乙肝病毒核心抗原二聚体形成相关氨基酸序列鉴定

发布时间：2018-07-04 23:24

本文选题：核心蛋白 + 二聚体　；参考：《重庆医科大学》2017年硕士论文

【摘要】：目的:乙肝病毒核心颗粒的基本构成单位是核心蛋白(HBc)二聚体,与二聚体形成相关的核心蛋白一级结构尚未被充分鉴定,造成该现状的一个重要原因,是缺乏一种简便有效的表征HBc二聚体形成的方法,本研究的目的,是建立一种可用于反映HBc二聚体形成的新方法,并用该方法来鉴定与乙肝病毒核心蛋白(HBc)二聚体形成相关的重要氨基酸序列。方法:我们首先构建了一种可反映乙肝病毒核心蛋白二聚体形成新方法,该方法主要基于分段的海肾荧光素酶(Rluc)互补原理。将Rluc基因分为N(1-229aa),C(229-311aa)两段,分别与核心蛋白基因通过长片段接头融合,当HBc二聚体形成时,这两段可通过互补部分恢复荧光素酶活性,因而该酶活性可以间接反映二聚体的形成情况。然后,我们构建一系列乙肝病毒核心蛋白缺失突变体,以鉴定对HBc二聚体形成较为重要的氨基酸序列。最后,在3x Flag-HBC系统上对某些缺失突变体是否能形成capsid进行了研究。结果:1.利用Golden gate克隆方法,成功构建了一系列质粒,包括过渡载体质粒GG1,质粒Rluc N-HBc,Rluc C-HBc,阴性对照质粒Rluc N-d HBc和Rluc C-d HBc,突变质粒Rluc N-HBc127Q,Rluc C-HBc127Q,Rluc N-HBc132A,Rluc C-HBc132A,Rluc N-HBc42A,Rluc C-HBc42A,Rluc N-HBc23A,Rluc C-HBc23A;2.转染实验表明,Rluc N-HBc与Rluc C-HBc共转染HEK293细胞后,与三种对照组Rluc C-d HBC+Rluc N-d HBC,Rluc C-HBC+Rluc N-d HBC,Rluc C-d HBC+Rluc N-HBC相比,Rluc C-HBC+Rluc N-HBC所产生的荧光素酶活性高出25倍以上;3.各种核衣壳形成缺陷型突变体,包括Rluc N-HBC127Q+Rluc C-HBC127Q,Rluc N-HBC132A+Rluc C-HBC132A,Rluc N-HBC23A+Rluc C-HBC23A,以及Rluc N-HBC42A+Rluc C-HBC42A共转染,产生的光信号与野生型Rluc N-HBC+Rluc C-HBC相比,均没有显著降低;4.成功构建了覆盖HBc N端,C端以及5个а螺旋区域的总共30余种缺失突变体质粒(基于质粒Rluc C-HBc);5.以上突变体质粒分别于Rluc N-HBC共转染后,Rluc荧光素酶活性检测结果显示,C端覆盖122-183aa、N端覆盖1-5aa的缺失突变保留阳性对照50-80%的荧光素酶活性;N端覆盖6-41aa、C端103-120aa的缺失突变体则接近阴性对照水平;覆盖6-17、30-41、75-81、86-90、121-143的缺失突变均获得阳性对照80%以上的荧光素酶活性,覆盖26-29、61-65、71-75、117-120的缺失突变约为阳性对照的40%左右,而覆盖18-25、50-60、66-70、81-85、91-116的缺失突变为阳性对照的20%一下。6.3x Flag-HBCd81-85、d86-90能形成capsid,3x Flag-HBCd38-41d50-55、d91-95、d106-110不能形成capsid。结论:1.成功构建能反映乙肝病毒核心蛋白二聚体形成的分段海肾荧光素酶报告系统;2.序列50-55、91-95、106-110对乙肝病毒核心蛋白二聚体的形成是必须的;3.序列18-25、56-60、66-70、96-105、111-116对乙肝病毒核心蛋白二聚体的形成可能是重要的。
[Abstract]:Objective: the basic unit of hepatitis B virus core particles is the core protein (HBc) dimer. The primary structure of the core protein related to the formation of the core protein has not been fully identified, which is an important reason for the present situation. The aim of this study is to establish a new method for reflecting the formation of HBc dimer. This method was used to identify the important amino acid sequences related to the formation of hepatitis B virus core protein (HBc) dimer. Methods: we first constructed a new method which can reflect the formation of hepatitis B virus core protein dimer, which is mainly based on the piecewise sea kidney luciferase (Rluc) complementary principle. The Rluc gene was divided into N (1-229aa) C (229-311aa) segments and fused with the core protein gene through long fragments respectively. When HBc dimer was formed, the luciferase activity could be recovered by complementary part. Therefore, the enzyme activity can indirectly reflect the formation of dimer. Then, we constructed a series of HBV core protein deletion mutants to identify the amino acid sequence of HBc dimer. Finally, we studied whether some deletion mutants can form capsid on 3x Flag-HBC system. The result is 1: 1. A series of plasmids were successfully constructed by using Golden gate cloning method, including G1, Rluc N-HBc+ Rluc C-HBc, Rluc N-d HBc and Rluc C-d HBc, and Rluc N-HBc127QN Rluc C-HBc122A, Rluc C-HBc132An Rluc N-HBc42An Rluc N-HBc23Ac, Rluc N-HBc23Ac and Rluc C-HBc23Ac. The transfection experiment showed that Rluc N-HBc and Rluc C-HBc co-transfected HEK293 cells, the luciferase activity of Rluc C-HBC Rluc N-HBC N-HBC Rluc C-HBC N-HBC Rluc N-HBC Rluc N-HBC was more than 25 times higher than that of Rluc C-HBC Rluc N-HBC N-HBC after co-transfection with Rluc C-HBc and Rluc C-HBc, and the luciferase activity of Rluc C-HBC Rluc N-HBC N-HBC was more than 25-fold higher than that of Rluc C-HBC N-HBC N-HBC. All kinds of nucleocapsid defective mutants, including Rluc N-HBC127Q Rluc C-HBC127Q, Rluc N-HBC132A Rluc C-HBC132A Rluc N-HBC23A Rluc C-HBC23A, and Rluc N-HBC42A Rluc C-HBC42A co-transfected, produced no significant reduction of light signal compared with wild-type Rluc N-HBC Rluc C-HBC. A total of more than 30 deletion mutants (based on plasmid Rluc C-HBc) 5 were successfully constructed covering the N-terminal C terminal and 5 helical regions of HBc. The results of detection of luciferase activity in Rluc N-HBC cotransfection showed that deletion mutants covering 122-183aaAU N-terminal 1-5aa of the positive control group retained 50-80% of the luciferase activity N terminal covering 6-41aaAU C-terminal 103-120aa deletion mutants. It was close to the level of negative control. More than 80% of luciferase activity was obtained in all the deletion mutants covering 6-17730-41-81-81-86-90121-143, and the deletion mutation covering 26-2961-61-61-75117-120 was about 40% of that of the positive control, while the deletion mutation covering 18-25A1-6066-7086-891-116 was 20% of the positive control. 6.3x Flag-HBCD81-85d86-90 could form capsidan3x Flag-HBCD38-41d50-5d95d106-110. Conclusion 1. To successfully construct a segmented sea kidney luciferase reporting system which can reflect the formation of hepatitis B virus core protein dimer. The formation of core protein dimer of hepatitis B virus (HBV) is essential to the formation of the core protein of hepatitis B virus (HBV) by sequence 50-55 91-95106-110. Sequence 18-2556-60 66-70 96-105111-116 may be important for the formation of hepatitis B virus core protein dimer.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R373

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