家蚕浓核病毒（镇江株）部分核苷酸序列分析及主要结构蛋白基因（vp）的表达
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摘要
本文对家蚕浓核病毒(Bombyx mori Densovirus,BmDNV)(镇江株)的部分核苷酸序列进行了研究，克隆并用大肠杆菌表达了主要结构蛋白基因，试图从分子水平、基因水平对它与山梨株的亲缘关系进行探讨。
一．根据已发表的日本山梨株的序列片段1(VD_1)设计了一对引物，克隆了1.5Kb的主要结构蛋白基因(vp)，测序并用Dna-Star软件分析了两者的同源性。该片段与山梨株VD_1-ORF_2的同源性为98.1％，有29处核苷酸发生了突变；推测的氨基酸序列与山梨株的同源性为98.6％，有7个氨基酸发生了替换。该序列已经在GeneBank登录，登录号为AY236978
二．根据已发表的日本山梨株的序列片段2(VD_2)在两个开放阅读框(ORFs)之间设计了一对引物，扩增得到了992bp位于两个开放阅读框(ORFs)之间的一个片段，测序并用Dna-Star软件分析了两者的同源性。该片段与BmDNV山梨株相应部分的同源性为98.5％，位于两个开放阅读框(ORFs)之间的序列有缺失或插入突变。
三．在NCBI数据库中用Blast软件在线检索，除家蚕浓核病毒山梨株DNA外，未发现其他同源序列。
四．将病毒的主要结构蛋白基因(vp)插入到表达质粒pET-28a多克隆位点BamH I与EcoR I之间，使其位于诱导型启动子LacZ之后，转化到BL21(DE3)菌株，进行了诱导表达。表达产物在SDS-聚丙烯酰胺凝胶电泳中呈现特异的条带，大小约为53KD，与预计的大小大致相近。用Western-blotting检测表达产物，可以与病毒的抗体产生特异的反应，表明其为病毒的结构蛋白基因。
五．上述结果表明，BmDNV镇江株和山梨株一样，病毒的基因组是由两个片段组成，并且两者的核苷酸序列有很高的同源性(高达98％以上)，而与其他浓核病毒的核苷酸序列没有同源性，可以认为两者是同种病毒的不同分离株。
An attempt to identify the relationship between Bombyx mori Densovirus (BmDNV) Zhenjiang strain and BmDNV Yamanashi strain, partial DNA sequences of BmDNV (Zhenjiang strain) were determined and compared with the corresponding region of BmDNV (Yamanashi strain). The major viral structural protein (vp) gene was expressed by Escherichia coli (E. coli) expression system and examined by Western-blotting assay.
I. Based on nucleotide sequences of BmDNV (Yamanashi strain) VD1 segment, oiigonuclotide primers was designed. Employing the designed-primers, a fragment of nucleotide sequences about 1.5 Kb, which is speculated to be the major structural protein (vp) gene, was amplified from the genomic DNA of BmDNV (Zhenjiang strain) by polymerase chain reaction (PCR).
Sequenced and analyzed with Dna-star software, the nucleotide sequence of the cloned structural protein (vp) gene and its deduced amino acid sequence are highly homologous with its homologue in BmDNV Yamanashi strain, with the identity of 98.1% and 98.6% respectively. Compared with Yamanashi strain, 29 bases and 7 amino acids of the vp gene were substituted in Zhenjiang strain, respectively. No reading frame shift mutant was observed. The newly determined sequence has been submitted to GeneBank and the accession number is AY 236978.
2. Based on nucleotide sequences of BmDNV (Yamanashi strain) VD2 segment, oiigonuclotide primers toward between two open reading frames (ORFs) on DNA of the virus was designed. Another fragment of nucleotide sequences about 1.0 Kb was generated from BmDNV (Zhenjiang strain) by polymerase chain reaction (PCR).
Sequenced and analyzed with Dna-star software, the similarity between the corresponding region of Zhenjiang strain and Yamanashi strain was 98.5%. 15 nucleotide mutants, including 3 gaps, were found in the segment.
3. On-line searching with Blast software in NCBI data base, no homologous sequences were found except DNA sequence of Densovirus Yamanashi strain.
4. The major structural protein (vp) gene of BmDNV (Zhenjiang strain) was inserted into between the EcoRI and BamHI sites of the multiple cloning sites (MCS) of the expression plasmid pET-28a under the inducible promoter LacZ. Transformed and expressed in E.coli BL21(DE3), a 53KD protein, identical to the predicted molecular mass, was demonstrated by SDS-PAGE analysis. The product can react with polyclonal antibody of virus specifically by Western-blotting assay. This indicated that the
cloned gene was viral structural protein gene.
5. The above results indicate that the genome of BmDNV Zhenjiang strain composes of two DNA segments as BmDNV Yamanashi strain does. The two BmDNVs are highly homologous in nucleotide sequences (about 98%) and no other homologous sequence was found in NCBI data base. These results reveal that the two BmDNVs are different isolate of the same virus.
一．根据已发表的日本山梨株的序列片段1(VD_1)设计了一对引物，克隆了1.5Kb的主要结构蛋白基因(vp)，测序并用Dna-Star软件分析了两者的同源性。该片段与山梨株VD_1-ORF_2的同源性为98.1％，有29处核苷酸发生了突变；推测的氨基酸序列与山梨株的同源性为98.6％，有7个氨基酸发生了替换。该序列已经在GeneBank登录，登录号为AY236978
二．根据已发表的日本山梨株的序列片段2(VD_2)在两个开放阅读框(ORFs)之间设计了一对引物，扩增得到了992bp位于两个开放阅读框(ORFs)之间的一个片段，测序并用Dna-Star软件分析了两者的同源性。该片段与BmDNV山梨株相应部分的同源性为98.5％，位于两个开放阅读框(ORFs)之间的序列有缺失或插入突变。
三．在NCBI数据库中用Blast软件在线检索，除家蚕浓核病毒山梨株DNA外，未发现其他同源序列。
四．将病毒的主要结构蛋白基因(vp)插入到表达质粒pET-28a多克隆位点BamH I与EcoR I之间，使其位于诱导型启动子LacZ之后，转化到BL21(DE3)菌株，进行了诱导表达。表达产物在SDS-聚丙烯酰胺凝胶电泳中呈现特异的条带，大小约为53KD，与预计的大小大致相近。用Western-blotting检测表达产物，可以与病毒的抗体产生特异的反应，表明其为病毒的结构蛋白基因。
五．上述结果表明，BmDNV镇江株和山梨株一样，病毒的基因组是由两个片段组成，并且两者的核苷酸序列有很高的同源性(高达98％以上)，而与其他浓核病毒的核苷酸序列没有同源性，可以认为两者是同种病毒的不同分离株。
An attempt to identify the relationship between Bombyx mori Densovirus (BmDNV) Zhenjiang strain and BmDNV Yamanashi strain, partial DNA sequences of BmDNV (Zhenjiang strain) were determined and compared with the corresponding region of BmDNV (Yamanashi strain). The major viral structural protein (vp) gene was expressed by Escherichia coli (E. coli) expression system and examined by Western-blotting assay.
I. Based on nucleotide sequences of BmDNV (Yamanashi strain) VD1 segment, oiigonuclotide primers was designed. Employing the designed-primers, a fragment of nucleotide sequences about 1.5 Kb, which is speculated to be the major structural protein (vp) gene, was amplified from the genomic DNA of BmDNV (Zhenjiang strain) by polymerase chain reaction (PCR).
Sequenced and analyzed with Dna-star software, the nucleotide sequence of the cloned structural protein (vp) gene and its deduced amino acid sequence are highly homologous with its homologue in BmDNV Yamanashi strain, with the identity of 98.1% and 98.6% respectively. Compared with Yamanashi strain, 29 bases and 7 amino acids of the vp gene were substituted in Zhenjiang strain, respectively. No reading frame shift mutant was observed. The newly determined sequence has been submitted to GeneBank and the accession number is AY 236978.
2. Based on nucleotide sequences of BmDNV (Yamanashi strain) VD2 segment, oiigonuclotide primers toward between two open reading frames (ORFs) on DNA of the virus was designed. Another fragment of nucleotide sequences about 1.0 Kb was generated from BmDNV (Zhenjiang strain) by polymerase chain reaction (PCR).
Sequenced and analyzed with Dna-star software, the similarity between the corresponding region of Zhenjiang strain and Yamanashi strain was 98.5%. 15 nucleotide mutants, including 3 gaps, were found in the segment.
3. On-line searching with Blast software in NCBI data base, no homologous sequences were found except DNA sequence of Densovirus Yamanashi strain.
4. The major structural protein (vp) gene of BmDNV (Zhenjiang strain) was inserted into between the EcoRI and BamHI sites of the multiple cloning sites (MCS) of the expression plasmid pET-28a under the inducible promoter LacZ. Transformed and expressed in E.coli BL21(DE3), a 53KD protein, identical to the predicted molecular mass, was demonstrated by SDS-PAGE analysis. The product can react with polyclonal antibody of virus specifically by Western-blotting assay. This indicated that the
cloned gene was viral structural protein gene.
5. The above results indicate that the genome of BmDNV Zhenjiang strain composes of two DNA segments as BmDNV Yamanashi strain does. The two BmDNVs are highly homologous in nucleotide sequences (about 98%) and no other homologous sequence was found in NCBI data base. These results reveal that the two BmDNVs are different isolate of the same virus.
引文
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