轮状病毒感染诱导MA104细胞感染相关蛋白的分析鉴定

发布时间：2018-06-02 12:30

本文选题：蛋白质组学 + 双向电泳　；参考：《第三军医大学》2007年硕士论文

【摘要】： 轮状病毒感染是全世界范围内婴幼儿腹泻的主要病因。MA104细胞是研究轮状病毒感染生物学的理想宿主细胞。对轮状病毒感染后宿主细胞基因表达的变化,通过RT-PCR、Northern blotting及cDNA微阵列的方法进行了很多研究,但是蛋白质才是生命活动的最终体现者。为了揭示轮状病毒的致病机理及宿主细胞对轮状病毒感染的反应特点,我们用蛋白组技术对轮状病毒感染前后胞内蛋白表达谱的变化情况进行研究。首先建立轮状病毒感染MA104细胞模型。我们代表性的选用非唾液酸(SA)依赖的人源轮状病毒Wa株和唾液酸依赖的猿猴轮状病毒SA11株,从对MA104细胞感染能力、胞内转录复制能力、诱导细胞凋亡坏死能力等方面进行观察,确定了轮状病毒感染MA104细胞的合适条件:轮状病毒Wa株以MOI=2,SA11株以MOI=8感染MA104细胞12h,既能保证较高的感染率,又使宿主细胞病变不过于严重,细胞结构保持完整,为进行感染后细胞蛋白表达谱研究的理想选择。为了分析感染病毒后宿主细胞蛋白表达谱的变化,用13cm pH3-11NL的IPG预制胶条做IEF,双向电泳分析感染了轮状病毒及模拟感染的MA104细胞蛋白谱表达情况。Wa株感染组分别可检测出772和798个蛋白点,SA11感染组分别可检测出1230和1312个蛋白点。通过图像分析及胶与图像的比对,重复3次,发现Wa株感染组,感染12h后,有14个蛋白点表达上调2倍以上,37个蛋白点表达下调50%以上;SA11株感染组,感染12h后,有24个蛋白点表达上调2倍以上,29个蛋白点表达下调50%以上。而在这些上调表达的蛋白点中,Wa株感染组与SA11感染组仅发现2个在等电点及分子量接近,提示可能为相同的蛋白。鉴于轮状病毒感染后会引起细胞蛋白表达的普遍下降,那些感染后表达反而上调的蛋白在轮状病毒于宿主细胞相互作用中似更有意义,所以我们选择在感染后表达上调的蛋白点用于蛋白鉴定。为了鉴定轮状病毒感染后宿主细胞内上调表达的蛋白质,我们采用MALDI-TOF-MS技术,并通过mascot用NCBInr数据库进行数据库检索。由于考虑到MA104细胞为猿猴来源,同时选取的蛋白中可能也会有病毒表达的蛋白质,我们对每个点同时搜索了灵长类和病毒两个库。轮状病毒wa株感染组,送检11蛋白点,检出阳性结果7个,排除蛋白质的种属特性,其中两个为同一蛋白HSP27,其它分别为与zeta晶体蛋白高度同源的未命名的蛋白产物(gi|90085272),理论上蛋白质(gi|114583139),环戊烷介导的运动受体(hyaluronan-mediated motility receptor (RHAMM)),轮状病毒非结构蛋白NSP3,角蛋白8。轮状病毒SA11株感染组,送检16个蛋白点,阳性点4个,除去种属差异性,分别为轮状病毒非结构蛋白NSP3,plectin 1 isoform 6, cortactin-binding protein 2与leucine-zipper-like transcription regulator, 1 isoform 1的复合物,KRT8 protein与reticulocalbin 1的复合物。这些蛋白可能与抑制宿主蛋白表达、胞内信号传递、胞内代谢、病毒复制与运输及宿主细胞发育分化相关,显示出轮状病毒感染后对宿主细胞的改造,使之适合于自身在胞内生存。为了对蛋白质谱鉴定的结果进一步确认,我们选取HSP27进行验证。Western blot显示,在被轮状病毒Wa株感染12后,MA104细胞HSP27表达显著上升。用SA11感染MA104,同样可发现HSP27表达明显上调,这可能是由于质谱鉴定的原因未能将该点鉴定出来,因为SA11感染组选取的蛋白点中,有两个点与Wa株感染组中两个鉴定为HSP27的点的位置相对应。换用HT-29细胞作为这两种毒株的感染宿主细胞时,发现HT-29细胞HSP27表达量显著低于MA104细胞,且轮状病毒感染不能诱导其增加表达,说明HSP27在轮状病毒感染中的上调表达可能是宿主细胞特异性的,提示其在轮状病毒感染中可能存在重要作用。其它的蛋白点则有待于进一步确证。
[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.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R725.1;R373

【共引文献】