| 摘要: |
| 【目的】建立一种利用CMV启动子获得拯救猪繁殖与呼吸综合征病毒（PRRSV）的方法，为进一步研究猪繁殖与呼吸综合征病毒提供操作平台。【方法】根据PRRSV的基因序列特征和CMV启动子的序列特征，对pBluescprict Ⅱ SK（+）载体进行多克隆序列改造；设计特异性引物，分6段扩增、组装出PRRSV的全长cDNA并扩增CMV真核启动子序列；采用酶切、连接、转化等方法将连接成功病毒的全长cDNA置于CMV真核启动子序列的下游，获得含有CMV启动子和猪繁殖与呼吸综合征病毒CH 1A株全长cDNA克隆的重组质粒pSK-CH-1A；再将重组质粒直接转染MARC 145宿主细胞，得到猪繁殖与呼吸综合征病毒CH-1A株病毒的拯救病毒。【结果】成功获得了19 112 bp含CH-1A全长cDNA的pSK-CH-1A载体，将其直接转染MARC-145宿主细胞后，经细胞体内转录合成病毒基因组RNA，获得了拯救病毒（rCH-1A）；病毒生长特性试验结果显示，拯救病毒与亲本病毒在MARC 145细胞上具有相似的增殖特性，且拯救病毒序列含有不同于亲本病毒的分子标记MluⅠ酶切位点。【结论】建立了一种方法简单，操作方便，节约时间和成本的PRRSV感染性cDNA克隆的体外拯救方法。 |
| 关键词: 猪繁殖与呼吸综合征病毒 CMV启动子 感染性克隆 |
| DOI： |
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| 基金项目:国家自然科学基金项目(31402208)；河南省科技攻关项目（162102110037） |
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| Construction of infectious clone of porcine reproductive and respiratory syndrome virus using CMV promoter |
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XU Yanzhao,WANG Qing,HU Jianhe,et al
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| Abstract: |
| 【Objective】A method to rescue porcine reproductive and respiratory syndrome virus (PRRSV) using CMV promoter was established to provide operation platform to future study on PRRSV.【Method】According to the sequence characteristics of PRRSV and CMV promoter,multiple cloning sequences of pBluescprict Ⅱ SK(+) were modification.Virus full-length cDNA and CMV eukaryotic promoter sequences were then amplified by six pairs of specific designed primers.Enzyme digestion,connection,and transformation were used to recombinant plasmid pSK-CH-1A,which contained CMV promoter and virus full length cDNA downstream.Then,the recombinant plasmid was transfected into the host cell MARC-145 to obtain rescue virus for CH 1A strain of PRRSV.【Result】The transcription of viral genomic RNA was synthesized and the infectious virus was rescued after the full-length cDNA clone of pSK-CH-1A containing 19 112 bp was directly transfected into MARC-145 cells.The virus growth characteristics test showed that the rescued virus was similar to parental virus in MARC-145 cells and it could be distinguished from the parental by the mutant genetic marker of MluⅠ enzyme site.【Conclusion】A simple,convenient,time saving and low-cost method for constructing infectious cDNA clone of PRRSV was established. |
| Key words: porcine reproductive and respiratory syndrome virus（PRRSV） CMV promoter infectious clone |
