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：由大肠杆菌产生的不耐热肠毒素 B 亚基 (LTB)，一种具有潜在生物学特性的无毒蛋白质亚基，是一种强大的粘膜和肠胃外佐剂，可诱导针对共同施用抗原的强烈免疫反应。

目的：在本研究中，讨论了由积雪草 (Centella asiatica) 中优化的 ltb（也称为合成 ltb，s-ltb）基因编码用作抗原的 LTB 蛋白。

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，然后通过基因枪转化将其导入积雪草植物。进行PCR扩增以确定转基因积雪草植物中s-ltb基因的存在。通过免疫印迹分析s-ltb基因的表达并通过ELISA定量。LTB蛋白的体外活性通过GM1-ELISA测定。

结果：PCR 扩增已发现七个转基因积雪草个体。然而，其中只有五个产生 LTB 蛋白。ELISA分析表明，在转基因积雪草叶片中检测到的LTB蛋白的最高含量约为总可溶性蛋白的0.8%。GM1-ELISA 检测表明植物 LTB 蛋白与 GM1-神经节苷脂特异性结合，表明 LTB 亚基形成了活性五聚体。

结论：通过基因枪法成功转化到积雪草植物中的 s-ltb 基因在五聚四级结构中产生了相对大量的植物 LTB 蛋白，该蛋白具有 GM1-神经节苷脂结合亲和力，是肠上皮膜上的受体。