乙型脑炎病毒基因组cDNA克隆在宿主菌中不稳定因子的初步研究

PRELIMINARY STUDY ON GENETIC INSTABILITY OF JAPANESES ENCEPHALITIS VIRUS CDNA CLONE TRANSFORMED IN ESCHERICHIA COLI

为了探求乙型脑炎病毒（Japanese encephalitis virus，JEV）基因组cDNA克隆在宿主菌中不稳定遗传的影响因子，本研究以猪源JEV HEN0701株基因组片段1-2913 bp为研究对象，分析基因组原核启动子和相关毒力基因在不稳定遗传中的作用。在5＇端，分析片段中潜在的原核启动子序列，通过PCR扩增得到不同长度的cDNA片段；在3＇端，通过内切酶酶切截断基因组prM蛋白或E蛋白编码区，获得3＇末端不同的cDNA片段。将各片段克隆入pCR-BluntII-TOPO载体，转化大肠杆菌，于37℃条件下培养。结果显示，片段73-2913 bp、97-2913 bp、355-2913 bp及1-933 bp、1-1275 bp能够在转化菌中稳定遗传；含有其他片段39-2913 bp、54-2913 bp及1-1800 bp、1-1971 bp的重组细菌不能在37℃条件下生长；片段1-1600 bp虽然能够在转化菌中传代，但是出现不规律突变。以上结果表明：软件所预测各原核启动子片段中，基因组73-118 bp区域与cDNA克隆稳定性有关，且上游的54 bp至73 bp的序列对该区域启动子存在影响；在JEV基因组1275 bp至1600 bp之间可能存在毒性蛋白编码序列。

In the present study, a series of fragments from 1 to 2913 bp were generated from Japanese encephalitis virus （JEV） HEN0701genome in order to explore factors affecting genetic instability of cDNA clone when propagated in E. coli. These cDNA fragments were obtained using either PCR starting at 5＇ terminus or restriction enzyme digestion at different positiosn of 3＇ terminus. Each fragment was cloned into pCR-BluntII-TOPO vector and transformed into E. coli cultures, then were incubated at 37℃. Fragments 73 to 2913 bp, 97 to 2913 bp, 355 to 2913 bp and 1 to 933 bp, 1 to 1275 bp showed stability during passages of the transformed bacteria. However, recombinant bacteria containing fragments of 39 to 2913 bp, 54 to 2913 bp and 1 to 1800 bp, 1 to 1971 bp did not grow at 37℃. In addition, fragment 1 to 1600 bp of JEV genome was passaged in transformed bacteria but mutation occurred. The above results indicated that the fragment 73 to 118 bp affected the stability of JEV cDNA clone. The upstream sequences at 54 to 73 bp might be involve in this process as well. The results also suggest that toxic protein coding sequence might exist between 1275 to 1600 bp of JEV genome.