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A Trial for a Creation of a New Protein Fiber by Modification of Fibroin Gene, Bombyx mori L.

通过修饰丝素基因（Bombyx mori L.）创建新蛋白纤维的试验。

基本信息

批准号：
62480046
负责人：
HIMENO Michio
金额：
$ 4.1万
依托单位：
University of Osaka Prefecture (1988)Tokyo University of Agriculture and Technology (1987)
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1987
资助国家：
日本
起止时间：
1987 至 1988
项目状态：
已结题

项目摘要

The silk fibroin is excellent materials as a fiber of cloth and it is expected as good materials for medical instrument and salso for immobilization of enzyme. This project is a trial for a ceration of new protein fiber by modification of fibroin gene, Bombyx mori L..Parts of a typical repeating amino acid sequence (- Ser - Gly - Ala - Gly - Ala - Gly -) in silk fibroin molecule are called a fibroin crystalline fraction (Fcp). If a radical amino acid be added in Fcp sequence, the character of fibroin protein will drastically changed. then in this project we attempt addition of the code of lysine and/or methionine in DNA of the Fcp sequence and expression of fibroin gene in escherichia coli.First, a expression vector of Fcp DNA sequence was made. When a passenger DNA sequence of 3n + 1 base pair is introduced into poly linker site of - galactosidase ( -gal) gene on plasmid p41 (made in this project) carring tac promoter. the introduced dna sequence will be express as a fussed protein with -gal in E.coli. Next, the Fcp sequence in plasmid pBmF6 harboring the fibroin gene (kindlly provided by Prof. Y. Suzuki) was obtained by Pst I, Hae II and Bal 31 treatment of the plasmid, and also by ALu I partially treatment, then we got plasmid pFCP and pFKB. Third, four DNA sequences were synthesized by a DNA synthesizer. The fragment were linked to p41 and also mixture of the fragments and polymer of one were introduced the plasmid, then plasmid pFMS was obtained. The pFCP, pFKB pr pFMS DNA easily deleted in E. coli. When E. coli JM109 carring pFCP, pFKB or pFMS was cultured on a plate containing IPTG and X-gal, the colony become blue. The blue colonies also reacted anti-Fcp rat serum. The protein extract isolated the blue colonies were applied on page, and determined by western blotting method using anti-Fcp rat serum and anti-rat IgG-HRPO conjugated rabbit IgG. The blue colonies produced a Fcp like protein but the molecular weight were not determined.

丝素蛋白是一种优良的布料纤维材料，有望成为医疗器械和酶固定化的良好材料。该项目是通过修饰丝素基因Bombyx mori L.来生产新型蛋白质纤维的试验。丝素蛋白分子中典型的重复氨基酸序列（-Ser-Gly-Ala-Gly-Ala-Gly-）的部分称为丝素结晶部分（Fcp）。如果在Fcp序列中添加一个自由基氨基酸，丝素蛋白的特性就会发生巨大的改变。然后在本项目中我们尝试在Fcp序列的DNA中添加赖氨酸和/或蛋氨酸的编码并在大肠杆菌中表达丝素基因。首先，制作Fcp DNA序列的表达载体。当将3n+1个碱基对的过客DNA序列引入携带tac启动子的质粒p41（本项目中制造）上的-半乳糖苷酶（-gal）基因的聚接头位点时。引入的DNA序列将在大肠杆菌中表达为与-gal的融合蛋白。接下来，通过对质粒进行Pst I、Hae II和Bal 31处理，并通过ALu I部分处理，获得带有丝心蛋白基因的质粒pBmF6(由Y. Suzuki教授友情提供)中的Fcp序列，然后得到质粒pFCP和pFKB。第三，通过DNA合成仪合成了四个DNA序列。将片段与p41连接，并将片段与其中之一的聚合物的混合物导入质粒，得到质粒pFMS。 pFCP、pFKB 或 pFMS DNA 在大肠杆菌中很容易被删除。当携带pFCP、pFKB或pFMS的大肠杆菌JM109在含有IPTG和X-gal的平板上培养时，菌落变成蓝色。蓝色菌落也与抗 Fcp 大鼠血清发生反应。将分离出的蓝色菌落的蛋白提取物涂在页面上，并使用抗Fcp大鼠血清和抗大鼠IgG-HRPO缀合的兔IgG通过蛋白质印迹法测定。蓝色菌落产生类似 Fcp 的蛋白质，但分子量未测定。