GPV VP1蛋白与宿主细胞蛋白SH3BP4互作的研究及其对GPV增殖的影响

发布时间：2018-06-25 04:50

本文选题：鹅细小病毒 + SH3BP4蛋白　；参考：《吉林农业大学》2017年硕士论文

【摘要】：鹅细小病毒属于细小病毒科依赖病毒属的成员。该病毒所引起的鹅传染病称为小鹅瘟。主要感染3-20日龄的雏鹅或雏番鸭,该病主要病理变化为全身败血性病变、纤维素性及渗出性肠炎,最后形成肠内栓塞。该病传播速度快,且具有高度传染性,病死率可高达95%以上,给我国水禽养殖业带来严重危害,造成了巨大的经济损失。该病主要通过带毒种鹅垂直传播给小鹅,因此,防止病毒的垂直传播是关键,研究鹅细小病毒治病机理,掌握病毒蛋白与宿主细胞蛋白之间的相互作用,对GPV的有效防控具有重要意义。本实验室在前期工作中,利用酵母双杂交技术筛选出3个与GPV-VP1蛋白相互作用的阳性克隆,其中的一个基因为479 bp,经过BLAST在线比对,与SH3BP4蛋白同源性为99%,为了进一步验证SH3BP4蛋白与VP1的相互作用,研究其对GPV增殖的影响,进行了本实验。PCR扩增完整的SH3BP4基因序列,构建原核表达载体pET-28aSH3BP4,IPTG诱导表达,纯化蛋白,产物进行SDS-PAGE分析,蛋白大小约为106kDa,同时,诱导表达实验室的pGEX-4T-1-VP1质粒,应用GST-pull down技术,在体外验证了SH3BP4蛋白与VP1蛋白可以相互作用。构建真核表达质粒pcDNA-3.0-SH3BP4,转染DEF,同时GPV感染DEF,以此为实验组,设置3组对照,分别为pcDNA-3.0+GPV,lip+GPV,DEPC+GPV,在作用12 h和24 h后,用荧光定量PCR检测病毒核酸的拷贝数,分析结果得知,在细胞内,转染12 h,各组之间GPV核酸拷贝数差异不明显,转染24 h,对照组的拷贝数是实验组的4倍,由此表明,SH3BP4在细胞内抑制了GPV的增殖;将纯化的SH3BP4蛋白以不同体积与GPV在细胞外感作,同时设置GPV与牛血清白蛋白为对照组,感染DEF,24 h后,荧光定量PCR检测GPV的核酸拷贝数,分析结果得知,随着蛋白量增加,GPV拷贝数逐渐降低。由此说明,在细胞外,SH3BP4蛋白抑制了GPV的增殖。
[Abstract]:Goose parvovirus is a member of the dependent genus of the parvoviridae. The goose infection caused by the virus is called goose plague. The main pathological changes were systemic sepsis, cellulose enteritis and exudative enteritis. The disease spreads rapidly and is highly infectious, and the mortality rate can be as high as 95%, which brings serious harm to the waterfowl breeding industry in China and causes huge economic losses. The disease is mainly transmitted to goslings vertically by the goose carrying virus. Therefore, preventing the vertical transmission of the virus is the key to study the therapeutic mechanism of goose parvovirus and to understand the interaction between virus protein and host cell protein. It is of great significance for the effective prevention and control of GPV. In our previous work, three positive clones interacting with GPV-VP1 protein were screened by yeast two-hybrid technique. The homology between SH3BP4 protein and SH3BP4 protein was 99. In order to further verify the interaction between SH3BP4 protein and VP1 protein and study the effect of SH3BP4 protein on GPV proliferation, we amplified the complete SH3BP4 gene sequence by PCR, constructed the prokaryotic expression vector pET-28aSH3BP4IPTG, and purified the protein. SDS-PAGE analysis showed that the protein size was about 106kDa. meanwhile, the plasmid pGEX-4T-1-VP1 was induced and expressed in the laboratory. The interaction between SH3BP4 protein and VP1 protein was confirmed by GST-pull down technique in vitro. Eukaryotic expression plasmid pcDNA-3.0-SH3BP4 was constructed and transfected with DEF. and GPV was infected with DEF. as experimental group, three groups of control groups were set up: pcDNA-3.0 GPVlip GPV-DEPC GPVP GPV.After 12 h and 24 h exposure, the copy number of viral nucleic acid was detected by fluorescence quantitative PCR. At 12 h after transfection, there was no significant difference in the number of GPV nucleic acid copies among the three groups. After 24 h transfection, the copy number of the control group was 4 times that of the experimental group, which indicated that SH3BP4 inhibited the proliferation of GPV in the cells. The purified SH3BP4 protein was treated with different volumes and GPV in vitro, and GPV and bovine serum albumin (BSA) were used as control group. After infected with DEF4 for 24 h, the nucleic acid copy number of GPV was detected by fluorescence quantitative PCR. The copy number of GPV decreased with the increase of protein content. Therefore, SH3 BP4 protein inhibited the proliferation of GPV.
【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.65

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