大肠杆菌不耐热肠毒素 B 亚单位在积雪草（Centella asiatica (L.) Urban）中的生物弹粒转化表达。
Expression of Escherichia coli Heat-Labile Enterotoxin B Subunit in Centella (Centella asiatica (L.) Urban) via Biolistic Transformation.
机构信息
Institute of Bioactive Compounds and Department of Biotechnology, University of Sciences, Hue University, Hue, Thua Thien Hue 530000, Vietnam.
Division of Biological Sciences and Research Center of Bioactive Materials, Chonbuk National University, Jeonju, Chonbuk 561-756, Korea.
出版信息
Curr Pharm Biotechnol. 2020;21(10):973-979. doi: 10.2174/1389201021666200226094150.
BACKGROUND
Heat-Labile enterotoxin B subunit (LTB) produced by Escherichia coli, a non-toxic protein subunit with potential biological properties, is a powerful mucosal and parenteral adjuvant which can induce a strong immune response against co-administered antigens.
OBJECTIVE
In the present study, LTB protein, encoded by the optimized ltb (also known synthetic ltb, s-ltb) gene in centella plant (Centella asiatica) for use as an antigen, has been discussed.
METHODS
The s-ltb gene was cloned into a plant expression vector, pMYO51, adjacent to the CaMV 35S promoter and was then introduced into centella plant by biolistic transformation. PCR amplification was conducted to determine the presence of s-ltb gene in the transgenic centella plant. The expression of s-ltb gene was analyzed by immunoblotting and quantified by ELISA. In vitro activity of LTB protein was determined by GM1-ELISA.
RESULTS
PCR amplification has found seven transgenic centella individuals. However, only five of them produced LTB protein. ELISA analysis showed that the highest amount of LTB protein detected in transgenic centella leaves was about 0.8% of the total soluble protein. GM1-ELISA assay indicated that plant LTB protein bound specifically to GM1-ganglioside, suggesting that the LTB subunits formed active pentamers.
CONCLUSION
The s-ltb gene that was successfully transformed into centella plants by the biolistic method has produced a relatively high amount of plant LTB protein in the pentameric quaternary structure that has GM1-ganglioside binding affinity, a receptor on the intestinal epithelial membrane.
背景
大肠杆菌产生的不耐热肠毒素 B 亚单位（LTB）是一种无毒的蛋白质亚单位，具有潜在的生物学特性，是一种强大的黏膜和非肠道佐剂，可诱导针对共同给予的抗原的强烈免疫反应。
目的
本研究讨论了植物中表达的 LTB 蛋白，该蛋白由 Centella 植物（积雪草）中的优化 ltb（也称为合成 ltb，s-ltb）基因编码，用作抗原。
方法
将 s-ltb 基因克隆到植物表达载体 pMYO51 中，与 CaMV 35S 启动子相邻，然后通过弹道转化引入 Centella 植物。通过 PCR 扩增确定转基因 Centella 植物中是否存在 s-ltb 基因。通过免疫印迹分析和 ELISA 定量分析来分析 s-ltb 基因的表达。通过 GM1-ELISA 测定 LTB 蛋白的体外活性。
结果
PCR 扩增发现了七个转基因 Centella 个体。然而，只有其中五个产生了 LTB 蛋白。ELISA 分析表明，转基因 Centella 叶片中检测到的 LTB 蛋白的最高量约为总可溶性蛋白的 0.8%。GM1-ELISA 测定表明植物 LTB 蛋白特异性结合 GM1-神经节苷脂，表明 LTB 亚基形成了活性五聚体。
结论
通过弹道方法成功转化到 Centella 植物中的 s-ltb 基因在具有 GM1-神经节苷脂结合亲和力的五聚体四级结构中产生了相对大量的植物 LTB 蛋白，这是肠上皮细胞膜上的一种受体。
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