香鱼主要组织相容性复合体MHCIIB的分子克隆及其在鳗弧菌感染下的功能鉴定

Molecular Cloning of The Major Histocompatibiliby Complex ΙΙB and its Function Identification in Ayu( Plecoglossus altivelis) on Vibrio anguillarum Infection

主要组织相容性复合体(major histocompatibility complex classⅡ, MHCⅡ)在脊椎动物免疫反应中发挥重要作用。本研究从香鱼(Plecoglossus altivelis)单核/巨噬细胞(monocytes/macrophages,MO/MФ)转录组中获得了MHC II基因β链(PaMHCIIB) cDNA序列。PaMHCIIB由920个核苷酸组成,包含一个大的开放阅读框,编码251个氨基酸,预测分子质量为28.23 kD。氨基酸序列分析表明,PaMHCIIB具有MHC IIB的典型特征,主要包括信号肽、2个胞外区和1个保守的connecting peptide/transmembrane/cytoplasmic (CP/TM/CYT)结构域,与北极红点鲑(Salvelinus alpinus) MHC IIB同源性最高,为65.04%;系统发育分析表明,PaMHCIIB与北极红点鲑MHC IIB进化相关性最高。实时荧光定量PCR(quantitative real-time PCR, qPCR)结果显示,PaMHCIIB mRNA主要在香鱼鳃、肠和脾中表达;鳗弧菌感染(Vibrio anguillarum)后香鱼肝中PaMHCIIB mRNA在感染后12 h(hours post infection, hpi)时上调显著,在24 hpi达到峰值,为对照组的3.18倍,脾、头肾、肠和鳃中PaMHCIIB mRNA均在4 hpi时上调显著,分别在4、8、24和12 hpi时达到峰值,分别为对照组的85.18、2.06、4.21和6.81倍(P<0.05)。香鱼MO/MФ经鳗弧菌感染后,PaMHCIIB mRNA的表达水平在4 hpi时上调显著,在12 hpi时达到峰值,为对照组的3.35倍(P<0.05)。原核表达了PaMHCIIB胞外区并制备其抗体。Western印迹分析结果表明,香鱼MO/MФ中PaMHCIIB具有N糖基化修饰,且鳗弧菌感染后其蛋白表达水平在12 hpi时显著增加,在24 hpi时达到峰值,为对照组的3.19倍(P<0.05)。抗体封闭PaMHCIIB后,香鱼MO/MФ吞噬活性被抑制,为对照组的0.28倍(P<0.05),而且鳗弧菌诱导的4个细胞因子TNF-α、IL-1β、IL-10和TGF-β表达均受到抑制,均在8 hpi时被抑制最明显,表达量分别为对照组的0.23、0.41、0.51和0.20倍(P<0.05)。本研究结果揭示PaMHCIIB可能参与香鱼MO/MФ抵抗病原菌感染的免疫防御。

The major histocompatibility complex class Ⅱ （MHC Ⅱ） plays pivotal roles in the immune system of vertebrates. We obtained the gene （PaMHCⅡB） encoding the β chain of MHC Ⅱ molecule in ayu （Plecoglossus altivelis） , with de novo transcriptome sequencing of ayu monocytes/macrophages （MO/ Mφ）. The full-length cDNA sequence of the gene was 920 nucleotides, containing a large open reading frame that encoded 251 amino acids, and the molecular mass was deduced to be 28.23 kD. Amino acid sequence analysis showed that PaMHCⅡB possessed the typical signatures of the MHC ⅡB, including the signal peptide, two extracellular domains, and one connecting peptide/transmembrane/cytoplasmie （CP/ TM/CYT） domain. PaMHCⅡB shared the highest amino acid sequence identity （65.04%） with that of Arctic charr （Salvelinus alpinus）. Phylogenetic analysis showed that PaMHCⅡB was most closely related to the MHC ⅡB of Arctic charr. Quantitative real-time PCR （qPCR） analysis showed that the PaMHCⅡB transcript was mainly expressed in the gill, intestine, and spleen. After infection by Vibrio anguillarum, PaMHCⅡB mRNA in the liver was significantly upregulated at 12 hours post infection （hpi） , reaching a peak value of 3.18-fold of at 24 hpi （P 〈 O. 05 ） ; PaMHCⅡB mRNA in the spleen, head kidney, intestine, and gill were all significantly upregulated at 4 hpi, reaching the peak values of 85. 18-, 2.06-, 4.21-, and 6. 81-fold, at 4, 8, 24, and 12 hpi （P 〈0.05） , respectively. The PaMHCⅡB transcription increased in MO/Mφ after in vitro stimulation with V. anguiUarum, reaching a peak value of 3.35-fold at 12 hpi （P 〈0.05）. The extracellular domain of PaMHCⅡB （PaMHCⅡBex） was overexpressed, and the antibodies against PaMHCⅡBex were prepared for detecting N-glycosylation of PaMHCⅡB or for neutralizing PaMHCⅡB in ayu MO/Mφ. Western blotting analysis showed that the native PaMHCⅡB protein was definitely N-glycosylated in ayu MO/Mφ, and the PaMHCⅡB protein level also increased in MO/Mφ after in vitro stimulation with V. anguillarum, reaching a peak value of 3. 19-fold at 24 hpi （P 〈 O. 05 ）. Furthermore, PaMHCⅡB neutralization remarkably restrained the MO/Mφ phagocytosis in ayu （0.28-fold） （P 〈0.05）, and the mRNA expression of TNF-α, IL-1β, IL-10, and TGF-β induced by V. anguillarum was also inhibited, reaching the minimum （ 0.23-, 0.41- 0.51-, and 0.20-fold, respectively） at 8 hpi （P 〈 0.05）. Overall, these results demonstrate the crucial role of PaMHCⅡB against bacterial challenge, and suggest its involvement in the host resistance to pathogen infection in ayu MO/Mφ.