Expression of Tandem Repeat Cecropin B in Chlamydomonas reinhardtii and Its Antibacterial Effect
Expression of Tandem Repeat Cecropin B in Chlamydomonas reinhardtii and Its Antibacterial Effect
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串联重复天蚕素B在莱茵衣藻中的表达及其抗菌作用
DOI：
10.3724/sp.j.1206.2010.00671
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发表时间：
2012-04-01
影响因子：
0.3
通讯作者：
Hu Zhang-Li
中科院分区：
文献类型：
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作者：
Mu Fei-Yun;Li Hui;Hu Zhang-Li
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（Chlamydomonas reinhardtii）核基因组的密码子偏好性，对抗菌肽Cecropin B基因进行了改造。将抗菌肽B基因的四个重复序列串联融合，并通过插入可裂解的接头肽序列（LWMRFA）将每个重复序列分开。将人工DNA（长度为522bp）插入hsp70-RBCS2启动子和RBCS2终止子之间的位点，以构建表达载体pCB124。本研究对一株C.用pCB124通过玻璃珠法转化莱茵衣藻CC-849。在含有10mg/L Zeomycin的Tris-乙酸盐-磷酸盐培养基上选择大量转化体。PCR和RT-PCR分析结果表明，Cecropin B基因已整合到拟南芥基因组中。并能在转录水平表达。Western blot结果证实了转基因藻细胞中存在重组抗菌肽Cecropin B。从转基因海藻中提取总蛋白并进行抑菌活性测试。结果表明，提取的转基因海藻蛋白对革兰氏阴性菌（E.大肠杆菌JM109）和革兰氏阳性菌（枯草芽孢杆菌和溶壁微球菌）。这一发现为抑菌饵料藻的生产和利用提供了一条新的途径。
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.