cDNA cloning, expression and antibacterial activity of lysozyme C in the blue shrimp (Litopenaeus stylirostris)  

作　　者：Weijun Mai Chaoqun Hu 

机构地区：[1]Applied Marine Biology Laboratory, South China Sea Institute of Oceanology, Science Academy of China, Guangzhou 510301, China [2]Graduate University of Chinese Academy of Sciences, Beijing 100049, China

出　　处：《Progress in Natural Science:Materials International》2009年第7期837-844,共8页自然科学进展·国际材料（英文版）

基　　金：supported by the National High Technology Development Project of China (Grant No.2006AA10A406)

摘　　要：The gene coding for lysozyme in blue shrimp （Litopenaeus stylirostris） was cloned, sequenced and expressed in pET-32a vector. The deduced amino acid sequence of F. merguiensi lysozyme showed 37-93% similarity with the mouse, human, chicken, and tiger prawn counterparts. The lysozyme was purified to homogeneity and observed as a band of approximately 15 kDa in 15% SDS-PAGE. Semi-quantitative RT-PCR analysis demonstrated that mRNA transcripts of lysozyme could be mainly detected in the tissues of haemocytes, gill, gonad and the lymphoid organ of unchallenged shrimps, whereas the expression of lysozyme transcripts was increased in all the tested tissues after the heat-killed Vibrio alginolyticus challenge. The temporal expression of lysozyme mRNA in haemolymph challenged by Micrococcus luteus and I4. alginolyticus was both up-regulated and reached the maximum level at 8 and 16 h post-stimulation, respectively, and then dropped back to the original level. Bacteriolytic activity of the lysozyme against different bacterial cultures was deter-mined by the solid phase and turbidimetric assays. The results demonstrated that the lysozyme we obtained was not only against Gram- positive and Gram-negative bacteria but also against shrimp pathogens V. alginolyticus and V. parahemolyticus. In addition, the study of the inhibition mechanism revealed that the antibacterial activity of the lysozyme was a result of the bactericidal effect.@2009 National Natural Science Foundation of China and Chinese Academy of Sciences. Published by Elsevier Limited and Science in China Press. All rights reserved.The gene coding for lysozyme in blue shrimp (Litopenaeus stylirostris) was cloned, sequenced and expressed in pET-32a vector. The deduced amino acid sequence of F. merguiensi lysozyme showed 37–93% similarity with the mouse, human, chicken, and tiger prawn counterparts. The lysozyme was purified to homogeneity and observed as a band of approximately 15 kDa in 15% SDS–PAGE. Semiquantitative RT-PCR analysis demonstrated that mRNA transcripts of lysozyme could be mainly detected in the tissues of haemocytes, gill, gonad and the lymphoid organ of unchallenged shrimps, whereas the expression of lysozyme transcripts was increased in all the tested tissues after the heat-killed Vibrio alginolyticus challenge. The temporal expression of lysozyme mRNA in haemolymph challenged by Micrococcus luteus and V. alginolyticus was both up-regulated and reached the maximum level at 8 and 16 h post-stimulation, respectively, and then dropped back to the original level. Bacteriolytic activity of the lysozyme against different bacterial cultures was determined by the solid phase and turbidimetric assays. The results demonstrated that the lysozyme we obtained was not only against Grampositive and Gram-negative bacteria but also against shrimp pathogens V. alginolyticus and V. parahemolyticus. In addition, the study of the inhibition mechanism revealed that the antibacterial activity of the lysozyme was a result of the bactericidal effect.

关 键 词：Lysozyme C Litopenaeus stylirostris PCR-cloning EXPRESSION Antibacterial activity 

分 类 号：S859.796[农业科学—临床兽医学] Q78[农业科学—兽医学]