应用聚合酶链反应克隆马立克氏病病毒A抗原基因  

MOLECULAR CLONING OF MAREK'S DISEASE VIRUS A ANTIGEN GENE WITH POLYMERASE CHAIN REACTION

作　　者：杨宝华 张鹰[2] 陈溥言[3] 王启松[2] 蔡宝祥[3] 

机构地区：[1]深圳动植物检疫局,518001 [2]复旦大学 [3]南京农业大学,南京210014

出　　处：《病毒学报》1993年第1期73-77,共5页Chinese Journal of Virology

摘　　要：根据马立克氏病病毒(MDV)A抗原基因的头尾DNA序列,设计并人工合成了两套用于聚合酶链反应(PCR)的引物(PCR PⅠ和PCR PⅡ、PCR PⅡ和PCR PⅢ)。以pBSA2.2k为模板DNA进行体外聚合酶链反应,选择性地使两引物间DNA片段扩增。将扩增的DNA片段纯化、酶切后与pBS载体连接克隆,经酶切分析得到能分别适用于真核和原核表达系统的MDV A基因。然后将用于原核表达的A抗原基因分步克隆于M13mp18,分别得到M13A0.4、正反向M13A1.0克隆。用双脱氧法进行DNA序列分析,在1408bp处比Causscns原文少1个A,1475bp处多1个A,但与Binus报道的一致。为此将pBSA2.2k质粒中EcoRI-NcoI片段(包含上述两位点)克隆于M13mp18测序,获得相同结果。Two pairs of primers used in polymerase chain reaction(PCR),PCR PI and PCR P I,PCR P! and PCR PI ,were designed and synthesized on the ground of DNA sequence of Marek's disease virus ( MDV ) A gene termini reported by Caussens. The desired MDV A antigen DNA frag-ment respectively for prokaryotic (1.4kb)and eukaryotic(1.5kb) expres-sion were obtained by PCR. After ligating A gene to pBS vector and transformed to E.coli JM109, we obtained pBSA 1.4k and pBSA 1.5k clones. Then, the 1.4kb MDV A antigen gene was subcloned into M13 mpl8 and sequenced. The result showed that i 408bp site ( deletion of one base A ) and l 475bp site ( an additional base A ) are different from Caussens' report, but consistent with Binns' report. Sequencing of Eco RI-NcoI DNA fragment ( including the two sites above ) from pBSA 2.2k which originated from MDV GA strain DNA fragments library, confir-med the above result. Thus, MDV A antigen gene should be 1440bp long, encoding 480 amino acids including the signal peptide.

关 键 词：A抗原基因 分子克隆 马立克病病毒 

分 类 号：S852.65[农业科学—基础兽医学]