猪表皮生长因子在植物乳杆菌中的表达及其活性检测

Expression and characterization of porcine epidermal growth factor in Lactobacillus plantarum

摘要:

为获得一株高效表达猪表皮生长因子 (pEGF) 的重组植物乳杆菌，并检测其生物活性，从仔猪肠道内容物中分离植物乳杆菌，选择其中生物活性最强的一株 (Lp-1) 为宿主。以商品化乳杆菌表达载体pIAβ8为骨架，干酪乳杆菌超强组成型启动子SCP、M6前体蛋白信号肽SP、猪表皮生长因子pEGF等为元件，构建表达pEGF的植物乳杆菌重组表达载体pSCPSE。用电转化方法 (2.0 kV，2 000 Ω，25 μF，4.0 ms) 将重组载体pSCPSE转化入宿主菌Lp-1中获得重组植物乳杆菌Lp-pSCPSE。以Tricine-SDS-PAGE法和pEGF ELISA特异性检测试剂盒检测目的蛋白的表达；用培养12 h的阳性转化子菌体给刚断奶的BALB/c小鼠灌胃，2次/天，连续10 d，以断奶小鼠的体重、肠绒毛长度和隐窝深度的变化为指标，检测目的蛋白的生物活性。Tricine-SDS-PAGE检测和小鼠实验结果显示，重组植物乳杆菌的培养上清中出现6 kDa左右pEGF的特异性条带，且表达pEGF的重组植物乳杆菌能显著增加 (P<0.05) 断奶小鼠的体重、肠绒毛高度和肠隐窝深度。以上结果表明，pEGF能在植物乳杆菌中成功表达，且具有良好的生物学活性。

Abstract:

Epidermal growth factor (EGF) is an epithelial cell growth factor that can stimulate intestinal development, repair the damage of epidermal cells as well as reduce the incidence of pathogen infection and diarrhea. In order to produce a recombinant Lactobacillus plantarum (L. plantarum) expressing porcine epidermal growth factor (pEGF), we constructed a recombinant vector stably expressing pEGF in L. plantarum strains. First, L. plantarum strain Lp-1 was isolated from intestinal contents of piglets. Then the functional domain of pEGF, M6 precursor protein signal peptide (SP) and super strong constitutive promoter (SCP) were connected with the backbone plasmid pIAβ8 to construct the recombinant vector that was transformed into Lp-1 by electroporation. Afterwards, pEGF was expressed in Lp-1 and detected by Tricine-SDS-PAGE and ELISA. After orally irrigated early-weaned BALB/c mice with the recombinant L. plantarum every morning and late afternoon for 10 consecutive days, body weight, villous height and crypt depth in the intestine were measured to examine the influence of the recombinant bacteria on the intestinal development of early-weaned mice in vivo. Finally, the results of our experiments demonstrated that pEGF was successfully expressed in Lp-1 and the molecular weight of pEGF was 6 kDa. In addition, the recombinant pEGF can enhanced the daily gain and exerted significance influence (P<0.05) to the small intestinal morphology of early-weaned BALB/c mice. In conclusion, pEGF could be expressed in L. plantarum and the recombinant pEGF possesses good biological activity.