舞毒蛾核型多角体病毒多角体蛋白基因的克隆、测序和表达

从东北林业大学实验林场采集并纯化了舞毒蛾核型多角体病毒（LdMNPV-NEFU)。用蛋白酶K消化，提取了病毒基因组DNA。用PCR方法，克隆出了该病毒的多角体蛋白（polyhedrin)基因，并对该基因进行了序列测定。结果显示，该基因序列是一个含有735个碱基对的开放阅读框（ORF），该阅读框编码245个氨基酸。有5对碱基与加拿大病毒株LdMNPV-G的多角体蛋白基因序列存在差异。LdMNPV-NEFU分离株的多角体蛋白基因的第54，109，379，508和701位（从起始密码子中的A开始）分别是C，G，T，C和G，而LdMNPV-G分离株的多角体蛋白基因（ORF）相应位置上的碱基分别是G，C，C，T和T，两个ORF编码的对应位置的氨基酸绝大多数相同，只有一对不同，即由LdMNPV-NEFU编码的天冬氨酸和由LdMNPV-G编码的对应位置的组氨酸。以质粒pT-7-7为载体，多角体蛋白基因在大肠杆菌BL21（DE3）菌株中进行了原核表达。

THE CLONING, SEQUENCING AND EXPRESSION OF THE LdMNPV POLYHEDRIN GENE

LdMNPV-NEFU isolate collected from the forestry farm of Northeast Forestry University was purified and the genomic DNA of LdMNPV was extracted. The LdMNPV polyhedrin gene was cloned by PCR. The results showed that the sequence was an open reading frame (ORF) of 735bp capable of encoding 245 amino acids. The polyhedrin gene sequences of the LdMNPV-NEFU isolate and a Canada strain, LdMNPV-G differed in 5 bases. The polyhedrin gene of the LdMNPV-NEFU isolate contained C, G, T, C and G at 54, 109,379, 508 and 701 sites from the start codon, but the LdMNPV-G isolate contained G, C, C, T and T at the corresponding sites respectively. The same amino acids were encoded by the two ORF sequences, with the exception that Asp and His are encoded by GAC on the polyhedrin gene sequence of the LdMNPV-NEFU isolate and by CAC in the LdMNPV-G isolate. The LdMNPV polyhedrin gene was expressed in E.coli BL21 (DE3) by the pT7-7 plasmid vector.