鸭β防御素10基因的克隆、遗传进化分析及其生物学特性的初步研究

畜牧兽医学报 ›› 2009, Vol. 40 ›› Issue (9): 1320-1326.doi:

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鸭β防御素10基因的克隆、遗传进化分析及其生物学特性的初步研究

廖文艳^1，2，马得莹¹*，王瑞琴^1，2，韩宗玺²，邵昙昊²，李慧昕²，刘胜旺²

1.东北农业大学动物营养研究所，哈尔滨 150030；2.中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室禽传染病研究室, 哈尔滨 150001

收稿日期:2008-08-07 修回日期:1900-01-01 出版日期:2009-09-24 发布日期:2009-09-24
通讯作者: 马得莹

mRNA Cloning, Evolutionary Analysis and Biological Characterization of Duck Avian Betadefensin 10

LIAO Wenyan^1,2,MA Deying ¹* , WANG Ruiqin ^1,2,HAN Zongxi ², SHAO Tanhao², LI Huixin ², LIU Shengwang ²

1.Institute of Animal Nutrition, Northeast Agricultural University, Harbin 150030, China; 2. Division of Avian Infectious Disease, National Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150001, China

Received:2008-08-07 Revised:1900-01-01 Online:2009-09-24 Published:2009-09-24
Contact: MA Deying

摘要/Abstract

摘要：

旨在从鸭肝脏组织中克隆鸭β防御素10（AvBD10）基因，并在原核表达重组鸭AvBD10蛋白，研究重组鸭AvBD10蛋白的体外抗菌活性与理化特性。试验采用RTPCR法，从鸭肝脏组织中扩增鸭AvBD10基因，测定该基因在鸭各组织器官中的分布；将鸭AvBD10基因克隆到大肠杆菌原核表达载体pGEX6p1上，构建重组表达质粒pGEXduck AvBD10，将重组质粒转化大肠杆菌BL21，用IPTG诱导表达；亲和层析纯化蛋白，利用薄层平皿琼脂糖孔穴扩散法测定重组鸭AvBD10蛋白的体外抗菌活性与理化特性。结果表明，鸭AvBD10 cDNA大小为169 bp，编码55个氨基酸残基，与鸡AvBD10氨基酸同源性为85.5%。该基因仅在不同日龄鸭肝脏与肾脏组织中大量表达。重组鸭AvBD10融合蛋白的分子量约为30 ku，重组蛋白占菌体总蛋白的34%。重组鸭AvBD10蛋白对多杀性巴氏杆菌、金黄色葡萄球菌和枯草芽孢杆菌有抗菌活性。结果，从鸭肝脏组织中扩增了鸭AvBD10基因，该基因仅在不同日龄鸭肝脏与肾脏组织中大量表达。重组鸭AvBD10融合蛋白具有广谱抗菌活性，该重组蛋白对温度和酸碱度有很高的稳定性。

Abstract:

The objective of the study was to clone avian βdefensin10 (AvBD10) gene from duck liver. In addition, expression of recombinant AvBD10 protein in E. coli and determination of its antimicrobial activity were investigated. The mRNA of duck AvBD10 was cloned from liver of duck by RTPCR. The mRNA differential expression of the AvBD10 gene has been demonstrated across a panel of tissues in duck. The cDNA of duck AvBD10 was subcloned into pGEX6p1 vector to construct recombinant plasmid pGEXduck AvBD10. The recombinant plasmid was transformed into E. coli BL21 and the bacteria were induced with IPTG. The recombinant fusion protein was purified by affinity chromatograph. Antimicrobial activity and physiochemical characteristics of the recombinant fusion protein were measured by inhibition zone assay. The results showed that the cDNA of duck AvBD10 consist of 169 bp encoding 55 amino acids. The duck AvBD10 had the highest amino acid homology (85.5%) with the chicken AvBD10. It was shown that the mRNA of duck AvBD10 just expressed in the liver and kidney of ducks at all ages investigated. The molecular weight of recombinant duck AvBD10 protein is about 30 ku (accounted for approximately 34% of the total proteins). The recombinant duck AvBD10 protein exhibit expected antimicrobial activity against Bacillus subtilis, Staphylococcus aureus, and Pasteurella multocida after purified. In conclusion, AvBD10 was cloned from duck liver. It was showed that the mRNA of duck AvBD10 expressed highly in the liver and kidney of duck at all ages investigated. The recombinant duck AvBD10 protein exhibit broadly antimicrobial activity, and the recombinant protein retained high antimicrobial activity under different temperatures and pH.

廖文艳;马得莹;王瑞琴;韩宗玺;邵昙昊;李慧昕;刘胜旺 . 鸭β防御素10基因的克隆、遗传进化分析及其生物学特性的初步研究[J]. 畜牧兽医学报, 2009, 40(9): 1320-1326.

LIAO Wenyan;MA Deying ;WANG Ruiqin;HAN Zongxi;SHAO Tanhao;LI Huixin;LIU Shengwang . mRNA Cloning, Evolutionary Analysis and Biological Characterization of Duck Avian Betadefensin 10[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2009, 40(9): 1320-1326.

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