本文選題：乙型肝炎病毒 + 冗余序列��； 參考：《重慶醫(yī)科大學(xué)》2013年碩士論文

【摘要】：目的：觀察乙型肝炎病毒（HBV）負(fù)鏈冗余序列區(qū)（r）堿基組成對復(fù)制及第三次模板轉(zhuǎn)換的影響。 方法：利用不依賴酶切和鏈接的分子克隆方法(RLIC)構(gòu)建負(fù)鏈3'r、5'r單堿基突變或者雙側(cè)r區(qū)單堿基同時(shí)突變的HBV1.1倍體質(zhì)粒，分別轉(zhuǎn)染HEK293細(xì)胞，提取細(xì)胞內(nèi)HBV復(fù)制中間體并用Southern Blot檢測；各突變質(zhì)粒分別與質(zhì)粒PEGFP-N1共轉(zhuǎn)染HEK293細(xì)胞，提取細(xì)胞總RNA，再用Northern Blot檢測HBV RNA。 結(jié)果：與野生型相比，3'r和至少部分5'r單堿基突變型的細(xì)胞內(nèi)HBV DNA總量及rcDNA的量沒有明顯改變；雙側(cè)r區(qū)T1821G突變體導(dǎo)致細(xì)胞內(nèi)各種形態(tài)的HBV DNA均顯著降低,而雙側(cè)r區(qū)nt1820、nt1822、nt1823堿基同時(shí)突變不顯著影響HBV DNA的復(fù)制水平；雙側(cè)r區(qū)T1821G突變體的pgRNA量為野生型的43.9％，而兩種單側(cè)T1821G突變體pgRNA轉(zhuǎn)錄水平與野生型相當(dāng)。 結(jié)論：負(fù)鏈3'r和至少部分5'r單個(gè)位置的堿基組成對HBV復(fù)制及第三次模板轉(zhuǎn)換沒有明顯影響；雙側(cè)r區(qū)T1821G突變能顯著降低HBVDNA復(fù)制水平，，這種效應(yīng)具有位置和堿基特異性；雙側(cè)r區(qū)T1821G突變降低HBV DNA復(fù)制的效應(yīng)至少部分可由pgRNA轉(zhuǎn)錄水平降低來解釋，而該種突變究竟通過何種機(jī)制導(dǎo)致pgRNA減少有待進(jìn)一步研究。 目的：觀察HBV中（M）蛋白N末端序列對S結(jié)構(gòu)域包裝功能的影響。 方法：利用不依賴酶切和鏈接的分子克隆方法(RLIC)構(gòu)建M蛋白和S蛋白表達(dá)缺失的HBV1.1倍體質(zhì)粒，利用酶切連接的方法構(gòu)建表達(dá)M蛋白、S蛋白、N末端不同程度截短的M蛋白的質(zhì)粒；將突變質(zhì)粒和表達(dá)質(zhì)粒共轉(zhuǎn)染HepG2細(xì)胞，用Southern Blot檢測培養(yǎng)上清中是否存在病毒復(fù)制中間體。 結(jié)果：成功構(gòu)建了M蛋白和S蛋白表達(dá)缺失的HBV1.1倍體質(zhì)粒，表達(dá)M蛋白、S蛋白、N末端不同程度截短的M蛋白的質(zhì)粒；在不能表達(dá)M蛋白和S蛋白的HBV培養(yǎng)上清中檢測到病毒復(fù)制中間體，與在反式提供S蛋白后培養(yǎng)上清的病毒復(fù)制中間體的檢測量相當(dāng)。 結(jié)論：核心顆�？梢圆灰蕾囃饽さ鞍锥尫懦霭晃覀冃枰⒎蛛x完整病毒顆粒的免疫沉淀方法，來檢測細(xì)胞培養(yǎng)上清中是否含有完成外膜包裝的病毒顆粒。
[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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：R512.62

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 黃媛;陶穎;張文露;黃愛龍;胡接力;;一種新的DNA分子克隆方法[J];中國科學(xué):生命科學(xué);2011年09期

本文編號(hào)：2037733