耐核糖核酸酶内含长片段嵌合体RNA的病毒样颗粒的构建和表达  

Construction and expression of ribonuclease-resistant virus-like particles containing long chimeric RNA sequence

作　　者：魏玉香[1] 张括[1] 魏葆珺[1] 王露楠[1] 张瑞[1] 李金明[1] 

机构地区：[1]卫生部北京医院卫生部临床检验中心,100730

出　　处：《中华检验医学杂志》2008年第3期280-286,共7页Chinese Journal of Laboratory Medicine

基　　金：欧盟第六框架（sp6）研究计划资助项目（Sp22-CT-2004-00383）;国家自然科学基金资助项目（30371365和30571776）;北京首都医学发展基金资助项目（2002-3041）

摘　　要：目的通过改变原噬菌体ms2包膜蛋白RNA包装位点（19碱基的茎环结构）的数量及亲和力，构建新的原核表达系统，探讨表达内含长片段（达到理论上的1900bp）RNA的耐RNase病毒样颗粒的可能性。方法首先设计含HindⅢ和NotⅠ酶切位点的引物，扩增ms2包膜蛋白的编码成熟酶蛋白和衣壳蛋白的1700bp序列，并将原来的19mer的包装位点序列改变为C-5变异体（19 bp stem-loop结构中-5位的尿嘧啶改变为胞嘧啶），HindⅢ和NotⅠ酶切后，与用同样酶切的表达载体pET-28（b）相连接，得到重组载体pET-ms2-pac。应用重叠PCR扩增3种病毒的5段嵌合体序列（包括3段SARS-CoV基因、一段HCV基因和一段H5N1基因），并在SARS-CoV3和HCV序列之间插入一个19mer的变异体包装位点序列，在设计引物时，使嵌合体两端含有NotⅠ酶切位点，与NotⅠ酶切的重组载体pET-ms2-pac相连接，构建得到具有2个变异包装位点的表达载体pET-ms2-3V。同时构建3种对照重组表达载体，分别测定N-P3V-pET-P、N-P3V-pET-C、P-3V-pET-P和pET-ms2-3 V 4种重组表达质粒表达产物的260nm吸光度（A260）值，根据公式A260=0．125mg／ml计算4种表达产物的表达效率。结果成功构建了4种原核表达载体：pET-ms2-3V、P-3V-pET-P、N-P3V-pET-P和N-P3V-pET-C。pET-ms2-3V和P-3V-pET-P经原核表达后得到含全长为1891的5段嵌合体RNA的病毒样颗粒；N-P3V-pET-P、N-P3V-pET-C其原核表达产物病毒样颗粒中仅包装了1200bp的目的嵌合体RNA。N-P3V-pET-P、N-P3V-pET-C、P-3V-pET-P和pET-ms2-3V的表达效率分别为0．23、0．35、0．35和0．51mg／ml。N-P3V-pET-C比N-P3V-pET-P表达效率高52％，而pET-ms2-3V比P-3V-pET-P表达效率高38％。所包装的RNA具有耐RNase和DNase消化的特性以及良好的不同温度条件下的稳定性。结论通过改变噬菌体ms2 RNA包装位点（19碱基的茎环结构）的数量，可构建能表达内含达到理论上的约1900bp外源RNA�Objective To construct and express ribonuclease-resistant virus-like particles containing long chimeric RNA sequence by changing quantity and affinity of ms2 19mer stem-loop. Methods 1.7 kb maturase and coat protein gene of ms2 were amplified. We used a C-variant aptamer of the wild-type stem-loop where uridine-5 has been substituted by cytosine. The 1 700 bp PCR-amplified DNA fragments was gel-purified and then digested with BamH Ⅰ and Hind Ⅲ and ligated to linearized pET28b vector to generate recombinant plasmid pET-MC-pac. The five target DNA sequences were spliced by overlapping extension（ including 3 fragment of SARS-CoV, one fragment of HCV and one fragment of H5N1 ）. PCR was carried out using primers contained Not Ⅰ site and then PCR were cloned into a pGEM T-easy vector and then excised with the NotⅠ restriction enzymes from the resulting recombinant plasmid, simoutaniously the pET-MC-pac plasmid was digested with NotⅠ restriction enzymes, fragments were ligated into the linearized pET-MC-pac plasmid to produce a new donor plasmids pET-MC-3V. Simoutaniously , we constructed three recombinant expression vector for control, including N-P3V-pET-P,N-P3V-pET-Cand P-3V-pET-P. N-P3V- pET-P contained one wild type pacsite located behind ms2 sequence; N-P3V-pET-C contained one C-variant pacsite located behind ms2 sequence; P-3V-pET-P contained two wild type pacsites that one is located behind ms2 sequence, another is located between SARS-CoV 3 and HCV sequence. Then these four expression vector were transformed into E. coli strain BL21 （DE3）, respectively. The 3V Armored RNA was expressed and purified. A260 absorbance value of expression product was determined. Results Four expression plasmids were constructed successfully. The Armored RNA with 1891 bases was successfully expressed by the two-pacsite expression system of pET-ms2-3V and P-3V-pET-P; for N-P3V-pET-P,N-P3V- pET-C, only 1 200 bp target sequence was packaged into virus-like particles. Expression efficiency of N- P3V-pET-P, N-P3V-pET-C,

关 键 词：轻病毒属 核糖核酸酶类 病毒粒子 病毒装配 病毒包膜蛋白质类 

分 类 号：R686[医药卫生—骨科学]