中华绒螯蟹蜕皮抑制激素基因全长cDNA克隆和重组表达

CLONING AND EXPRESSION ANALYSIS OF MOLT-INHIBITING HORMONE GENE (Es-MIH) IN ERIOCHEIR SINENSIS

根据实验室分离自中华绒螯蟹(Eriocheir sinensis)的一种蜕皮抑制激素(Molting-inhibiting hormone,MIH)N端氨基酸测序结果设计简并引物,采用RACE方法,首次从中华绒螯蟹眼柄中克隆到蜕皮抑制激素基因全长cDNA(Es-MIH,GenBank登录号:DQ341280),该基因全长为1457 bp,开放阅读框为330 bp,编码110个氨基酸(含有35个氨基酸的信号肽);其成熟肽包含C7-C44、C24-C40和C27-C53三个二硫键,有典型的CHH家族结构域。该cDNA编码的氨基酸序列与地蟹(Gecarcinus lateralis)MIH同源性最高,达到了85%。Northern杂交和半定量RT-PCR显示蜕皮间期成体蟹仅在眼柄中有MIH基因表达,提示该基因的表达具有一定组织特异性。利用pCR T7/NT TOPO TA系统重组表达MIH成熟肽,纯化的重组蛋白得率为0.3 g/L,纯化产物经质谱鉴定为中华绒螯蟹MIH。研究解决了CHH家族神经肽在机体中的表达量少,直接纯化较难的问题,为深入研究MIH的作用机制和在生产上控制中华绒螯蟹蜕皮和生长奠定了基础。

Periodic molting is essential for growth and development in crustaceans.Molting is triggered by steroid hormones（ecdysteroids） which secreted by paired endocrine glands,the Y-organs.The synthesis of ecdysteroids by Yorgans is negatively regulated by a peptide neurohormone,moltinhibiting hormone（MIH）,a polypeptide neurohor-mone released from neurosecretory cells in the X-organ/sinus gland complex of the eyestalks.To clone a full length cDNA of molt-inhibiting hormone gene from Eriocheir sinensis by RACE-PCR,degenerate primers was designed ac-cording to the partial amino acid sequences of MIH which was isolated by our lab.A novel MIH（Es-MIH,GenBank accession No.DQ341280） of 1457 bp was successfully cloned from Chinese mitten crab.It was consisted of a 330bp open reading frame,the untranslation region of 5′ and 3′ end were 189 and 938 nucleotides,respectively.Deduced pro-tein contained a putative signal peptide of 35 amino acids and a mature peptide of 75 amino acids.Es-MIH contains 6 conserved cysteines which formed three disulfide bonds（C7-C44,C24-C40 and C27-C53）.A typical Crust_neurohorm do-main（position 2—74 nt in mature peptide）（E-value=2.80e-33） was identified by SMART（Simple Modular Architecture Research Tool） in Expasy.There was an arthropod CHH/MIH/GIH neurohormones family signature in this domain.Multiple alignment results showed that Es-MIH has the highest identity with Gecarcinus lateralis MIH（85%）,it also shared high identities with Carcinus maenas（66%） and Portunus trituberculatus（62%）,moreover,it showed highly identity with MIH from shrimps,such as Metapenaeus ensis MIH（44%）,Fenneropenaeus chinensis MIH（43%）,Penaeus monodon MIH（43%） and Litopenaeus vannamei MIH（42%）.Northern blotting reveled that transcripts of Es-MIH were only found in eyestalks,no bands could be observed in heart,muscle,ventral nerve cord,brain and haemocytes lanes.Semi-quantitative RT-PCR gave similar results.It indicated that Es-MIH was specifically expressed in eyestalk.The recombinant Es-MIH（rEs-MIH） was expressed by pCRT7/NT TOPOTA expression system.The op-timal time for isopropyl β-D-thiogalactopyranoside induction was 5 hours.After collection and lyses of host E.coli,rEs-MIH was purified by immobilized metal affinity chromatography column.The yield of rEs-MIH could reach 0.3 g/L.The LC-ESI-MS analysis showed that two peptide fragments of the recombinant protein were identical to the corre-sponding sequence of Eriocheir sinensis MIH1（GenBank accession No.GI34597340）.Low content and difficulties of purification of polypeptide neurohormone in crustacean was solved in this research by means of genetic engineering,which could facilitate understanding the probable role of MIH in development process of crab and would be helpful to crustacean culture.