马尾松毛虫CPV基因组S7的序列分析及部分序列的原核表达

Nucleotide Sequence Analysis and Prokaryotic Expression of S7 cDNA from Dendrolimus punctatus CPV Hunan Strain

马尾松毛虫质多角体病毒(湖南株)基因组S7节段(AY180908)cDNA克隆及序列分析结果表明:S7由1501个碱基组成,编码由448个氨基酸组成的分子量为49.8 kDa的多肽P50。5′末端和3′末端具有5′-AGTAA-3′和5′-GTTAGCC-3′末端保守序列。该基因组与舞毒蛾质多角体病毒1型和家蚕质多角体病毒1型S7节段有很高的同源性,核苷酸序列同源性分别为97.2％和87.0％,氨基酸序列同源性分别为98.7％和92.8％。P50多肽与人型支原体的DnaK样蛋白在C-末端有相似性。本文报道了编码P50C259的cDNA序列的克隆并作了原核表达,当用1.0 mmol/L IPTG诱导2h,分子量约为37.3 kDa的融合蛋白在大肠杆菌BL21中得到大量表达。

The nucleotide sequence of genome S7 cDNA (AY 180908) from Dendrolimus punctatus cypovirus 1 Hunan strain ( DpCPV-HN ) was determined. S7 cDNA consists of 1501 nucleotides and encodes a protein of 448 amino acids with a molecular mass of 49.8 kDa. The 5'- and 3'- terminus conserved sequences are 5'-AGTAA-3'and 5'-GTTAGCC-3', respectively. When compared with S7 and its putative protein of Lymantria dispar cypovirus 1 and Bombyx mori cypovirus 1, 97.2% and 87.0% identities in the nucleotide level, and 98.7% and 92.8% identities in the amino acid level were found, repectively. BLAST search program revealed some similarity between DpCPV-HN P50 and DnaK-like protein of Mycoplasma hominis. The cDNA fragment encoding DpCPV-HN P50 C259 was also cloned and inserted into pET-28a expressive vector after being digested with BamH Ⅰ and Hind Ⅲ to construct pET-28a/S7(591-1 371) recombinant expression vector. Expression of the fusion protein was induced by IPTG in E. coli BL21. The results indicated that the fusion protein with a molecular mass of 37.3 kDa containing P50C259 was expressed abundantly, and the fusion protein had an optional level when induced by 1.0mmol/L IPTG over about 2 hours.