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半乳糖凝集素(Galectin)是动物凝集素家族的一员,在脊椎动物和无脊椎动物中都参与了重要的免疫防御反应,是一种重要的免疫因子。通过RT-PCR和RACE技术克隆获得了三疣梭子蟹(Portunus trituberculatus)半乳糖凝集素基因全长,并命名为PtGal。该基因全长875bp,开放阅读框为669bp,编码222个氨基酸,包含一个糖识别结构域CRD(位于16—142位氨基酸),预测蛋白的分子量(Mw)为23529.4Da,理论等电点(p I)为5.21。同源性比对结果显示,PtGal编码的氨基酸序列与中华绒螯蟹、南美白对虾、日本囊对虾galectin的同源性分别为77%、66%、58%。实时荧光定量PCR(qRT-RCR)结果表明,PtGal在三疣梭子蟹肝胰腺、肌肉、肠、胃、鳃、心脏、性腺中都有表达,其中在性腺中的表达量最高,在肝胰腺中的表达量最低,但人工感染溶藻弧菌(Vibrio alginolyticus)后,肝胰腺的表达呈先升高后降低的趋势。利用原核表达系统对三疣梭子蟹PtGal进行了重组表达,并通过纯化复性获得了重组目的蛋白rPtGal,ELISA检测rPtGal与不同细菌及真菌的结合活性,结果表明,该重组蛋白与革兰氏阳性菌、阴性菌和真菌均有较强的结合。本研究为进一步深入研究三疣梭子蟹半乳糖凝集素的功能奠定了基础。
Galectin, a member of the animal lectin family, is an important immunological factor involved in both important immune defenses in both vertebrates and invertebrates. The full-length galectin gene of Portunus trituberculatus was cloned by RT-PCR and RACE techniques and named as PtGal. The full length of this gene is 875bp and contains an open reading frame of 669bp and encodes a polypeptide of 222 amino acids with a sugar recognition domain CRD (amino acids 16-142). The predicted molecular weight (Mw) is 23529.4Da. The theoretical isoelectric point I) is 5.21. Homology comparison showed that the deduced amino acid sequences of PtGal were 77%, 66% and 58% homologous with galectin of Chinese mitten crab, Penaeus vannamei and Japanese shrimp respectively. Real-time quantitative PCR (qRT-RCR) results showed that PtGal was expressed in hepatopancreas, muscle, intestine, stomach, gill, heart and gonad of Portunus trituberculatus, with the highest expression in gonads The lowest expression, but the artificial infection of Vibrio alginolyticus, hepatopancreas expression increased first and then decreased. The prokaryotic expression system was used to express PtGal of Portunus trituberculatus. The recombinant protein rPtGal was obtained by purification and renaturation. The binding activity of rPtGal with different bacteria and fungi was detected by ELISA. The results showed that the recombinant protein interacts with Gram-positive bacteria , Negative bacteria and fungi have a strong combination. This study lays the foundation for further study on the function of glandulactin in Portunus tritubus.