《厦门大学学报（自然科学版）》

采用Piscidin基因串联表达的方案,通过Eco31Ⅰ限制性内切酶定向连接方法合成Pseudosciaena crocea-Sciaenops ocellatus-piscidin(PSP)串联基因.以毕赤酵母(Pichia pastoris)的穿梭表达载体pPICZαA构建重组表达质粒pPICZαA-PSP,转化入毕赤酵母SMD1168菌株中.应用实时荧光定量PCR方法进行多拷贝克隆筛选,实现了PSP重组串联肽的诱导表达和纯化,并对其抑菌活性进行初步鉴定,为piscidin抗菌肽的生产应用奠定基础.

In this study, the Pseudosciaena crocea-Sciaenops ocellatus-piscidin(PSP)tandom gene was constructed using a directional ligation method with Eco31Ⅰ restriction endonuclease. The recombinant expression plasmid pPICZαA-PSP was constructed based on shuttle expression vector PPICZαA for Pichia pastoris, and then transformed into the P. pastoris strain SMD1168. The multi-copy transformants were screened using real-time fluorescent quantitative PCR, and the recombinant PSP tandom peptide was successfully induced expressed and purified, and its antimicrobial activity was preliminarily identified as well, providing basis for production and application of piscidin antimicrobial peptide.

引言
1 材料与方法
2 结果与分析
3 讨论

图1 PSP串联基因设计<br/>Fig.1 Design of PSP tandom gene

图1 PSP串联基因设计
Fig.1 Design of PSP tandom gene

表1 本研究所使用的引物<br/>Tab.1 Primers used in this study

表1 本研究所使用的引物
Tab.1 Primers used in this study

图2 pPICZαA-PSP质粒PCR检测(a)与双酶切验证(b)<br/>Fig.2 PCR detection(a)and double enzyme digestionconfirmation(b)of pPICZαA-PSP plasmid

图2 pPICZαA-PSP质粒PCR检测(a)与双酶切验证(b)
Fig.2 PCR detection(a)and double enzyme digestionconfirmation(b)of pPICZαA-PSP plasmid

图3 阳性克隆筛选<br/>Fig.3 Positive clone screening

图3 阳性克隆筛选
Fig.3 Positive clone screening

图4 GAP基因和PSP串联基因的标准曲线<br/>Fig.4 Standard curves of GAP gene and PSP tandom gene

图4 GAP基因和PSP串联基因的标准曲线
Fig.4 Standard curves of GAP gene and PSP tandom gene

图5 PSP串联肽表达产物的tricine-SDS-PAGE检测(a)和Western blot分析(b)<br/>Fig.5 Tricine-SDS-PAGE detection(a)and Western blot analysis(b)of the PSP tandom peptide expression product

图5 PSP串联肽表达产物的tricine-SDS-PAGE检测(a)和Western blot分析(b)
Fig.5 Tricine-SDS-PAGE detection(a)and Western blot analysis(b)of the PSP tandom peptide expression product

表2 PC-piscidin、SO-piscidin和PSP的MIC比较<br/>Tab.2 Comparison of MIC of PC-piscidin, SO-piscidin and PSP

表2 PC-piscidin、SO-piscidin和PSP的MIC比较
Tab.2 Comparison of MIC of PC-piscidin, SO-piscidin and PSP

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引言

1 材料与方法

2 结果与分析

3 讨论

学报简介

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引言

1 材料与方法

2 结果与分析

3 讨 论

学报简介

3 讨论