马立克氏病病毒糖蛋白B基因克隆及其在家蚕细胞中的表达

Gene Clone of the Glycoprotein B Antigen of Marek's Disease Virus (MDV) and Its Expression in Bombyx mori (Bm) Cells

将马立克氏病病毒（ＭＤＶ）ＧＡ株基因文库中的ＢａｍＨＩＩ３和Ｋ３克隆质粒分别用双酶切消化，获得２．８ｋｂ的ＢａｍＨＩ－ＳａｌⅠ片段和１．１ｋｂ的ＢａｍＨＩ－ＥｃｏＲＩ片段，然后将这２个片段定向克隆于载体ｐＵＲ２２２中，构建了含ＭＤＶ糖蛋白Ｂ（ｇＢ）基因的重组质粒。为了基因操作方便和高效表达，设计了１对带有酶切位点的引物，用ＰＣＲ扩增了ＭＤＶｇＢ基因部分片段，并按正确方向插入去磷酸化的载体质粒中，得到起始密码子前带有ＥｃｏＲＩ酶切位点的ＭＤＶｇＢ基因克隆，再将该基因插入去磷酸化的家蚕核型多角体病毒（ＢｍＮＰＶ）转移载体ｐＢＦ１４中，ＤＮＡ序列分析确证了克隆基因及阅读柜架的正确性。以重组转移载体与野生型ＢｍＮＰＶ共转染家蚕细胞，用有限稀释法点杂交结合空斑技术纯化重组病毒。用荧光抗体试验检查重组病毒感染的家蚕细胞。

The I 3 and K 3 plasmids from MDV strain GA genomic Bam HI library were digested with double enzyme so that a 2.8 kb Sal Ⅰ Bam HI fragment and a 1.1 kb Bam HI Eco RI fragment were obtained. A recombinant plasmid containing the gB gene of MDV was constructed by orientation cloning the two fragments into vector pUR222. In order to manipulate gene easily and expresstion effectively, a pair of primers with enzyme sites were designed. Partial gene of MDV gB was amplified by PCR, then inserted into dephosphorized vector plasmid in correct direction. As a result, a Eco RI site increased in front of start code of MDV gB gene. The gB gene excised with Eco RI from recombinant plasmid was cloned into deophosphorized BmNPV transfer vector pBF14. DNA sequencing demonstrated that the cloned gene and reading frame were all right. After co transfection of Bm cells with wild type BmNPV and recombinant transfer vector, recombinant viruses were selected by using combination of limit dilution dot hybridization and plaque techniques. Fluorescent antibody test indicated that on the surface of the plasma membrane and plasma of Bm cells infected with recombinant virus, a strong fluorescent reaction occurred.