【摘要】 利用反转录多聚酶链式反应(RT-PCR)技术从烟夜蛾Helicoverpa assulta(Guene)雌虫卵巢中扩增得到了组织蛋白酶B酶原基因(cathepsin B,CB)cDNA片段,将其克隆至pMD19-T载体。测序结果表明,该片段长度为1017bp,含有组织蛋白酶B酶原基因完整开放阅读框架(ORF)(Gen Bank登录号:EF154237)。序列分析结果显示:烟夜蛾组织蛋白酶B(HassCB)编码338个氨基酸残基,预测N-末端含有长度为21个氨基酸残基的信号肽序列;去除信号肽序列后,预测成熟蛋白分子量为35.5kDa,等电点为5.96。氨基酸序列比对结果表明,HassCB与其他昆虫的组织蛋白酶B酶原氨基酸序列有较高的一致性。将去除信号肽序列的HassCB(HassCBa)重组到表达载体pGEX-4T-1中,并转入原核细胞中表达,SDS-PAGE和Western印迹分析表明:该基因能在大肠杆菌BL21中表达,电泳检测到一条大约61kDa的目的条带,与预测的融合蛋白分子量相符。用该基因制备多克隆抗体并测得该抗体对重组表达的HassCBa的效价为1∶51200。通过免疫印迹检测证实,此抗体既能识别重组表达的HassCBa,又能识别烟夜蛾卵巢匀浆液中的HassCB。更多还原

【Abstract】 The cathepsin B cDNA from ovary of Helicoverpa assulta (Guene) was cloned by reverse transcription polymerase chain reaction (RT-PCR). The results of cloning and sequencing showed that the full-length open reading frame (ORF) of HassCB is 1 017 bp (registered with GenBank accession no. EF154237),encoding 338 amino acid residues,and the predicted N-terminus hydrophobic region contains 21 amino acid residues displaying the characteristic features of a signal peptide. The predicted molecular weight (MW) and isoelectric point (pI) are 35.5 kDa and 5.96,respectively. The HassCBa gene without signal peptide was then constructed into the expression vector pGEX-4T-1 for expression in prokaryotic cells. The SDS-PAGE and Western blot analysis showed that the HassCB was expressed in Escherichia coli BL21,and the expressed product has the MW of 61 kDa,nearly equal to the predicted. Furthermore,the mice were immunized with recombinant HassCB,and the results indicated that the antibody liter was 1∶51 200 against the recombinant HassCBa. Importantly,the immunoblotting result showed that the antibodies could identify HassCB both recombinant and from natural ovary of H. assulta.更多还原