| 摘要: |
| 为了实现基因工程高效制备tachyplesin Ⅰ, 采用tachyplesinⅠ基因串联表达的方法, 以穿梭表达载体pSBPTQ 为表达载体, 通过RT-PCR 扩增tachyplesinⅠ基因(tac)和采用直接退火的方法合成tachyplesinⅠ串联基因(2tac), 构建重组表达载体pSBPTQ-TAC 和pSBPTQ-2TAC, 转化到枯草杆菌BS168 实现高效表达。实验结果表明: 基因tac和2tac在枯草杆菌中表达成功, TAC 和2TAC 抗菌肽表达量分别约为8.5％、15.8％, 经高效液相色谱分析表达产物在发酵上清液中的含量约为5.46、10.36mg/L; 表达产物TAC 和2TAC 对大肠杆菌K88 和金黄色葡萄球菌都具有明显的抑菌作用, 2TAC 经BrCN 水解产物抑菌活力高于TAC, 而2TAC 的表达产量大约是TAC 的1.89 倍。这表明通过tachyplesinⅠ串联表达产物降低了自身对宿主的毒性, 可以提高tachyplesinⅠ的表达产量。 |
| 关键词: 抗菌肽tachyplesinⅠ 表达载体构建 串联表达 抗菌活性 |
| DOI： |
| 分类号: |
| 基金项目:安徽省高校自然科学研究资助项目(KJ2009B014); 安徽省高等学校省级食品科学与工程特色专业建设点资助项目(20101091);安徽省食品科学与工程教学团队项目资助(20101094); 蚌埠学院优秀人才基金项目及院级教育教学研究项目(JYLC1015); 高校省级优秀人才基金项目(2011SQRL166) |
|
| High expression of antimicrobial peptide tachyplesinⅠ in Bacillus subtilis (BS168) |
|
|
| Abstract: |
| To produce tachyplesinⅠ, an antimicrobial peptide, by a stable and efficient gene engineering approach, cDNAs containing single tachyplesin gene sequence (tac) and tandem repeat of tachyplesin gene sequence (2tac) were, respectively, developed by annealing two synthesized complementary single-stranded DNAs and constructed into pSBPTQ shuttle vector. The vectors containing the target gene sequence were then transformed into Bacillus subtilis BS168. The secreted TAC and 2TAC were about 5.46 and 10.36 mg/L in the supernatant, respectively. Purified supernatant at different induced times was analyzed with SDS-PAGE. Then 2TAC was fragmented by BrCN. The antimicrobial activities of TAC, 2TAC and fragmented 2TAC were measured by the size of bacteriostatic circle of the fermentation supernatants against Escherichia coli K88. Ultrastructural alterations of E.coli K88 and S.aureus were observed under transmission electron microscopy. The results showed that in comparison with TAC, 2TAC was expressed at a higher level and also indicating strong antimicrobial activity in vitro. The amount of expression product of 2TAC was higher than TAC. BrCN-fragmented 2TAC showed higher activity than TAC. |
| Key words: tachyplesinⅠ construction of expression vector expression of tandem-arranged gene antimicrobial activity |
