【作者】 卢士英；

【导师】 赵广英； 华育平； 柳增善；

【作者基本信息】 东北林业大学 ， 野生动植物保护与利用， 2004， 硕士

【摘要】 从构建的大腹园蛛主壶腹腺cDNA文库中筛先到Avgl(基因登录号为AY302573)完整cDNA克隆，为一新的CB相关基因。CB为溶酶体内一种重要的半胱氨酸内切蛋白水解酶，具有广谱的蛋白酶活性，参与机体多种肿瘤细胞浸润转移等多种生理和病理过程，因此越来越受到人们的广泛关注。 本实验根据AvglcDNA序列特点，设计合成引物(AvgS,AvgX)，提取大腹园蛛主壶腹腺总RNA并以此为模板，RT-PCR方法扩增AvglcDNA去除信号肽的部分序列(Avg)，将目的片段Avg与原核表达载体pET-28a(+)，pET-20b(+)分别用EcoR Ⅰ，Nco Ⅰ酶切处理，回收纯化后，连接构建两个重组载体，转化克隆宿主菌E．coli DH5a中，经酶切及PCR鉴定为阳性克隆后进一步测序鉴定，结果碱基、预测氨基酸序列均与Avgl(去除信号肽)100％一致，并具有正确的读码框，获得的重组质粒Avg-28a、Avg-20b可以用于下步表达。 大量提取重组质粒Avg-28a、Avg-20b，将其转化到表达宿主菌E．coli BL21(DE3)中，鉴定为阳性克隆后，用IPTG 37℃诱导表达，SDS-PAGE电泳分析，Avg-28a-DE3在近36kDa处出现了明显的表达带，其大小与预计分子量(35459.13Da)相当，经薄层扫描表达外源蛋白量占全菌体蛋白总量的39.6％，且表达形式主要为包涵体，将表达的包涵体蛋白进行变性、复性处理后测定蛋白酶活性，未见明显的酶活性；而Avg-20b-DE3未出现明显的表达带，用RT-PCR方法检测到了Avg mRNA的转录。将Avg-20b-DE3诱导，超声波破菌后上清液测定蛋白酶活性，显示了明显的酶活性。 制备包涵体蛋白，电洗脱纯化后，常规方法免疫吉戎獭兔，结束后颈动脉采血，经ELISA检测，呈明显的阳性反应。Western blot在36kDa处显示了特异蛋白结合带，Avg原核表达蛋白能够有效地刺激机体产生抗体，具有良好的免疫原性，为CB的进一步应用研究奠定了基础。更多还原

【Abstract】 Avgl cDNA (GenBank AY302573) screened from the major ampullate gland of Araneus Ventricousus cDNA library is a new cathepsin B-like gene. Cathepsin B is a lysosomal cysteine endoproteinase, which is implicated, in many physiological and pathological events, especially in tumor invasion and metastasis. So the relate studies on CB are increasingly focused on.The Avg was amplified from the tatal RNA of the major ampullate gland of Araneus Ventricousus by RT-PCR with the primer AvgS and AvgX designed according to Avgl cDNA. The PCR product and The prokaryotic expression vector pET-28a(+) and pET-20b(+) were digested by the restriction enzyme of EcoR I and Nco I .Two recombinant expression plasimids were constructed by T4 ligase and transformed into host strain E.coli DH5a. The Avg was completely identical to free signal peptide part of Avgl by sequencing and had the correct open reading frame. The two recombinant expression plasimids Avg-28a and Avg-20b were successfully constructed.Avg-28a in E.coli BL21 (DE3) was induced by 1PTG and the special protein (about 36 kDa) was detected by SDS-PAGE. It amounted to 39.6%of the total protein of E.coli DE3 by thin-layer scan. The pattern of expression was mainly inclusion body. SDS-PAGE of inducing Avg-20b-DE3 by IPTG did not show the special protein but the Avg mRNA transcription was detected by RT-PCR. The experiment of assaying the expression protein enzyme activity was carried on. The result indicated that the inclusion body protein treated in denaturing and renaturing agent was not shown the activity of cathepsin. But the supernatant of Avg-20b-DE3 by supersonic treatment showed the high activity.The inclusion body protein was purified by SDS-PAGE and electrophoresis elution and immunized rabbits to prepare anti-serum. The result of EL1SA showed obvious positive reaction. The special band of 36kDa appeared in Western-blot. It indicated that the inclusion body protein effectively induced the body to produce the antibody and had excellent antigenicity. The findings of this experiment will lay foundations for further studying the application of CB.更多还原