马立克氏病病毒(MDV)糖蛋白D基因的分离克隆、定序及其在大肠杆菌中的表达

Amplification, Cloning, Sequencing and Expression of Marek′s Disease Virus Glycoprotein D gene

用聚合酶链式反应（ＰＣＲ）技术，从马立克氏病病毒（ＭＤＶ）ＧＡ株感染的鸡胚成纤维细胞（ＣＥＦ）基因组ＤＮＡ中扩增出ＭＤＶ糖蛋白Ｄ（ｇＤ）抗原基因１２０９ｂｐ编码序列。将该ＰＣＲ扩增产物于ＥｃｏＲＩ和ＫｐｎⅠ位点克隆进ｐＵＣ１８质粒载体中。将ｇＤ重组ｐＵＣ１８质粒ＤＮＡ用Ｄｉｇｏｘｉｇｅｎｉｎ（Ｄｉｇ）标记后，在Ｓｏｕｔｈｅｒｎｂｌｏｔ中，该探针能识别ＭＤＶ基因组ＤＮＡ的ＢａｍＨＩ－Ａ克隆中的Ａ片段ＤＮＡ（含ｇＤ全基因）。重组质粒序列分析结果表明，ＭＤＶＧＡ株ｇＤ与ＭＤＶＲＢ１Ｂ株ｇＤ在ＤＮＡ和氨基酸序列组成上略显差异。将ｇＤ基因从重组的ｐＵＣ１８质粒中切出，插入表达性质粒ｐＥＺＺ１８载体中的葡萄球菌Ａ蛋白（ＳＰＡ）的信号肽基因的下游。对含ｇＤ重组ｐＥＺＺ１８质粒插入序列进行部分测序，结果表明，ｇＤ阅读框与ｐＥＺＺ１８中ＳＰＡ信号肽的阅读框是吻合的。ｇＤ重组ｐＥＺＺ１８质粒在大肠杆菌中以融合蛋白的形式表达ｇＤ，即ｇＤ与ＳＰＡ的信号肽（１４０００）相连。根据ＳＰＡ的信号肽可与ＩｇＧ结合的特性，用兔ＩｇＧ结合的Ｓｅｐｈａｒｏｓｅ４Ｂ制备的亲和凝胶柱来纯化表达的ｇＤ融合蛋白。纯化的表达产物经ＳＤＳ－ＰＡＧＥ鉴定后，?

Marek′s Disease Virus (MDV) glycoprotein D (gD) gene was amplificated from Chicken Embryo Fibroblast (CEF) genomic DNA infected with GA strain MDV by polymerase chain reaction (PCR). PCR product of the gD gene was labeled with digoxigenin (dig) as probe, which could only detect the MDV genomic DNA Bam HIA fragment DNA from the MDVCEF genomic DNA, but not from the uninfected CEF DNA by dot. PCR product of the gD gene was cloned into pUC18 plasmid, and recombinant plasmid was screened by in situ hybridization with the probe of gD PCR product. The recombinant plasmid was also labeled with dig as probe, which could detect gD PCR product of MDV genomic DNA Bam HIA fragment DNA by hybridization in Southern blot. The recombinant plasmid was sequenced, showing GA MDV gD gene to be 1 209 nucleotides long encoding 403 amino acids (45 000). GA MDV gD gene cleaved off from recombinant plasmid was cloned into pEZZ18 protein A gene fusion vector. The gD was expressed as fusion protein linked with "ZZ" domains of protein A (14 000). The "ZZ" domains tightly bound by IgG sepharose 4B, enable onestep purificatin of the expressed proteins, its molecular weight being approximately 58 000 by SDSPAGE.