HBV负链冗余序列区碱基组成对复制的影响

发布时间：2018-06-19 01:31

本文选题：乙型肝炎病毒 + 冗余序列　；参考：《重庆医科大学》2013年硕士论文

【摘要】：目的：观察乙型肝炎病毒（HBV）负链冗余序列区（r）碱基组成对复制及第三次模板转换的影响。方法：利用不依赖酶切和链接的分子克隆方法(RLIC)构建负链3'r、5'r单碱基突变或者双侧r区单碱基同时突变的HBV1.1倍体质粒，分别转染HEK293细胞，提取细胞内HBV复制中间体并用Southern Blot检测；各突变质粒分别与质粒PEGFP-N1共转染HEK293细胞，提取细胞总RNA，再用Northern Blot检测HBV RNA。结果：与野生型相比，3'r和至少部分5'r单碱基突变型的细胞内HBV DNA总量及rcDNA的量没有明显改变；双侧r区T1821G突变体导致细胞内各种形态的HBV DNA均显著降低,而双侧r区nt1820、nt1822、nt1823碱基同时突变不显著影响HBV DNA的复制水平；双侧r区T1821G突变体的pgRNA量为野生型的43.9％，而两种单侧T1821G突变体pgRNA转录水平与野生型相当。结论：负链3'r和至少部分5'r单个位置的碱基组成对HBV复制及第三次模板转换没有明显影响；双侧r区T1821G突变能显著降低HBVDNA复制水平，，这种效应具有位置和碱基特异性；双侧r区T1821G突变降低HBV DNA复制的效应至少部分可由pgRNA转录水平降低来解释，而该种突变究竟通过何种机制导致pgRNA减少有待进一步研究。目的：观察HBV中（M）蛋白N末端序列对S结构域包装功能的影响。方法：利用不依赖酶切和链接的分子克隆方法(RLIC)构建M蛋白和S蛋白表达缺失的HBV1.1倍体质粒，利用酶切连接的方法构建表达M蛋白、S蛋白、N末端不同程度截短的M蛋白的质粒；将突变质粒和表达质粒共转染HepG2细胞，用Southern Blot检测培养上清中是否存在病毒复制中间体。结果：成功构建了M蛋白和S蛋白表达缺失的HBV1.1倍体质粒，表达M蛋白、S蛋白、N末端不同程度截短的M蛋白的质粒；在不能表达M蛋白和S蛋白的HBV培养上清中检测到病毒复制中间体，与在反式提供S蛋白后培养上清的病毒复制中间体的检测量相当。结论：核心颗粒可以不依赖外膜蛋白而释放出胞；我们需要建立分离完整病毒颗粒的免疫沉淀方法，来检测细胞培养上清中是否含有完成外膜包装的病毒颗粒。
[Abstract]:Aim: to observe the effect of the base composition of the negative redundancy region of hepatitis B virus (HBV) on replication and third template conversion. Methods: HBV1.1 times mass fragments were constructed by using RLIC-independent molecular cloning method with either negative chain 3 rrrr1 r1 or double r region single base mutation, respectively transfected into HEK293 cells, and HBV replication intermediates were extracted from the cells and detected by Southern Blot. The results were as follows: (1) the HBV1.1 times were transfected into HEK293 cells, and HBV replicating intermediates were extracted from the cells and detected by Southern blot. The mutant plasmids were cotransfected with PEGFP-N1 into HEK293 cells, and the total RNAs were extracted. HBV RNAs were detected by Northern blot. Results: compared with wild type, the total amount of HBV DNA and the amount of rcDNA in the cells of T1821G mutant and at least some of the 5kr single base mutants were not significantly changed, while the T1821G mutants of bilateral r region resulted in a significant decrease of HBV DNA in various forms of cells, and no significant changes were found in the total amount of HBV DNA and the amount of rcDNA in the cells. However, the simultaneous mutation of nt1820nt1822 and nt1823 in bilateral r region did not significantly affect the replication level of HBV DNA, while the pgRNA content of T1821G mutant in bilateral r region was 43.9 of that of wild type, while the transcription level of pgRNA in two single-sided T1821G mutants was similar to that of wild type. Conclusion: there is no significant effect on HBV replication and third template conversion in the single site of negative chain 3r and at least part of 5r.T1821G mutation in bilateral r region can significantly reduce the level of HBV DNA replication, and this effect is location-specific and base-specific. The effect of bilateral r region T1821G mutation on reducing HBV DNA replication can be explained at least in part by the reduction of pgRNA transcription level, and the mechanism by which the mutation leads to the reduction of pgRNA remains to be further studied. Objective: to observe the effect of N-terminal sequence of MN protein on the packaging function of S domain in HBV. Methods: RLIC-independent molecular cloning method was used to construct 1. 1-fold mass of HBV with missing expression of M protein and S protein. Plasmids expressing M protein and S protein N terminal truncated M protein were constructed by enzyme digestion. The mutant plasmids and expression plasmids were co-transfected into HepG2 cells, and Southern blot was used to detect the presence of viral replication intermediates in the supernatants. Results: the plasmids containing 1. 1 times of M protein and 1. 1 times of S protein were successfully constructed, and the plasmids expressing M protein with truncated N end of M protein S protein in different degree were constructed. Viral replication intermediates were detected in HBV supernatants which could not express M protein and S protein, which was similar to that of viral replication intermediates in the culture supernatants after S protein was provided. Conclusion: the core particles can release cells independently of outer membrane proteins, and we need to establish an immunoprecipitation method for the isolation of intact viral particles to detect whether there are viral particles in the supernatant of cell culture that complete the outer membrane packaging.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R512.62

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