中国组织工程研究

中国组织工程研究 ›› 2011, Vol. 15 ›› Issue (24): 4549-4552.doi: 10.3969/j.issn.1673-8225.2011.24.044

• 组织构建基础实验 basic experiments in tissue construction • 上一篇    下一篇

抗菌肽Cecropin A基因原核表达及表达产物的鉴定

陈  磊   

  1. 河南中医学院第一附属医院心内科，河南省郑州市  450000
  • 收稿日期:2011-01-09 修回日期:2011-05-20 出版日期:2011-06-11 发布日期:2011-06-11
  • 作者简介:陈磊★，女，1978年生，河南省郑州市人，2006年郑州大学毕业，硕士，主治医师，主要从事冠心病的诊断与治疗方面的研究。

Prokaryotic expression of antibiotic peptide Cecropin A gene and identification of expression products

Chen Lei   

  1. Department of Cardiology, First Affiliated Hospital, Henan University of Chinese Medicine, Zhengzhou  450000, Henan Province, China
  • Received:2011-01-09 Revised:2011-05-20 Online:2011-06-11 Published:2011-06-11
  • About author:Chen Lei★, Master, Attending physician, Department of Cardiology, First Affiliated Hospital, Henan University of Chinese Medicine, Zhengzhou 450000, Henan Province, China sqh@hpu.edu.cn

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405

被引次数

8

摘要：

背景：Cecropins是一种具有抗菌活性的小分子蛋白质。采用真核细胞表达或人工合成Cecropins，效率低、成本高。
目的：克隆表达家蝇抗菌肽基因Cecropin A，并对其重组表达产物进行鉴定。
方法：依据GenBank中家蝇Cecropin A基因序列设计特异性引物，用RT-PCR从家蝇幼虫组织中扩增Cecropin A成熟肽基因，将其克隆入原核表达载体pET32a中，与表达载体中的Thioredoxin基因构成融合基因，并转化E.coli BL21。经异丙基-β-D硫代半乳糖苷诱导表达。采用家蝇幼虫血淋巴免疫家兔获得抗血清；Western blot和三羟甲基氨基甘氨酸-十二烷基硫酸钠-聚丙烯酰胺凝胶电泳鉴定诱导的重组蛋白质。
结果与结论：经异丙基-β-D硫代半乳糖苷诱导，E.coli BL21可表达Cecropin A成熟肽，兔抗家蝇幼虫血清、三羟甲基氨基甘氨酸-十二烷基硫酸钠-聚丙烯酰胺凝胶电泳及Western blot结果证实表达产物为Cecropin A成熟肽。说明使用原核表达系统可以获得天然Cecropin A成熟肽。

关键词: 家蝇, Cecropin A, 多克隆抗体, 融合表达, 三羟甲基氨基甘氨酸-十二烷基硫酸钠-聚丙烯酰胺凝胶电泳

Abstract:

BACKGROUND: Cecropins are a kind of micromolecule protein with antibacterial activity. Eukaryotic cell-expressed or artificially synthesized Cecropins is characterized by low efficiency and high cost.
OBJECTIVE: To clone and express an antibacterial peptide gene of Musca domestica Cecropin A, and to identify recombinant expression product.
METHODS: Mature Musca domestica Cecropin A encoding nucleotide sequence was searched from the GenBank and amplified by RT-PCR. The gene of Musca domestica Cecropin A was cloned into prokaryotic expression vector pET32a and fused with gene of Thioredoxin (Trx) and expressed in E.coli BL2l (DE3). After induction by isopropyl-β-D-thiogalactoside, the sera of the immunized rabbits were collected after rabbits were immunized with the hemolymph of housefly larvae. Recombinant protein was identified by western blot analysis and N-[Tris(hydroxymethyl)methyl]glycine-sodium dodecylsulfate-polyacrylamide gel electrophoresis.
RESULTS AND CONCLUSION: After induction by isopropyl-β-D-thiogalactoside, E.coli BL21 expressed mature Cecropin. Rabbit anti- housefly larvae sera, N-[Tris(hydroxymethyl)methyl]glycine-sodium dodecylsulfate-polyacrylamide gel electrophoresis and western blot analysis results confirmed that expression products were mature Cecropin. These suggest that prokaryotic expression system can be utilized to obtain natural mature Cecropin.

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