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珊瑚礁生态系统是重要的海洋生态系统之一,栖息着全球多达四分之一的海洋生物,被誉为“海洋中的热带雨林”,为人类提供了食物、生态和社会经济文化等服务[1-2]。造礁石珊瑚是珊瑚礁生态系统的支柱生物,其通过与虫黄藻和细菌等微生物共生来适应强光照寡营养的礁区环境,因此维持健康的微生物群落结构对造礁石珊瑚的健康尤为重要。当前全球气候变暖导致的海水升温迫使造礁石珊瑚外排共生虫黄藻,珊瑚因营养短缺而免疫力下降,机会致病菌[例如:溶珊瑚弧菌(Vibrio coralliilyticus)]的致病力相对显著提升,造成了某些珊瑚疾病的爆发率显著升高[3]。因此,亟需开展造礁石珊瑚免疫防御机制的相关研究,尤其是高温对珊瑚免疫的影响。造礁石珊瑚与其他无脊椎动物一样,依靠固有免疫应答来识别和清除入侵的病原菌等微生物[4]。此外,珊瑚还能通过固有免疫应答控制共附生菌群中病原菌的数量和比例,进而降低病原菌的潜在威胁[5]。珊瑚的凋亡、自噬、抗菌肽、氧化还原系统、酚氧化酶系统和补体系统等均参与了致病微生物的控制和清除[6-7]。抗菌肽是生物中普遍存在的一类阳离子活性多肽,是机体非特异性免疫的关键因子之一,目前已从多种无脊椎动物中分离出具有活性的抗菌肽[8]。近年来,分子生物学和基因组学的迅猛发展促进了免疫相关基因的挖掘,更好地推动珊瑚固有免疫应答和共附生菌群调控的深入研究。溶菌酶属于抗菌肽家族,是生物体内一种具有杀菌作用的重要抗菌蛋白,其通过催化水解细胞壁中N−乙酰氨基葡萄糖和N−乙酰胞壁酸之间形成的β−1,4糖苷键而破坏细菌的细胞壁[9-12]。目前,已有许多研究聚焦于无脊椎动物的溶菌酶,但对造礁石珊瑚,甚至刺胞动物中溶菌酶的了解还不清楚,仅有类溶菌酶活性的相关报道,且缺乏确切的分子证据[13-14]。例如,有研究表明受黄带病影响的珊瑚(Orbicella faveolata)中具有较高的类溶菌酶活性[15];海扇可能通过增加类溶菌酶和壳多糖酶活性抵抗微生物入侵[16];华丽黄海葵(Anthopleura elegantissima)和等指海葵(Actinia equina)的粘液均具有类溶菌酶活性[17-18]。鹿角杯形珊瑚(Pocillopora damicornis)隶属动物界(Animalia)、刺胞动物门(Cnidaria)、珊瑚纲(Anthozoa)、石珊瑚目(Scleractinia)、杯形珊瑚科(Pocilloporidae)、杯形珊瑚属(Pocillopora),广泛分布于太平洋和印度洋的热带和亚热带浅海海域。鹿角杯形珊瑚已作为模式生物用于探究造礁石珊瑚对病原菌,以及病原菌和高温交互作用的响应研究中。例如,BEN-HAIM等[19]分离出一种温度依赖型的溶珊瑚弧菌,该病原菌在水温超过26 ℃时,可在2周内迅速破坏鹿角杯形珊瑚的组织[19]。笔者克隆了鹿角杯形珊瑚的溶菌酶基因PdLYZ,随后体外诱导表达并纯化PdLYZ重组蛋白,同时测定PdLYZ重组蛋白对大肠杆菌(Escherichia coli),变异链球菌(Streptococcus mutans)和溶珊瑚弧菌(V. coralliilyticus)的抑菌活性,还探究了PdLYZ重组蛋白在高温条件下对病原菌溶珊瑚弧菌抑制活力的变化,从而揭示溶菌酶在珊瑚免疫应答中扮演的重要作用。
Recombinant Protein Expression and Bacteriostatic Activity of Pocillopora damicornis Lysozyme in Vitro
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摘要: 珊瑚疾病的大规模爆发已严重威胁到珊瑚礁生态系统的结构和功能。溶菌酶是无脊椎动物固有免疫应答中的重要效应分子之一。本研究从鹿角杯形珊瑚(Pocillopora damicornis)中克隆得到了1个溶菌酶基因(PdLYZ),随后体外诱导表达并纯化PdLYZ重组蛋白,同时测定PdLYZ重组蛋白对大肠杆菌(Escherichia coli)、变异链球菌(Streptococcus mutans)和溶珊瑚弧菌(Vibrio coralliilyticus)的抑菌活性,还探究了PdLYZ重组蛋白在高温条件下对病原菌溶珊瑚弧菌抑制活性变化。研究结果:PdLYZ的cDNA开放阅读框长648 bp,编码215个氨基酸残基,其序列与其他生物的溶菌酶相似性为25.54%~49.15%;生物信息学预测PdLYZ推导蛋白具有1个类溶菌酶超家族结构域(Ile31-Gly214);PdLYZ重组蛋白能显著抑制大肠杆菌、变异链球菌和溶珊瑚弧菌的生长;PdLYZ重组蛋白在高温条件下会在前期显著抑制溶珊瑚弧菌的生长。这些结果表明,溶菌酶PdLYZ可能参与珊瑚免疫防御。本研究为进一步理解珊瑚免疫机制提供了理论参考。Abstract: Large-scale outbreaks of coral diseases have been seriously threatening the health of coral reef ecosystems. Lysozyme is one of the important effectors in the innate immune response of invertebrates. A lysozyme gene, PdLYZ, was cloned from Pocillopora damicornis. The recombinant protein of PdLYZ (rPdLYZ) was expressed and purified in vitro, and its bacteriostatic activities against the Gram-positive bacteria Streptococcus mutans and Gram-negative bacteria Escherichia coli were determined. The effect of high temperature on the bacteriostatic activity of rPdLYZ against the pathogenic bacteria Vibrio coralliilyticus was further explored. The identified PdLYZ was comprised of 215 amino acid residues and predicted to contain one lysozyme-like superfamily domain (Ile31-Gly214), and its sequence was 25.54%−49.15% similar to those of lysozymes from other organisms. Results showed that rPdLYZ had lytic activities against E. coli, S. mutans and V. coralliilyticus. The rPdLYZ inhibited the growth of V. coralliilyticus at the early stage under high temperature. These results suggest that PdLYZ be involved in the immune defense of corals, and this study provides a theoretical reference for further understanding of the immune mechanism of corals.
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Key words:
- Pocillopora damicornis /
- lysozyme /
- prokaryotic expression /
- bacteriostatic activity
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图 2 不同生物溶菌酶的系统进化树
Fig. 2 Phylogenetic tree of lysozymes from different organisms
Sp:柱状珊瑚Stylophora pistillata; Of:星珊瑚Orbicella faveolate; Am:鹿角珊瑚Acropora millepora; Nv:海葵Nematostella vectensis; Ed:海葵Exaiptasia diaphana; At:海葵Actinia tenebrosa; Dg:软珊瑚 Dendronephthya gigantea; Ta:锦蟾Thalassophryne amazonica; Sf:硬骨舌鱼Scleropages formosus; Gm:鳕鱼Gadus morhua.
图 3 PdLYZ重组蛋白的表达与纯化
M:蛋白marker;1:未诱导组蛋白的表达;2:诱导组蛋白的表达;3:镍柱纯化后的PdLYZ蛋白。
Fig. 3 SDS-PAGE analysis of recombinant PdLYZ protein.
Lane M: protein molecular weight marker. Lane 1: protein expression of control group, not induced; Lane 2: protein expression of induced group; Lane 3: PdLYZ protein purified by Ni2+ chelating Sepharose colum.
图 4 PdLYZ重组蛋白对大肠杆菌的抑菌活性,
rLYZ:PdLYZ重组蛋白;BSA:牛血清蛋白(CK)。数据以平均值±标准差表示(n=3),*代表显著性差异(P<0.05)。
Fig. 4 Inhibitory activity of recombinant PdLYZ protein against Escherichia coli.
rLYZ: recombinant PdLYZ protein; BSA:bovine serum albumin (CK). Values are presented as the mean ± SD (n=3), and * represents significant difference (P < 0.05).
图 5 PdLYZ重组蛋白对变异链球菌的抑菌活性,
rLYZ:PdLYZ重组蛋白;BSA:牛血清蛋白(CK)。数据以平均值±标准差表示(n=3),*代表显著性差异(P<0.05)。
Fig. 5 Inhibitory activity of recombinant PdLYZ protein against Streptococcus mutansi.
rLYZ: recombinant PdLYZ protein; BSA: bovine serum albumin (CK). Values are presented as the mean ± SD (n=3), and * represents significant difference (P<0.05).
图 6 在26 ℃和32 ℃下,PdLYZ重组蛋白对溶珊瑚弧菌的抑菌活性
rLYZ:PdLYZ重组蛋白;BSA:牛血清蛋白(CK)。数据以平均值±标准差表示(n=3),*代表显著性差异(P<0.05)。
Fig. 6 Inhibitory activity of recombinant PdLYZ protein against Vibrio coralliilyticus at 26 ℃ and 32 ℃.
rLYZ: recombinant PdLYZ protein; BSA:bovine serum albumin (CK). Values are presented as the mean ± SD (n=3), and * represents significant difference (P<0.05).
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