苏州大学本科生毕业设计（论文）
目 录
摘要 ........................................................................................................................... 1 前言 ........................................................................................................................... 3 1 材料和方法 ........................................................................................................... 3 1.1 材料及主要试剂 ................................................................................................ 4 1.2 方法 .................................................................................................................... 4 1.2.1 引物设计 ......................................................................................................... 4 1.2.2 PCR扩增目的片段 ........................................................................................ 4 1.2.3 目的片段的分离和回收（Takara回收试剂盒） ........................................ 5 1.2.4 酶切反应 ......................................................................................................... 5 1.2.5 连接反应 ......................................................................................................... 5 1.2.6 感受态细胞制备 ............................................................................................. 6 1.2.7 连接产物的转化 ............................................................................................. 6 1.2.8 试剂盒抽提质粒DNA ................................................................................... 6 1.2.9 重组质粒的鉴定及测序 ................................................................................. 6 1.2.10 原核表达 ....................................................................................................... 7 1.2.11 SDS-PAGE蛋白电泳 ................................................................................... 7 2 结果 ....................................................................................................................... 8 2.1 野桑蚕Serpin-2基因的cDNA序列克隆 ....................................................... 8 2.2 野桑蚕Serpin-2基因的cDNA序列分析 ....................................................... 9 2.3 Serpin-2重组表达载体酶切鉴定 ................................................................... 12 2.4 蛋白质表达与SDS-PAGE分析 ..................................................................... 13 2.5 Western Bloting分析........................................................ 错误！未定义书签。 3 讨论 ..................................................................................................................... 14 参考文献： ............................................................................................................. 15 致 谢 ..................................................................................................................... 16 附录1. .................................................................................................................... 19 附录2. .................................................................................................................... 20
苏州大学本科生毕业设计（论文）
家蚕丝氨酸蛋白酶抑制剂基因serpin-2的克隆、序列分析及表达及表达
摘 要
本研究以家蚕中大造品种为材料，以其 cDNA为模板克隆家桑蚕丝氨酸蛋白酶抑制剂Serpin-2基因；采用大肠杆菌原核表达系统（BL21）进行家蚕Serpin-2基因的表达研究。主要结果如下：
根据已知野蚕Serpin-2（GenBank登录号：AF242200.1）序列设计合适的引物，采用PCR方法克隆了家蚕Serpin-2基因。序列分析表明，家蚕Serpin-2基因编码区长度为990bp，编码329个氨基酸，相对分子量约为43.75 kDa，等电点约为4.67。与家蚕
Serpin-2比较发现，两者基因序列相似度高达99.29%，氨基酸序列相似度高达98.93%，
推测家蚕Serpin-2与野桑蚕Serpin-2基因起着相同或类似的作用。
将家蚕大造Serpin-2基因克隆进原核表达载体pET-28a(+)，转化到大肠杆菌BL21中，经1 mmol/L IPTG诱导蛋白质表达。SDS-PAGE电泳分析结果表明，经IPTG诱导转化家蚕Serpin-2的BL21菌与对照BL21菌、空pET-28a(+)转化BL21菌相比出现了一条特异性的蛋白质条带，相对分子量约44 kDa，与预计的大小相符，证实家蚕
Serpin-2在大肠杆菌中得到表达。
家蚕Serpin-2基因的成功克隆和原核表达研究为从分子水平上解析Serpin在野桑蚕体内的功能打下了基础。
关键词：家蚕；Serpin-2；基因克隆；原核表达
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苏州大学本科生毕业设计（论文）
Cloning and prokaryotic expression of Serpin-2 gene of Bombyx
mori
Abstract
In this study，the gene of Serpin in Bombyx mori (Serpin-2) was cloned by PCR using the whole body cDNAs as templates. E.coli prokaryotic expression system was used for expression study of serpin-2 gene. The main results are as follows:
According to the sequence of Bombyx mandarina serpin-2 gene, proper primer was designed and Serpin-2 gene of Bombyx mori（The GenBank accession numbers：AF242200.1）was cloned by PCR. According to sequence analysis, the 990bp coding region of Serpin-2 encoding 329 amino acid residues results in theoretical molecular weight of 43.75 kDa, isoelectric point of 4.67. Comparied to the Serpin-2 gene of Bombyx mori and Bombyx mandarina，the gene identity and amino acid identity reach 98.7%，respectively. It reveals that Serpin-2 of Bombyx mori and Bombyx mandarina may play a same or similar role.
The Serpin-2 gene was constructed to the prokaryotic expression vector pET-28a（+） and then transformed into E. coli BL21(DE3). And 1mmol/L IPTG was used to induce the target protein to express. SDS-PAGE analysis indicated that there was a specific band with relative molecular weight of 44 kDa on PAGE profiles for the BL21 with Serpin-2 induced by IPTG, comparied to the contract BL21 and BL21 with empty pET-28a(+)，which confirmed that the Serpin-2 gene was successfully expressed.
The successful cloning and prokaryotic expressing research of Serpin-2 gene in Bombyx mori has established the foundation for analying the fuction of Serpin in Bombyx mori.
Key words: Bombyx mandarina; Serpin-2; cloning; prokaryotic expression
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苏州大学本科生毕业论文
