兔出血症病毒基因组末端序列对蛋白表达与调控的影响
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摘要
【目的与意义】目前，关于兔出血症病毒（Rabbit hemorrhagic disease virus，RHDV）的基因组结构、基因功能和致病机理等还了解甚少，而阐明此类问题则是有效防控RHD发生的前提。本论文首次对RHDV基因组末端序列的功能进行了系统研究和分析，为进一步阐明RHDV基因组的表达和调控机制以及致病机理等提供了有益线索。
【方法】首先，克隆了RHDV基因组的5’/3’末端序列（含非编码区non-codingregion，NCR)，然后将其插入双顺反子载体中，进行体外翻译，通过检测其下游报告基因（萤火虫荧光素酶基因）的表达，分析5’/3’末端序列对报告基因表达的调控作用；为了进一步验证5’/3’末端序列在细胞水平对报告基因表达的调控作用，又构建了含有5’/3’末端序列不同组合的单顺反子，然后转染细胞，通过检测荧光素酶的表达活性判断靶序列对报告基因表达的影响；为了从基因水平分析5’/3’末端序列对RHDV基因组表达的影响，先构建了携带LUC基因的RHDV复制子；在此基础上，又构建了缺失5’末端序列的RHDV复制子突变体，然后转染细胞，分析缺失突变对LUC表达的影响。
【结果】RHDV基因组的5’末端序列具有内部起始下游蛋白表达的功能，无论是体外翻译，还是细胞水平，置于RHDV5’末端序列下游的LUC都能得到良好的表达，而3’末端序列具有增强5’末端序列功能的作用，将其与5’末端序列串联，可以提高LUC的表达水平。此外，本论文还成功构建了携带LUC基因的RHDV复制子，将其转染细胞以后，LUC基因在12h就能得到表达，36h可以达到表达高峰；在该复制子的基础上，我们又分别构建了仅缺失5’NCR、缺失NSP1以及同时缺失5’NCR和NSP1的复制子突变体，为在基因水平上分析5’末端序列对RHDV复制和表达的影响奠定了坚实基础。
【结论】 RHDV基因组的5’/3’末端序列对蛋白的表达有增强作用，此外，5’末端序列中的5’NCR和NSP1序列是维持RHDV5’末端序列内部起始蛋白翻译功能的关键性区域。
【Objective】At present, we know less about the genome structure, function andpathogenic mechanism of Rabbit hemorrhagic disease virus（RHDV），which are the basis ofeffective control RHD. Therefore, this article is trying to explore the expression andregulation mechanism of RHDV genome through analysis the function of the extremesequence of RHDV genome, and hope to provide some useful clues for further exploring thetranslation mechanism of RHDV.
【Method and Result】In order to evaluate the translation initiation function of the5’endsequence of RHDV genome, a bicistronic reporter plasmid was constructed by the insertion ofa cDNA corresponding to the RHDV5’ extreme sequence (nts1to429) between two reportergene sequences, the first encoding CAT protein and the second encoding LUC protein. Thebicistronic plasmids were individually added to TNT rabbit reticulocyte lysate (RRL) system(Promega)，and then the reporter proteins translated in vitro were evaluated by measuringLUC activity. Internal initiation activity of the different sequences (5’/3’) included betweentwo reporters genes was estimated by the accumulation of LUC. Our results showed that allplasmids containing the RHDV5’ extreme sequence in sense orientation allowed theexpression of LUC, indicating that the5’ extreme sequence of RHDV could direct translationinitiation in vivo.
To confirm and extend the results from these assays in vitro， diffent types ofmonocistrons were constructed and tested by transient-expression experiments in mammaliancells. Our results showed that the5’extreme sequence of RHDV could produce efficient LUCexpression in host cells. Though the expression level of5’ end sequence of RHDV was lowerthan that of EMCV IRES clearly, these results still indicated that RHDV5’end sequence had translation initiation function in vivo.
To further characterize the regulation of3’ NCR on the function of RHDV genome,additional bicistronic reporter plasmids were constructed, containing RHDV3’NCR. Thebicistronic plasmids were individually added to TNT rabbit reticulocyte lysate (RRL) system(Promega)，and then the reporter proteins translated in vitro were evaluated by measuringLUC activity. Internal initiation activity of the different sequences included between the tworeporter genes was estimated by the expression of LUC. Moreover, the bicistronic plasmidswere transfected into vFT7infected BHK-21cells, in order to evaluate internal initiationactivity in vivo, and the expression of LUC protein expressed from the different constructswas determined through detecting LUC activity. Our results indicated that the presence of3’NCR was not required for translation initiation，but it indeed positively modulated thefunction of the5’ end sequence of RHDV.
To evaluate the influence of the5’/3’ extreme sequence on the expression of RHDVthrough gene level, the RHDV replicon carrying LUC gene was constructed, and then seriesreplicon mutants were also constructed. RK13cells were transfected with these repliconmutants, and the affection of deleted mutation on the expression of RHDV genome wasanalyzed through measuring LUC activity. The experimental results showed that5’endseqnence (429bp) took a key role during the expression of RHDV genome, which isconsistent to above results.
【Conclusion】RHDV5’ extreme sequence indeed positively regulated the expression ofLUC，and then，it could direct translation initiation in vivo.3’NCR was not required fortranslation initiation，but it indeed positively modulated the function of the5’ end sequence ofRHDV.5’NCR and NSP1sequence was the key sequence,which could matain translationinitiation function of the5’end sequence in vivo.
【方法】首先，克隆了RHDV基因组的5’/3’末端序列（含非编码区non-codingregion，NCR)，然后将其插入双顺反子载体中，进行体外翻译，通过检测其下游报告基因（萤火虫荧光素酶基因）的表达，分析5’/3’末端序列对报告基因表达的调控作用；为了进一步验证5’/3’末端序列在细胞水平对报告基因表达的调控作用，又构建了含有5’/3’末端序列不同组合的单顺反子，然后转染细胞，通过检测荧光素酶的表达活性判断靶序列对报告基因表达的影响；为了从基因水平分析5’/3’末端序列对RHDV基因组表达的影响，先构建了携带LUC基因的RHDV复制子；在此基础上，又构建了缺失5’末端序列的RHDV复制子突变体，然后转染细胞，分析缺失突变对LUC表达的影响。
【结果】RHDV基因组的5’末端序列具有内部起始下游蛋白表达的功能，无论是体外翻译，还是细胞水平，置于RHDV5’末端序列下游的LUC都能得到良好的表达，而3’末端序列具有增强5’末端序列功能的作用，将其与5’末端序列串联，可以提高LUC的表达水平。此外，本论文还成功构建了携带LUC基因的RHDV复制子，将其转染细胞以后，LUC基因在12h就能得到表达，36h可以达到表达高峰；在该复制子的基础上，我们又分别构建了仅缺失5’NCR、缺失NSP1以及同时缺失5’NCR和NSP1的复制子突变体，为在基因水平上分析5’末端序列对RHDV复制和表达的影响奠定了坚实基础。
【结论】 RHDV基因组的5’/3’末端序列对蛋白的表达有增强作用，此外，5’末端序列中的5’NCR和NSP1序列是维持RHDV5’末端序列内部起始蛋白翻译功能的关键性区域。
【Objective】At present, we know less about the genome structure, function andpathogenic mechanism of Rabbit hemorrhagic disease virus（RHDV），which are the basis ofeffective control RHD. Therefore, this article is trying to explore the expression andregulation mechanism of RHDV genome through analysis the function of the extremesequence of RHDV genome, and hope to provide some useful clues for further exploring thetranslation mechanism of RHDV.
【Method and Result】In order to evaluate the translation initiation function of the5’endsequence of RHDV genome, a bicistronic reporter plasmid was constructed by the insertion ofa cDNA corresponding to the RHDV5’ extreme sequence (nts1to429) between two reportergene sequences, the first encoding CAT protein and the second encoding LUC protein. Thebicistronic plasmids were individually added to TNT rabbit reticulocyte lysate (RRL) system(Promega)，and then the reporter proteins translated in vitro were evaluated by measuringLUC activity. Internal initiation activity of the different sequences (5’/3’) included betweentwo reporters genes was estimated by the accumulation of LUC. Our results showed that allplasmids containing the RHDV5’ extreme sequence in sense orientation allowed theexpression of LUC, indicating that the5’ extreme sequence of RHDV could direct translationinitiation in vivo.
To confirm and extend the results from these assays in vitro， diffent types ofmonocistrons were constructed and tested by transient-expression experiments in mammaliancells. Our results showed that the5’extreme sequence of RHDV could produce efficient LUCexpression in host cells. Though the expression level of5’ end sequence of RHDV was lowerthan that of EMCV IRES clearly, these results still indicated that RHDV5’end sequence had translation initiation function in vivo.
To further characterize the regulation of3’ NCR on the function of RHDV genome,additional bicistronic reporter plasmids were constructed, containing RHDV3’NCR. Thebicistronic plasmids were individually added to TNT rabbit reticulocyte lysate (RRL) system(Promega)，and then the reporter proteins translated in vitro were evaluated by measuringLUC activity. Internal initiation activity of the different sequences included between the tworeporter genes was estimated by the expression of LUC. Moreover, the bicistronic plasmidswere transfected into vFT7infected BHK-21cells, in order to evaluate internal initiationactivity in vivo, and the expression of LUC protein expressed from the different constructswas determined through detecting LUC activity. Our results indicated that the presence of3’NCR was not required for translation initiation，but it indeed positively modulated thefunction of the5’ end sequence of RHDV.
To evaluate the influence of the5’/3’ extreme sequence on the expression of RHDVthrough gene level, the RHDV replicon carrying LUC gene was constructed, and then seriesreplicon mutants were also constructed. RK13cells were transfected with these repliconmutants, and the affection of deleted mutation on the expression of RHDV genome wasanalyzed through measuring LUC activity. The experimental results showed that5’endseqnence (429bp) took a key role during the expression of RHDV genome, which isconsistent to above results.
【Conclusion】RHDV5’ extreme sequence indeed positively regulated the expression ofLUC，and then，it could direct translation initiation in vivo.3’NCR was not required fortranslation initiation，but it indeed positively modulated the function of the5’ end sequence ofRHDV.5’NCR and NSP1sequence was the key sequence,which could matain translationinitiation function of the5’end sequence in vivo.
引文
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