克氏原螯虾一种新型甲壳素基因的鉴定和抑菌功能研究

IDENTIFICATION OF A NEW CRUSTIN AND STUDY OF ITS ANTIBACTERIAL FUNCTION FROM PROCAMBARUS CLARKII

  • 摘要: 抗菌肽是无脊椎动物非特异性免疫系统中重要的免疫因子,抗菌肽具有良好且广谱的抗菌活性,并且不易产生耐药性,有望替代传统抗生素药物发挥功能。甲壳素(Crustin)是目前甲壳类动物中研究比较广泛的一类抗菌肽家族。研究鉴定了来自克氏原螯虾( Procambarus clarkii )中的一种新型Crustin基因,命名为 Pc-CruL 。通过 Pc-CruL 抵抗病原性细菌的免疫防御反应的初步探究,发现 Pc-CruL 对克氏原螯虾的天然免疫系统至关重要,为深入研究克氏原螯虾奠定了一定的基础,在克氏原螯虾的人工养殖过程中的疾病的预防和治疗中具有一定的指导意义且提供了一定的理论基础。 Pc-CruL 包含330 bp,编码109个氨基酸,与已知的克氏原螯虾甲壳素同源性较低,为Ⅰ型甲壳素蛋白家族新成员。 Pc-CruL 在健康的克氏原螯虾的血淋巴、鳃、肠道、胃、心和肝胰腺中均存在,在血淋巴中的含量最高,而在肝胰腺中含量最低。同时进行了体外抑菌实验,结果表明Pc-CruL重组蛋白显著地抑制了几种常见的病原性细菌的生长,具有良好的广谱抑菌活性。体内实验表明,对健康的克氏原螯虾注射Pc-CruL重组蛋白可以保护克氏原螯虾,在副溶血弧菌感染后,提高存活率。 Pc-CruL 是克氏原螯虾中关键的免疫因子,在克氏原螯虾抵抗外界病原菌入侵和感染过程中起着重要的作用。

     

    Abstract: The crayfish (Procambarus clarkii) is one of the important economic shrimp species which is widely distributed in the southeastern and central regions of China. In recent years, with the continous rise of peoples’ demand and the continous development of aquaculture industry, various diseases in the aquaculture have frequently outbroken, including bacterial and viral diseases, causing huge economic losses to the crayfish culture industry. As an invertebrate Procambarus clarkii lacks an adaptive immune system, which mainly resists the invasion of pathogens through the innate immune system. Antimicrobial peptide (AMP), as an important effector in the invertebrate’s innate immune system, is one of the substitutes for traditional antibiotics due to its broad-spectrum antibacterial activity and resistance to drug resistance. Crustin is one of the most widely studied antibacterial peptide in Procambarus clarikii. This study amplified and identified a new type of Crustin from Procambarus clarikii, named Pc-CruL. In order to reveal the function of Pc-CruL in Procambarus clarikii, antibacterial activity was determined by means of recombinant expression in vitro. It was hoped to reveal its relationship with pathogens in the form of the prevention and control of the disease and the opening of medicines for Procambarus clarikii.  Amplifying the Pc-CruL gene in Procambarus clarikii and ligating this gene to the PGEX-4T-1 vector to construct a prokaryotic expression plasmid. The recombinant plasmid was induced and purified in competent BL21 (DE3) for in vitro and in vivo antibacterial experiment. The CDS region of Pc-CruL contains 330 bp and encodes 109 amino acids. Pc-CruL is expressed in all tissues of normal Procambarus clarikii, with higher expression levels in hemocytes and relatively lower expression levels in hepatopancreas. The results of liquid antibacterial assay showed that Pc-CruL protein significantly restricted the growth of Vibrio parahaemolyticus, Staphylococcus aureus, Aeromonas hydrophila, and Aeromonas veronii, and had broad-spectrum antibacterial activity. The injection of exogenous recombinant Pc-CruL protein could improve the survival rate of infection with Vibrio parahaemolyticus. In general, Pc-CruL is an important immune molecule in Procambarus clarikii and plays a vital role in defending against bacterial infections.

     

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