家蚕杆状病毒半胱氨酸蛋白酶基因的序列分析及表达

通过PCR扩增技术克隆了家蚕核型多角体病毒 (BombyxmoriNuclearPolyhedrosisVirus ,BmNPV)ZJ8株的半胱氨酸蛋白酶 (cysteineproteinase ,CP)基因 (BmNPVZJ8 CP)。序列分析表明 ,该蛋白酶基因的ORF为 972个核苷酸 ,编码 32 3个氨基酸。同源性分析表明 ,BmNPVZJ8 CP在DNA水平上与其它来源的CP具有较高的同源性 ,根据已知的杆状病毒半胱氨酸蛋白酶序列构建了该基因的进化树 ,发现BmNPVZJ8 CP与人的木瓜蛋白酶超家族的CP在同一进化分支上 ,与BmNPV T3株、AcNPV的半胱氨酸蛋白酶基因的核苷酸同源性最高。该基因在大肠杆菌中表达的产物对大肠杆菌具有生理毒性 ,从而限制了该基因的表达。

Sequence Analysis and Expression of Cysteine Proteinase Gene From Bombyx mori Baculovirus

The gene encoding the cysteine proteinase (CP) was cloned from Bombyx mori nucleopolyhedrosis virus ZJ8 by polymerase chain reaction (PCR).Nucleotide sequence analysis of CP revealed that an open reading frame of 972 bp codes for 323 amino acids.Compared to the CP gene from other nuclear polyhedrosis viruses,it was shown that the identity on DNA level was high.The phylogenetic tree of CP gene was constructed according to the sequences of other cysteine proteinase genes,and it is found that the CP gene was on the same branch with papain upper family,and the identity with the BmNPV-T3 and AcNPV is the highest.The CP gene was cloned into E.coli expression plasmid pET-28a(+).The result of the CP expressed in E.coli showed that the CP has physiological toxicity to the host BL21,which blocked the expression of itself.