In order to produce tachyplesin I with stable construction by efficient gene engineering approach, the tachyplesin 1 gene (tac) was obtained by RT-PCR and the tandem repeat of tachyplesin I gene sequence (2tac) was developed by annealing two synthesized complementary single-stranded DNAs and constructed into pSBPTQ shuttk vector. The vectors containing thQ target gene sequence were then transformed into Bacillus subtilis WB800. Both expression of tac and 2tac were induced by 2% sucrose. The fermentation supernatant was purified by the means of salt precipitation, dialyse, ion-exchange chromatography in sequence. Then 2TAC was cleaved by BrCN. The antimicrobial activities of TAC, 2TAC and hydrolytic 2TAC were measured by the size of bacteriostatic circle of the fermentation supernatants against Eschenchia coli K88. Ultrastructural alterations of E. coli K88 and Salmonella typhimurium were observed under transmission electron microscopy. The results show that, in comparison with TAC, 2TAC is expressed at a higher level and both of them have strong antimicrobial activity against E. coli K88 and Salmonella typhimurium in vitro. The secreted TAC and 2TAC are about 8.25 and 17.36 mg-L"1 in the fermentation supernatant, respectively. 2TAC cleaved by BrCN has higher antimicrobial activity than TAC.%为了实现通过基因工程方法制备具有稳定结构的抗菌肽tachyplesin Ⅰ,采取了tachyplesinⅠ基因串联表达的方法.实验以高拷贝穿梭表达载体pSBPTQ为表达载体,通过RT-PCR扩增tachyplesin Ⅰ基因(tac)和采用直接退火的方法合成tachyplesin Ⅰ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WB800实现高效表达,经2％蔗糖诱导获得表达串联产物(2TAC)和TAC.通过离子交换柱层析纯化后得表达产物2TAC和TAC,2TAC经BrCN水解后,对表达产物抑菌活性分析,观察发酵液上清对大肠杆菌(E.coli K88)和伤寒沙门氏菌(Salmonella typhi)的抑菌圈和细胞微观形态的变化.实验结果表明:基因tac和2tac在枯草杆菌中表达成功,表达产物在发酵上清液中的含量分别约为8.25、17.36 mg·L-1;表达产物TAC和2TAC对大肠杆菌K88和伤寒沙门氏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.
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