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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.
In order to cope with the side effects of various antibiotics in aquaculture, we attempt to use the microalgae for the feasibility study of the expression of an antimicrobial peptide.According to the preference codon of the genome of Chlamydomonas reinhardtii, the antimicrobial peptide Cecropin B And the four modified Cecropin B genes were connected in series with LWMRFA of Chlamydomonas reinhardtii in the middle to synthesize a tandem Cecropin B gene with a total length of 522 bp.The tandem Cecropin B gene Cloned into pH105 vector containing hsp70-RBCS2 promoter and RBCS2 terminator, and then ligated with the expression frame carrying the ble screening gene to construct the recombinant expression vector pCB124. The vector pCB124 was introduced into Chlamydomonas reinhardtii cc-849 by glass bead transformation , And the transgenic Chlamydomonas reinhardtii could be screened to express the antimicrobial peptide Cecropin B. After six months of keeping culture, the antibacterial activity of the extract of transgenic algae was further analyzed, and it was found that the transgenic alga has significant inhibitory effect on Gram-negative bacteria ) And Gram-positive bacteria (Bacillus subtilis and Micrococcus lysarium) growth of the characteristics of the results for the antibacterial activity of feed algae students Production and application provides a new way.