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

丝素蛋白是一种优良的布用纤维材料，有望成为医疗器械和酶固定化的良好材料。本课题是通过对家蚕丝素蛋白基因进行改造，制备新型蛋白纤维的一次尝试。丝素蛋白分子中典型的重复氨基酸序列（-Ser-Gly-Ala-Gly-Ala-Gly-）的一部分称为丝素蛋白结晶部分（Fcp）.如果在Fcp序列中加入一个自由基氨基酸，丝素蛋白的性质将发生显著变化。本课题尝试在Fcp序列的DNA中添加赖氨酸和/或蛋氨酸的编码序列，并在大肠杆菌中表达丝素蛋白基因。将一段3 n + 1碱基对的过客DNA序列引入到本课题制备的携带tac启动子的质粒p41上的-半乳糖苷酶基因的多聚接头位点。导入的DNA序列将在大肠杆菌中表达为与-gal融合的蛋白。接着，将含有丝心蛋白基因的质粒pBmF 6中的Fcp序列（由Y.用Pst Ⅰ、Hae Ⅱ和Bal 31处理质粒，再用ALu Ⅰ部分处理质粒，得到质粒pFCP和pFKB。第三，通过DNA合成仪合成四个DNA序列。将该片段与p41连接，并将该片段与其中一个片段的聚合物的混合物引入质粒，获得质粒pFMS。pFCP、pFKB和pFMS DNA在E.杆菌当E.将携带pFCP、pFKB和pFMS的大肠杆菌JM 109接种于含有IPTG和X-gal的平板上，菌落殖民地变蓝。蓝色菌落也与抗Fcp大鼠血清反应。将从蓝色菌落中分离的蛋白质提取物应用于PAGE上，并使用抗Fcp大鼠血清和抗大鼠IgG-HRPO缀合的兔IgG通过蛋白质印迹法进行测定。蓝色菌落产生Fcp样蛋白，但未测定分子量。

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.