一种凡纳滨对虾新的C型凝集素基因(LvLc2)的克隆及免疫应答特征
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Q786;S945.1

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天津市自然科学基金(19JCYBJC29700,15JCZDJC33800);天津市水产生态及养殖重点实验室开放基金(TJAE2015005);国家自然科学基金(31472299)


Molecular cloning and characterization in immune response of a novel C-type lectin gene (LvLc2) from Litopenaeus vannamei
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    摘要:

    为有效地进行凡纳滨对虾病害防治和健康养殖,探究对虾天然免疫机制,为其免疫防控提供新思路,实验根据本课题组前期转录组结果提示,在凡纳滨对虾血细胞中克隆获得了一个新的C型凝集素基因(LvLc2, GenBank登录号 KR020738)。生物信息学分析显示,LvLc2的ORF区全长465 bp,编码154个氨基酸(aa),5′-UTR 为11 bp,3′-UTR 为126 bp;其预测的分子量为17.16 ku,理论等电点为4.54;推断其氨基端含有一个由17个aa组成的信号肽序列,羧基端含有一个保守的糖识别结构域(CRD)。该基因在凡纳滨对虾各种组织中有不同程度的表达,其中在肠道中表达量最高,其次为胃、肝胰腺、血细胞等组织或细胞,在肌肉中表达量最低。利用脂多糖(LPS)和肽聚糖(PGN)刺激对虾后,比较分析了LvLc2的表达变化特征,不同类型的病原表面多糖可在刺激后不同时期引发LvLc2基因的应答改变,表明其具有潜在的广谱应答模式,同时针对不同病原又具有不同的表达特征,显示了其识别结合和间接免疫效应的差异。对其作用特征的探讨有助于更好地了解对虾C型凝集素在病原感染过程中的功能和作用机制。研究结果为深入探讨对虾天然免疫调控机制提供了参考,在对虾健康养殖及病害防治方面具有潜在的应用价值。

    Abstract:

    Litopenaeus vannamei is the largest economic shrimp breed in the world at present. However, with the expansion of shrimp culture, diseases and other problems are exposed gradually. Shrimp culture industry was affected seriously by diseases all over the world. Shrimp belongs to invertebrate, and has no typical acquired immune system. They mainly rely on the innate immune system for defense and protection when pathogens invade. In innate immune response, crustaceans recognize non-self components through pattern recognition receptors (PRRs). The components recognized by PRRs are mainly constitutive and conserved polysaccharide molecules on the surface of microorganisms, which are not produced by eukaryotic cell hosts and are necessary for the survival of pathogenic microorganisms. They are called pathogen associated molecular patterns (PAMPs). The recognition and binding of PRRs to PAMPs is the beginning of humoral immunity. The recognition of PRRs to pathogens can directly or indirectly activate proteases, hemocytes and intracellular signal pathways related to humoral immunity. A variety of immune defense reactions were triggered. It produces antibacterial substances and effective molecules to inhibit and eliminate pathogens. As a pattern recognition receptor and terminal effector, C-type lectin participates in the process of “non-self” recognition and elimination of invasive pathogens, which is crucial in the innate immune defense mechanism of crustaceans. In this study, a novel C-type lectin gene (LvLc2, GenBank accession number: KR020738) was cloned from hemocytes of L. vannamei according to our previous transcriptome results. Bioinformatics analysis showed that the ORF region of LvLc2 gene was 465 bp encoding 154 aa with 11 bp 5'-UTR and 126 bp 3'-UTR. The predicted molecular weight was 17.16 ku and the theoretical isoelectric point was 4.54. The deduced amino acid sequence contained a signal peptide sequence with 17 amino acids at the amino end and a conserved carbohydrate recognition domain (CRD) at the carboxyl end, in which four conserved cysteines formed two disulfide bonds. The motif associated with PAMP binding specificity is EPS (Glu113-Pro114-Ser115). Blast homology analysis showed that LvLc2 had high similarity with mannose receptor C-type lectin from different species, and 38% similarity with the mannose receptor of Rimicaris exoculata. LvLc2 gene was expressed in different tissues of L. vannamei. The highest expression was in intestine, followed by stomach, hepatopancrease, hemocytes, and the lowest expression level was in muscle. After shrimps were challenged by LPS and PGN, the expression profiles of LvLc2 gene in hemocytes were analyzed by qPCR. Compared with the control group, the expression of LvLc2 showed a slight down-regulated expression profile at the initial stage of LPS injection (0.5 h), and then reached the highest value at 12 h after LPS injection, which was about 2.2 times of the initial expression level. At 24-72 h after injection, the expression of LvLc2 basically returned to the initial level, and there was no significant change. At the early stage after PGN injection, the expression of LvLc2 fluctuated slightly. At the 48th hour, the expression of LvLc2 was significantly up-regulated, which was about 4.5 times of that in the control group and then fell back to slightly lower than the initial level. Different pathogen-associated molecular patterns (PAMP) could induce response changes of LvLc2 gene after stimulation, which indicated that it had a potential broad-spectrum response pattern. At the same time, it had different expression profiles for different pathogens, which showed the differences of recognition, combination and indirect immune effects. The research of its characterization and function are helpful to better understand the mechanism of shrimp C-type lectin during the process of pathogen infection.

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郝少彦,尹丝雨,龚晨,张亦陈,耿绪云,刘逸尘,孙金生.一种凡纳滨对虾新的C型凝集素基因(LvLc2)的克隆及免疫应答特征[J].水产学报,2021,45(6):948~957

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  • 收稿日期:2021-01-16
  • 最后修改日期:2021-03-12
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  • 在线发布日期: 2021-06-16
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