日本鳗鲡肝脏表达抗菌肽2基因的克隆与表达
MOLECULAR CLONING AND EXPRESSION ANALYSIS OF A LIVER EXPRESSED ANTIMICROBIAL PEPTIDE-2 IN JAPANESE EEL, ANGUILLA JAPONICA
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摘要: 为探讨鱼类抗菌肽基因的生物学功能,研究应用RACE方法克隆获得了日本鳗鲡 (Anguilla japonica) 肝脏表达抗菌肽2基因 (Liver-Expressed Antimicrobial Peptide 2,LEAP-2),即AJLEAP-2的cDNA序列,全长为450 bp,开放阅读框编码89个氨基酸。其成熟肽含有LEAP-2保守基序C-X5-C-X4-C-X4-C。AJLEAP-2基因组结构与其他脊椎动物LEAP-2相同,都包含有三个外显子。利用荧光定量PCR检测了AJLEAP-2在日本鳗鲡不同组织/器官中的表达,发现其转录子在肝脏中表达量最高,是内参基因 (-actin) 的6倍; 其次是肠道,但其表达量仅为肝脏的1/130。此外,还检测了AJLEAP-2在日本鳗鲡玻璃鳗(Glass eel)阶段的转录表达水平,结果显示,玻璃鳗中AJLEAP-2的转录表达量仅低于黑仔期的肝脏,为黑仔鳗肠道表达量的2倍。LPS和迟缓爱德华菌 (Edwardsiella tarda) 刺激能显著上调鳗鲡血液中AJLEAP-2的转录表达,刺激16h后上调倍数最高,分别为对照组的86倍和12倍。此外,LPS刺激72h和E. tarda 刺激8h后,肠道中AJLEAP-2显著上调表达(P0.05),为对照组的8倍。Poly I:C刺激24h后,血液中AJLEAP-2转录表达显著下调。结果表明,AJLEAP-2在日本鳗鲡抗细菌感染过程中起重要的作用。Abstract: Liver Expressed Antimicrobial Peptide 2 (LEAP-2), a cysteine-rich cationic antimicrobial peptide, plays a vital role in the host innate immune system. In the present study, the AJLEAP-2 gene was cloned from Japanese eel (Anguilla japonica) by RACE. The length of AJLEAP-2 cDNA was 450 bp including a 270 bp ORF that encodes a puta-tive 89-peptide. The peptide contained the conserved motif MTPFWR and the characteristic motif C-X5-C-X4-C-X4-C of LEAP-2. AJLEAP-2 has three exons similar to the counterpart from other vertebrates. The expression of AJLEAP-2 from tissue/organs of Japanese eel elver indicated the highest expression in liver that was five times higher than that of -actin, and then in intestine that was only 1/130 the amount in liver. Additionally, AJLEAP-2 was highly expressed in glass eel, which was almost twice the amount in elver intestine. LPS and Edwardsiella tarda increased the expression of AJLEAP-2 in blood and reached the peak level at 16h post challenge increasing 86 folds and 12 folds, respectively. Both LPS and E. tarda significantly up-regulated the expression of AJLEAP-2 in intestine. Meanwhile, the transcript level of AJLEAP-2 in blood was significantly down-regulated at 24h challenged with Poly I:C. These results suggest that AJLEAP-2 may play an important role in innate immunity of Japanese eel against bacterial infection.
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Keywords:
- Anguilla japonica /
- LEAP-2 /
- Antimicrobial peptide /
- Edwardsiella tarda
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