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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 2 4 5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 5 4、10 9、379、5 0 8和 70 1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pT7 7为载体 ,多角体蛋白基因在大肠杆菌BL2 1(DE3)菌株中进行了原核表达。
Gypsy moth nuclear polyhedrosis virus (LdMNPV-NEFU) was collected and purified from experimental forestry farm of Northeast Forestry University. The viral genomic DNA was extracted by protease K digestion. The polyhedrin gene of the virus was cloned by PCR and the gene was sequenced. The results showed that the gene sequence was an open reading frame (ORF) with 735 base pairs, encoding 245 amino acids. Five pairs of bases differ from the polyhedrin gene sequences of the Canadian strain LdMNPV G. The polyhedrin gene of LdMNPV NEFU isolates were located at positions 5 4, 10 9, 379, 50 8 and 70 1 (starting from A in the start codon) of C, G, T, C and G The bases at the corresponding positions of the polyhedrin gene (ORF) of LdMNPV G isolates are G, C, C, T and T, respectively. Most of the amino acids at the corresponding positions of the two ORF codes are the same, with only one pair being different, that is, aspartic acid encoded by LdMNPV NEFU and histidine encoded by LdMNPV G at the corresponding position. The polyhedrin gene was expressed in E. coli BL21 (DE3) strain by using plasmid pT7 7 as a vector.