Zheng Wenbiao, Tian Chen, Chen Xinhua
Key Laboratory of Marine Biogenetic Resources, Third Institute of Oceanography, State Oceanic Administration, 178 Daxue Road, Xiamen, PR China.
Immunol Lett. 2007 Nov 15;113(2):107-16. doi: 10.1016/j.imlet.2007.08.001. Epub 2007 Aug 27.
Lysozyme acts as an innate immunity molecule against the invasion of bacterial pathogens. Here, the cDNA of a goose-type lysozyme (g-lysozyme) was cloned from large yellow croaker (Pseudosciana crocea) by expressed sequence tags (EST) and RACE-PCR techniques. The full-length cDNA of large yellow croaker g-lysozyme (LycGL) is 716 nucleotides (nt) encoding a protein of 193 amino acids (aa), with a theoretical molecular weight of 21.3kDa. The deduced LycGL possessed the typical structural features of g-lysozyme, including three catalytic residues (E71, D84, D101) and four substrate binding sites (L97, L121, L128, G152). Genomic analysis revealed that the LycGL gene consisting of 2383nt, contained five exons interrupted by four introns and exhibited a similar exon-intron organization to its homologues in Japanese flounder and Chinese perch, except for having a much longer intron 1 in the LycGL gene. Recombinant LycGL produced in Pichia pastoris exhibited obvious lytic activity against Micrococcus lysodeikticus and several fish pathogenic bacteria such as Aeromonas sobria, Vibrio alginolyticus, Vibrio parahaemolyticus and Vibrio vulnficus. Tissue expression profile analysis showed that LycGL mRNA was constitutively expressed in all tissues examined, such as spleen, head kidney, intestine, liver, gills and heart, although at a different level. Upon stimulation with trivalent bacterial vaccine, LycGL mRNA levels in intestine, spleen and head kidney were quickly up-regulated and had 10.32-, 10.2- and 8.26-fold increases, respectively, and LycGL transcripts in intestine and head kidney reached their peak levels at 24h post-induction and then decreased gradually while LycGL mRNA in spleen increased to its highest level at 48h. These results suggest that LycGL may be involved in antibacterial immune response activated by bacterial vaccine as an acute-phase molecule.
溶菌酶作为一种先天性免疫分子，可抵御细菌病原体的入侵。在此，通过表达序列标签（EST）和RACE-PCR技术，从大黄鱼（Pseudosciana crocea）中克隆出鹅型溶菌酶（g-溶菌酶）的cDNA。大黄鱼g-溶菌酶（LycGL）的全长cDNA为716个核苷酸（nt），编码一个由193个氨基酸（aa）组成的蛋白质，理论分子量为21.3kDa。推导的LycGL具有g-溶菌酶的典型结构特征，包括三个催化残基（E71、D84、D101）和四个底物结合位点（L97、L121、L128、G152）。基因组分析表明，LycGL基因由2383nt组成，包含五个外显子，被四个内含子打断，并且与牙鲆和鲈鱼中的同源物相比，具有相似的外显子-内含子组织，只是LycGL基因中的内含子1长得多。在毕赤酵母中产生的重组LycGL对溶壁微球菌以及几种鱼类病原菌，如温和气单胞菌、溶藻弧菌、副溶血性弧菌和创伤弧菌，表现出明显的裂解活性。组织表达谱分析表明，LycGL mRNA在所有检测的组织中，如脾脏、头肾、肠道、肝脏、鳃和心脏中均组成性表达，尽管表达水平不同。在用三价细菌疫苗刺激后，肠道、脾脏和头肾中的LycGL mRNA水平迅速上调，分别增加了10.32倍、10.2倍和8.26倍，肠道和头肾中的LycGL转录本在诱导后24小时达到峰值水平，然后逐渐下降，而脾脏中的LycGL mRNA在48小时增加到最高水平。这些结果表明，LycGL可能作为急性期分子参与由细菌疫苗激活的抗菌免疫反应。
