本文選題：核心蛋白 + 二聚體　； 參考：《重慶醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:乙肝病毒核心顆粒的基本構(gòu)成單位是核心蛋白(HBc)二聚體,與二聚體形成相關(guān)的核心蛋白一級(jí)結(jié)構(gòu)尚未被充分鑒定,造成該現(xiàn)狀的一個(gè)重要原因,是缺乏一種簡(jiǎn)便有效的表征HBc二聚體形成的方法,本研究的目的,是建立一種可用于反映HBc二聚體形成的新方法,并用該方法來鑒定與乙肝病毒核心蛋白(HBc)二聚體形成相關(guān)的重要氨基酸序列。方法:我們首先構(gòu)建了一種可反映乙肝病毒核心蛋白二聚體形成新方法,該方法主要基于分段的海腎熒光素酶(Rluc)互補(bǔ)原理。將Rluc基因分為N(1-229aa),C(229-311aa)兩段,分別與核心蛋白基因通過長(zhǎng)片段接頭融合,當(dāng)HBc二聚體形成時(shí),這兩段可通過互補(bǔ)部分恢復(fù)熒光素酶活性,因而該酶活性可以間接反映二聚體的形成情況。然后,我們構(gòu)建一系列乙肝病毒核心蛋白缺失突變體,以鑒定對(duì)HBc二聚體形成較為重要的氨基酸序列。最后,在3x Flag-HBC系統(tǒng)上對(duì)某些缺失突變體是否能形成capsid進(jìn)行了研究。結(jié)果:1.利用Golden gate克隆方法,成功構(gòu)建了一系列質(zhì)粒,包括過渡載體質(zhì)粒GG1,質(zhì)粒Rluc N-HBc,Rluc C-HBc,陰性對(duì)照質(zhì)粒Rluc N-d HBc和Rluc C-d HBc,突變質(zhì)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.轉(zhuǎn)染實(shí)驗(yàn)表明,Rluc N-HBc與Rluc C-HBc共轉(zhuǎn)染HEK293細(xì)胞后,與三種對(duì)照組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所產(chǎn)生的熒光素酶活性高出25倍以上;3.各種核衣殼形成缺陷型突變體,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共轉(zhuǎn)染,產(chǎn)生的光信號(hào)與野生型Rluc N-HBC+Rluc C-HBC相比,均沒有顯著降低;4.成功構(gòu)建了覆蓋HBc N端,C端以及5個(gè)а螺旋區(qū)域的總共30余種缺失突變體質(zhì)粒(基于質(zhì)粒Rluc C-HBc);5.以上突變體質(zhì)粒分別于Rluc N-HBC共轉(zhuǎn)染后,Rluc熒光素酶活性檢測(cè)結(jié)果顯示,C端覆蓋122-183aa、N端覆蓋1-5aa的缺失突變保留陽性對(duì)照50-80%的熒光素酶活性;N端覆蓋6-41aa、C端103-120aa的缺失突變體則接近陰性對(duì)照水平;覆蓋6-17、30-41、75-81、86-90、121-143的缺失突變均獲得陽性對(duì)照80%以上的熒光素酶活性,覆蓋26-29、61-65、71-75、117-120的缺失突變約為陽性對(duì)照的40%左右,而覆蓋18-25、50-60、66-70、81-85、91-116的缺失突變?yōu)殛栃詫?duì)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。結(jié)論:1.成功構(gòu)建能反映乙肝病毒核心蛋白二聚體形成的分段海腎熒光素酶報(bào)告系統(tǒng);2.序列50-55、91-95、106-110對(duì)乙肝病毒核心蛋白二聚體的形成是必須的;3.序列18-25、56-60、66-70、96-105、111-116對(duì)乙肝病毒核心蛋白二聚體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.
【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】：2017
【分類號(hào)】：R373

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 甘春楊;劉亞;羅英英;張文露;黃愛龍;蔡雪飛;胡接力;;一種適用于片段替換/插入突變掃描的克隆方法[J];中國(guó)生物工程雜志;2016年08期

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本文編號(hào)：2097906