含SRSV U3区的重组Moloney小鼠白血病病毒的构建及其致病特性

GENERATION OF A RECOMBINANT MOLONEY MURINE LEUKEMIA VIRUS CONTAINING SRSV U3 REGION AND A STUDY ON ITS PATHOGENICITY

质粒pSF11含有小鼠白血病病毒(SRSV)基因组5kb Bam H Ⅰ片段。应用限制性内切酶分析及Southern印迹杂交技术,确定了长末端重复区(Long terminal repeats,LTR)在pSF11上的位置。pSF11经Bg1 Ⅱ酶解,Klenow酶补平5’粘性末端,碱性磷酸酯酶脱磷后与EcoR Ⅰ Linker连接,转化大肠杆菌C600,经EcoRⅠ酶切筛选得到重组子 pER 1。pER 1经ClaⅠ+EcoR Ⅰ酶切,电泳回收含SRSV LTR的2.55kbDNA,与p63-2(含Moloney小鼠白血病病毒(leukemia virus,M-MuLV)完整基因组)的ClaⅠ+EcoRⅠ14.1kbDNA连接,转化C600受体菌,得到一个M-MuLV 3’LTR被SRSV LTR取代的重组质粒pMS。纯化后转染NIH 3T3细胞,经XC合胞试验及逆转录酶活性测定证明,转染细胞含有M-MuLV与SR-SV的重组病毒MSV,将MSV经皮下接种到新生的NIH Swiss小鼠,通过对小鼠发病阶段血像、大体解剖以及组织病理学检查,发现该重组病毒MSV具有与SRSV极其相似的致病特征。

pSF11 is a pBR322 plasmid containing 5 kb BamH I fragment of mouse SRSV proviral DNA. pSFll was digested with Bgl II and its 5' cohesive ends were filled, then treated with CIP and ligated with EcoRI linker. The recombinant named pER1 was obtained. Ligation of the 2.55kb ClaI-EcoRI fragment containing SRSV LTR from pERl with the 14.1kb Clal-EcoRI fragment from p63-2 which is a pBR322 plasmid containing a complete M-MLV proviral integration gave the final construction, containing a SRSV 3' LTR and a M-MLV 5' LTR, named pMS. It was able to induce XC plaques and had a reverse transcriptase activity after transfection in unin-fected mouse fibroblasts. The recombined MSV was inoculated into newborn NIH Swiss mice subcutaneously.The disease caused by MSV is very similar to that of SRSV under the observation of blood smear, gross pathology and histopathology.