中文摘要：

丝素重链是家蚕丝素蛋白的主要组成部分,位于丝素重链中间的核心区域由12个重复区肽段和11个非重复区肽段组成,非重复区是含有大量极性侧基的亲水性肽段。为了研究丝素重链各重复区组成序列的结构及其在生物医学领域的适用范围,需要对其进行高纯度分离。克隆了丝素重链非重复区编码基因片段f（1）及其延伸片段f（4）和f（8）,并构建重组质粒在大肠埃希菌（Escherichia coli）中诱导表达融合蛋白GST-F（1）、GST-F（4）和GST-F（8）。通过三点设计法优化诱导剂IPTG浓度、起始菌密度和诱导时间,采用SDS-PAGE电泳和BCA蛋白浓度检测法定性、定量分析融合蛋白的表达水平,确定了融合蛋白GST-F（1）、GST-F（4）和GST-F（8）的最佳诱导表达条件：起始菌密度D（600 nm）值分别为1.5、1.2和0.9,诱导剂浓度分别为0.2 mmol/L、0.2 mmol/L和0.3 mmol/L,诱导时间分别为3 h、4 h和5 h。在上述最佳诱导表达条件下,3种融合蛋白的表达量达到50～90 mg/L,可满足后续研究的需求。

英文摘要：

Silk fibroin heavy chain is the major component of Bombyx mori fibroin. The core region of fibroin heavy chain is composed of 12 repetitive regions and 11 non-repetitive hydrophilic polypeptides which contain a large number of polar side group. In order to have a clear view on the sequence structure of various repetitive regions of fibroin heavy chain and its application in biomedical field, high-purity fibroin heavy chain is necessary. In present study, we cloned the gene sequence f（1 ） encoding the non-repetitive domain of fibroin heavy chain and the extended segment quad- ruple f（4） and octuple f（8）, and constructed recombinant expression vectors to express fusion proteins GST-F（ 1 ）, GST-F（4） and GST-F（8） in Escherichia coil ceils. Three-point design method was used to optimize the induced ex- pression conditions including inducer IPTG concentration, initial bacterial cell density and induction time. SDS-PAGE and BCA method were used to analyze the expression levels of fusion proteins. The optimal induced expression condi- tions of fusion proteins GST-F（ 1 ）, GST-F（4） and GST-F（8） were as following： initial bacterial cell density D（600 nm） values were 1.5, 1.2 and 0.9, IPTG concentrations were 0.2 mmol/L, 0.2 mmol/L and 0.3 mmoVL, and induction time were 3 h, 4 h and 5 h, respectively. Under the optimized induced expression conditions, the yields of three fusion proteins reached 50 to 90 mg/L, which can meet the need of future research.