中国明对虾T7噬菌体展示文库的构建与免疫相关基因的淘选

刘宁; 杜欣军; 赵小凡; 王金星

中国明对虾T7噬菌体展示文库的构建与免疫相关基因的淘选

1.
山东大学, 生命科学学院, 山东, 济南, 250100;山东泉港药业有限公司, 山东, 济南, 250014
2.
山东大学, 生命科学学院, 山东, 济南, 250100

基金项目: 国家自然科学基金资助(30470222);高等学校博士学科点专项科研基金(20060422034)资助

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出版历程
- 收稿日期: 2006-03-08
- 修回日期: 2007-01-29

Panning for immune-related genes from T7 phage display library constructed using Chinese shrimp’s hemocyte RNA

1.
School of Life Sciences Shandong University, Jinan 250100, China;Shandong Quangang Pharmaceutical Con Led Jinan 250014, China
2.
School of Life Sciences Shandong University, Jinan 250100, China

摘要

摘要: 利用淘选噬菌体展示文库的方法,获取中国明对虾(Fenneropenaeus chinensis)的免疫相关基因.首先用中国明对虾的血细胞mRNA构建了其T7噬菌体展示文库,文库滴度达到1.1×10¹¹pfu/cm³,然后用不同的配体(脂多糖/几丁质/不同的细菌)进行文库淘选.淘选3轮后随机挑取数个噬菌斑,用T7 select 10-3b载体引物扩增插入片段,将不同长度的片段克隆入pMD 18-T载体后进行测序.把基因序列在GenBank数据库进行BLAST搜索比对,同时在ExPASy进行编码蛋白质的结构和性质的预测.经不同配体淘选,获得了16个基因片段,其中围食膜蛋白、蛋白质精氨酸N端转甲基酶、血小板反应素、RNA结合蛋白、核自身免疫精蛋白等基因,首次在中国明对虾中发现.
- T7噬菌体展示 /
- 先天免疫 /
- 病原相关分子模式 /
- 中国明对虾
Abstract: The objective was to find and identify immune-related genes of Chinese shrimp (Fenneropenaeus chinensis) by panning phage display library T7 phage display libraries were constructed with the shrimp hemocyte mRNA following the manufactures instruction.The titer of the Library was 1.1×10¹¹pfu/cm³.The library was panning by various ligands, including lipopoly saccharide, chitin and different kinds of bacteria, such as gram positive bacteria, Staphylococcusaureus, Bifidobacteriumlactis and Micrococcusluteus, and gramnegative bacteria, Escherichia coli, Vibrio anguillarum.After three rounds of phage panning, the inserts in the phage was amplified by PCR with T7 select 10-3b vector primers, and subcloned into pMD 18T vecter for sequencing.The sequences were analyzed by BLAST Sixteen fragments were obtained.Among them, peritrophin, Proteinarg inine N-methyltransferase, thr mbospondin, RNA binding protein, and nuclear autoantigenic sperm protein which were firstly reported in Chinese shrimp.The expression profile of peritrophin was analyzed by Reverse Transcription-PCR (RT-PCR), the results showed that it was reducing expression in hemocytes, heart, stomach, gut, gill and sperm in infected shrimp.This surgested that the peritriphin may be involved in innate immunity.Other genes panned from the library need further study.
- T7 phage display /
- innate immunity /
- pathogen associated molecular patterns /
- Chinese shrimp

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参考文献(15)

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