串联抗菌肽Cecropin B基因在莱茵衣藻中的表达及其抗菌活性分析(英文)

Expression of Tandem Repeat Cecropin B in Chlamydomonas reinhardtii and Its Antibacterial Effect

为了应对各种抗生素在水产养殖业所带来的副作用,我们在本文中尝试利用微藻对一种抗菌肽进行表达的可行性研究.根据莱茵衣藻核基因组偏爱密码子对抗菌肽Cecropin B基因进行改造,并将4个经改造的Cecropin B基因依次串联起来,中间加上莱茵衣藻的自剪切连接肽序列LWMRFA,人工合成总长度为522 bp的串联Cecropin B基因.将串联Cecropin B基因克隆到含hsp70-RBCS2启动子和RBCS2终止子的pH105载体上,再与携带ble筛选基因的表达框架连接,构建重组表达载体pCB124.采用玻璃珠转化法将载体pCB124导入莱茵衣藻cc-849中,筛选得到能表达抗菌肽Cecropin B的转基因衣藻.经过6个月的保持培养后,进一步对转基因藻细胞提取液进行抗菌活性分析,发现转基因藻具有明显的抑制革兰氏阴性菌(大肠杆菌)和革兰氏阳性菌(枯草芽孢杆菌和溶壁微球菌)生长的特征.这一结果为具有抗菌活性的饵料藻的生产和应用提供了新的途径.

To overcome the negative effects of antibiotics commonly employed in most aquaculture,here we present a study to examine the feasibility of expressing an antimicrobial peptide by microalga as alternative.An antimicrobial peptide Cecropin B gene was modified according to the codon bias of the nuclear genome in Chlamydomonas reinhardtii.Four repeats of the Cecropin B gene were fused in tandem and each repeat was separated by inserting a cleavable linker peptide sequence（LWMRFA）.The artificial DNA（522 bp in length） was inserted into a site between hsp70-RBCS2 promoter and RBCS2 terminator for constructing the expression vector pCB124.A cell-wall deficient strain of C.reinhardtii CC-849 was transformed by using glass bead method with pCB124.A large number of transformants were selected on Tris-acetate-phosphate media containing 10 mg/L Zeomycin.PCR and RT-PCR analyses on the transformants revealed that tandem repeated Cecropin B gene had been integrated into the genome of C.reinhardtii and could express at transcriptional level.The Western blot results confirmed the presence of recombinant antimicrobial peptide Cecropin B in the transgenic algal cells.The total protein was extracted from transgenic algae and its antimicrobial activity was tested.The results indicated that the extracted proteins from transgenic alga showed very strong antimicrobial activity against both Gram negative bacterium（E.coli JM109） and Gram positive bacteria（Bacillus subtilis and Micrococcus lysodeikticus）.This finding has provided a new approach for production and utilization of antibacterial bait-algae.