells, intracellular transcriptional replication ability, and the ability to induce apoptosis and necrosis. The appropriate conditions for the rotavirus infection of MA104 cells were determined: the rotavirus Wa strain was MOI=2, SA11 strain infected MA with MOI=8. The 104 cell 12h, which can not only guarantee high infection rate, but also makes the host cell pathological changes not too serious, the cell structure remains intact, which is the ideal choice for the study of the protein expression profile after infection.
In order to analyze the changes in the protein expression profiles of host cells after infection, IEF was made with 13cm pH3-11NL IPG prefabricated glue strips. The protein expression of rotavirus and simulated infected MA104 cells was detected by two-dimensional electrophoresis. 772 and 798 protein spots were detected in the infection group of.Wa strain, and 1230 and 1312 eggs were detected in SA11 infection group respectively. By image analysis and the comparison between the image and the image, 3 times were repeated, and the Wa infection group was found. After infection 12h, the expression of 14 protein points was up to 2 times, and the expression of 37 protein points was down 50%. After the infection of 12h, the expression of 24 protein points was up 2 times up, and the expression of 29 protein points was down 50%. In the protein spots of the Wa, only 2 in the infection group and the SA11 infection group were found to be close to the isoelectric point and the molecular weight, suggesting that it might be the same protein. In view of the infection of rotavirus, the expression of cell protein is generally reduced. So we chose to use the protein points expressed after infection to identify proteins.
In order to identify the protein expressed in the host cells after the rotavirus infection, we used MALDI-TOF-MS technology and retrieved the database using the NCBInr database through mascot. Considering that the MA104 cells are the source of the ape, we may also have the protein of the virus in the selected protein. We searched each point at the same time. Two libraries of primates and viruses, rotavirus wa infection group, 11 protein points and 7 positive results, excluding the characteristics of protein species, two of which are the same protein, the other are unnamed protein products (gi| 90085272), which are highly homologous to Zeta crystal protein (gi| 90085272), and theoretically protein (gi|114583139), cyclopentane mediated Exercise receptor (hyaluronan-mediated motility receptor (RHAMM)), rotavirus non structural protein NSP3, and keratin 8. rotavirus SA11 strain infected group, 16 protein spots were tested and 4 positive points were detected, except for the difference of species, including rotavirus non structural protein NSP3, plectin 1 isoform 6, cortactin-binding protein 2 and leucine-zipper-lik. E transcription regulator, 1 isoform 1 complex, complex of KRT8 protein and reticulocalbin 1. These proteins may be associated with inhibition of host protein expression, intracellular signaling, intracellular metabolism, viral replication and transport and development of host cells, indicating the transformation of host cells after the infection of rotavirus. Self living in the cell.
In order to further confirm the results of protein mass spectrometry identification, we selected HSP27 to verify.Western blot showing that the expression of HSP27 in MA104 cells increased significantly after the infection of rotavirus Wa strain 12. SA11 infection of MA104 also found that the HSP27 expression was obviously up-regulated. This may be caused by the identification of mass spectrometry. In the protein points selected for the SA11 infection group, two points were corresponding to the location of two HSP27 points in the Wa infection group. When HT-29 cells were used as the two host cells to infect the host cells, the HSP27 expression of HT-29 cells was significantly lower than that of the MA104 cells, and the rotavirus infection could not induce the increased expression of the HT-29 cells, indicating that HSP27 was in the wheel. The up-regulated expression of the virus in the virus may be specific to the host cell, suggesting that it may play an important role in the infection of rotavirus. Other protein points need to be further confirmed.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R725.1;R373

【共引文獻(xiàn)】

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本文編號：1968770