摘要
应用RT PCR、NestedPCR和Half nestedPCR技术从实验感染兔脾组织的总RNA中得到了覆盖猪瘟病毒(CSFV)C 株全基因组的7个cDNA重叠片段,分别克隆于pMD18 T或pGEM TEasy载体后进行测序。运用T A克隆和多片段一步连接法,将cDNA重叠片段F1、F2、F3、F4与F5、F6、F7分别克隆到pGEM 5Zf(+)载体后,得到两个半长cDNA亚克隆重组质粒pGEM 5Zf(+)/F1 4和pGEM 5Zf(+)/F5 7,再将两个半长cDNA重叠片段连接并克隆到pGEM 5Zf(+)载体,构建出了CSFVC 株全长cDNA重组质粒pGEM 5Zf(+)/F1 7。全长cDNA的成功构建为进一步研究CSFV分子生物学提供了良好的工具。
Utilizing the RTPCR,nested and halfnested PCR techniques,7 overlaping cDNA fragments covering the full genome of Cstrain were amplified from total RNA extracted from spleens of experimentally infected rabbits.These fragments were sequenced and spliced into fulllength cDNA after being cloned into pMD18T or pGEMT Easy vectors.Skillfully using TA cloning technique and theory of onestep multifragment ligation,halflength cDNA,recombinant plasmids pGEM5Zf(+)/F14 was obtained when cDNA overlapping fragments F1,F2,F3,F4 were cloned into pGEM5Zf(+),and pGEM5Zf(+)/F57 when cDNA fragments F5,F6,F7 were cloned into pGEM5Zf(+).Then the fulllength cDNA pGEM5Zf(+)/F17 of CSFV Cstrain were obtained when the target fragments in recombinant plasmids pGEM5Zf(+)/F14 and pGEM5Zf(+)/F57 were cloned into pGEM5Zf(+).Successful construction of fulllength cDNA provided an excellent tool for further study on CSFV molecular biology.
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2003年第5期490-493,共4页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
国家重点基础研究发展规划(G19990119)
