鸡痘病毒载体启动子的优化  

Construction and Selection of Strong Promoter for Fowlpox Virus Vector

作　　者：朱爱华[1] 彭大新[1] 刘秀梵[1] 张如宽[1] 

机构地区：[1]扬州大学农业部畜禽传染病学重点开放实验室,江苏扬州225009

出　　处：《病毒学报》2000年第4期347-351,共5页Chinese Journal of Virology

基　　金：国家863计划基金!(NO 96 0 332 0 2 5 ) ;江苏省应用基础研究基金!(BJ95 10 7) ;江苏省自然科学基金!(BK970 6 9);江苏省教委基金

摘　　要：要提高外源基因在鸡痘病毒 (FPV)载体的表达量 ,必须使用强启动子来控制表达。为构建较强的启动子组合 ,根据痘苗病毒天然启动子P7 5的结构 ,人工合成 4条寡核苷酸 ,形成一个早期启动子和一个晚期启动子 ,其首尾可相互连接。利用分子生物学常用实验方法把合成的启动子进行不同组合 ,最终形成 10个较有代表性的启动子组合PS1～ 10。在其下游插入大肠杆菌 β 半乳糖苷酶基因LacZ ,并进一步构建成鸡痘病毒转移载体 pFBS1～10 ,同时以P7 5作对照构建成 pFB7 5。以脂质体介导转染鸡痘病毒中国疫苗株 2 82E4感染 3h～ 4h后的原代鸡胚成纤维细胞 (CEF)单层。用蓝斑筛选法纯化病毒 4次 ,最终得到稳定的重组病毒。将纯化的重组病毒感染次代CEF ,分别于感染后 10h、2 4h、36h、48h、72h收获细胞 ,制备提取物 ,检测提取物中 β 半乳糖苷酶的活性。通过计算比较 ,筛选出其中较强的启动子组合LLEE ,其表达活性约为P7 5的 3 5倍 ,为进一步构建高效表达的鸡痘病毒通用载体打下了良好的基础。For constructing strong combined promoter to enhance the foreign gene expression in Fowlpox virus(FPV) vector, four oligonucleotides were synthesized according to the essential structure of the vaccinia virus nature promoter P7.5 and were used for generating early and late promoters. Ten artificial promoters designated PS1～10 were constructed by using different combination of early and late promoters, which were recognized by restriction endonuclease analysis and sequence analysis. LacZ gene transfer vectors of FPV with these promoters and P7.5 were generated by a series of molecular biology manipulation. DOSPER liposome mediated transfection with these transfer vectors was performed on chicken embryo fibroblast (CEF) monolayers, which was infected with FPV Chinese vaccine strain 282E4 3～4 hours earlier, to produce recombinant viruses. Recombinant FPVs with blue plaque were selected and purified 4 times on secondary CEF subcultures overlaid with agarose containing X Gal. The level of the β galactosidase expressed by these recombinant viruses with LacZ gene under the control of various synthetic promoters and P7.5 were assayed and compared in the presence or absence of cytosine arabinoside (Arc C) at 10, 24, 36, 48, 72 hours post infection respectively. The LLEE combination was the strongest synthetic promoter selected out which drived expression of β galactosidase three more times higher than the P7.5, and appeared to be valuable for the construction of more efficient expression FPV vector.

关 键 词：人工合成 强启动子 鸡痘病毒 重组病毒载体 

分 类 号：S858.312.6[农业科学—临床兽医学] Q782[农业科学—兽医学]