剑尾鱼IgZ基因的克隆及疫苗免疫对其在组织中表达的影响

Identification and expression analysis of IgZ gene in swordtail fish(Xiphophorus hellri) responses to vaccine challenges

为研究剑尾鱼igZ基因序列及其在疫苗免疫后的表达规律，本实验根据已知的EST库设计引物，利用RT-PCR等方法，获得剑尾鱼冶z基因cDNA全长序列，并进行生物信息学分析和疫苗免疫后组织表达分析。结果显示，剑尾鱼堙z基因全长序列为5420bp，包含4个外显子和3个内含子；cDNA序列全长1527bp，包含一个1299bp的完整ORF框，该基因编码432个氨基酸，预测其编码蛋白的分子量大小为47．48ku，并具有IgZ的基本结构，与其他硬骨鱼类IgZ氨基酸序列一致性为28％～54％。荧光定量PCR分析结果显示，增z基因主要在剑尾鱼的头肾、脾脏和肠中分布，且疫苗免疫后11天内，lgZ基因在头肾、脾脏和肠组织中均表现为先上升后下降的趋势。头肾中IgZ基因的表达量在第4天时最高，为对照组的2．12倍；脾脏中IgZ基因在第4天时呈现最高峰，为对照组的4．65倍；肠组织中坛z基因的表达量在12h时有一个小高峰，第2天时最高，为对照组的11．41倍。本研究获得了剑尾鱼坛z基因cDNA全长序列，并对其表达规律进行了初步研究，发现IgZ在肠道黏膜免疫中具有重要作用，这将为进一步验证其在黏膜免疫中的作用以及剑尾鱼作为疾病研究模式动物和疫苗免疫评价模型奠定基础。

The full length DNA and cDNA of IgZ gene from swordtail fish Xiphophorus hellri were cloned. The obtained DNA of IgZ is 5 420 bp in length, which is composed of four exons and three introns. The length of cDNA is 1 527 bp,containing a 1 299 bp open reading frame（ORF） which encoded a protein of 432 amino acids with deduced protein molecular weight 47.48 ku. Alignment based on amino acid sequence indicates that swordtail fish IgZ shared similarities of 28% -54% with other fishes. IgZ mRNA expression of swordtail fish was predominantly detected in head kidney, spleen and intestines. The expression levels of IgZ in the head kidney and spleen both demonstrated peaks at 4 days post immunization, increasing of approximately 2 fold and 4 fold in comparison with control group, respectively. Moreover, the expression level of IgZ in intestines varied more sensitively with increasing about 11 fold in comparison with control in the second day after immunization. The results further reveal the IgZ gene plays an important role in the intestinal mucosal immunity. Furthermore, this study also indicates that the swordtail fish could be utilized as a useful fish model for the research of fish diseases and vaccine evaluation.