依博素生物合成基因ste22的异源置换研究

摘要

Objective Ebosin is a novel exopolysaccharide produced by Streptomyces and evidenced to possess an anti-rheumatic arthritis activity. The ebosin biosynthesis gene cluster has been identified. Here, we aim to generate derivatives of ebosin for better activity, and replace ebosin biosynthesis gene ste22 by heterologous gene. Methods Gene ste22 has previously been demonstrated to code for a rhamnosyltransferase. With PCR amplification, a gene encoding glucosyltransferase was isolated from Streptococcus thermophilus. Using a double crossover via homologous recombination, the gene ste22 in Streptomyces sp.139 was replaced by the gene encoding glucsyltransferase originated from Streptococcus thermophilus. With the assay of IL-1β-converting enzyme (ICE) and acute inflamed mice model induced by croton oil, primary results of activity for novel ebosin derivative produced by the heterologous gene replacement strain was identified. Results The heterologous gene replacement strain Streptomyces sp.139 (gtf-22) was constructed, which produced a novel ebosin derivative EPS-22g. This alteration resulted in EPS-22g with its monosaccharide composition dramatically changed, especially in the proportion of glucose, which increased from 2.14% (ebosin) to 48.64% (EPS-22g). Comparing with ebosin, EPS-22g exhibited a higher inhibitory effect on the activity of IL-1β-converting enzyme (ICE). Experiments with acute inflamed mice induced by croton oil showed higher anti-inflammatory activity of EPS-22g than that of ebosin. Conclusion To generate derivatives of ebosin for better activity, the strategy of ebosin biosynthesis gene replaced by heterologous gene may be more useful.%目的依博素是由链霉菌产生的一种新型胞外多糖，具有显著抗类风湿性关节炎的作用，我们研究已确定了该多糖的生物合成基因簇（ste）。为了对依博素结构进行改造以提高其生物活性，对其生物合成基因 ste22进行了异源置换研究。　　方法采用 PCR 扩增方法从乳酸菌获得了编码葡萄糖糖基转移酶的基因 gtf，通过基因同源重组双交换，从链霉菌中置换了 ste22基因。采用白细胞介素-1合成酶活性测定方法及小鼠巴豆油急性炎症模型，初步确定了基因置换株的依博素衍生物生物活性。　　结果获得了葡萄糖糖基转移酶基因置换株 Streptomyces sp.139（gtf-22），产生了依博素新衍生物 EPS-22g。研究表明该衍生物与依博素相比，葡萄糖比例从2.14%显著上升到48.64%，体外活性实验显示，其对炎症细胞因子白细胞介素-1合成关键酶 ICE 抑制率高于依博素，小鼠巴豆油急性炎症体内活性分析结果证明，该衍生物抑制活性亦高于依博素。　　结论采用异源基因置换生物合成基因的方法改造依博素产生菌，可能是获得生物活性较好新多糖衍生物的一种有效手段。