CgAATase with specific expression pattern can be used as a potential surface marker for oyster granulocytes
CgAATase with specific expression pattern can be used as a potential surface marker for oyster granulocytes
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具有特定表达模式的CgAATase可作为牡蛎粒细胞的潜在表面标记物
DOI：
10.1016/j.fsi.2019.01.003
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发表时间：
2019-04
影响因子：
4.7
通讯作者：
Song Linsheng
中科院分区：
文献类型：
--
作者：
Dong Miren;Song Xiaorui;Wang Min;Wang Weilin;Zhang Peng;Liu Yu;Li Meijia;Wang Lingling;Song Linsheng
粒细胞是牡蛎的主要免疫活性血细胞，在牡蛎的免疫防御中起着重要作用。本研究从牡蛎C中鉴定出一种具有特异表达模式的醇酰基转移酶（CgAATase）。gigas，可作为分离牡蛎粒细胞的潜在标记。CgAATase的开放阅读框（ORF）为1431 bp，编码476个氨基酸，具有典型的保守结构域。CgAATase mRNA在血细胞中的表达量最高，在肝胰腺、外套膜、性腺、鳃、神经节、闭壳肌和唇须中的表达量较低。在灿烂弧菌刺激后3-12 h，血细胞中CgAATase mRNA表达水平显著上调，6 h时达到最高水平，为对照组的27.40倍（p< 0.05）。Percoll®密度梯度离心法将血细胞分为粒细胞、半粒细胞和无粒细胞，粒细胞中CgAAT酶的表达量占优势，分别是无粒细胞和半粒细胞的8.26倍（p< 0.05）和2.80倍（p< 0.05）。制备了抗CgAAT酶的单克隆抗体，用免疫磁珠分离粒细胞，发现CgAAT酶蛋白主要分布在粒细胞膜上。用抗CgAAT酶单克隆抗体包被的免疫磁珠进行流式细胞术可成功分离出85.7 ± 4.60%的粒细胞CgAAT酶阳性，其余97.7 ± 1.01%的无粒细胞和半粒细胞CgAAT酶阴性。分离的原代粒细胞在体外培养中可保持细胞活性一周以上，并有大量丝状伪足。这些结果表明，CgAATase是牡蛎粒细胞的潜在标志物，用抗CgAATase单克隆抗体包被的免疫磁珠可以有效地从总循环血细胞中分离出粒细胞。
Granulocytes are known as the main immunocompetent hemocytes that play important roles in the immune defense of oyster Crassostrea gigas. In the present study, an alcohol acyltransferase (designed asCgAATase) with specific expression pattern was identified from oysterC. gigas, and it could be employed as a potential marker for the isolation of oyster granulocytes. The open reading frame (ORF) ofCgAATase was of 1431 bp, encoding a peptide of 476 amino acids with a typically conserved AATase domain. The mRNA transcripts ofCgAATase were highest expressed in hemocytes, lower expressed in hepatopancreas, mantle, gonad, gill, ganglion, adductor muscle, and labial palp. The mRNA expression level ofCgAATase in hemocytes was significantly up-regulated at 3–12 h and reached the highest level (27.40-fold compared to control group,p< 0.05) at 6 h after Vibrio splendidus stimulation. The total hemocytes were sorted as granulocytes, semi-granulocytes and agranulocytes by Percoll®density gradient centrifugation.CgAATase transcripts were dominantly observed in granulocytes, which was 8.26-fold (p< 0.05) and 2.80-fold (p< 0.05) of that in agranulocytes and semi-granulocytes, respectively. The monoclonal antibody againstCgAATase was produced and employed for the isolation of granulocytes with the immunomagnetic bead.CgAATase protein was mainly detected on the cytomembrane of granulocytes. About 85.7 ± 4.60% of the granulocytes were positive forCgAATase and they could be successfully separated by flow cytometry with immunomagnetic bead coated with anti-CgAATase monoclonal antibody, and 97.7 ± 1.01% of the rest hemocytes (agranulocytes and semi-granulocytes) were negative forCgAATase. The isolated primary granulocytes could maintain cell activity for more than one weekin vitroculture that exhibited numerous filopodia. These results collectively suggested thatCgAATase was a potential marker of oyster granulocytes, and the granulocytes could be effectively isolated from total circulating hemocytes by immunomagnetic bead coated with the anti-CgAATase monoclonal antibody.
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