草蝦之蝦激素基因選殖、定性及功能分析

Abstract

血球在甲殼類動物的免疫反應中扮演著重要的角色，因此造血作用為維持體內平衡所必需的重要機制。脊椎動物中，不同種類的細胞所分泌之細胞激素可調控造血及免疫反應；然而無脊椎動物中，與造血相關的細胞激素卻鮮少得知。在本篇論文中，我們從草蝦(Penaeus monodon)血球cDNA中選殖出一個全長1,509 bp的蝦激素(astakine)分子，其中包含143 bp的5'-UTR和375 bp的編碼區，以及991 bp的3'-UTR，且此分子的3'-UTR序列比已發表於NCBI之草蝦蝦激素cDNA序列(GenBank accession no. AY787657)多插入一段長671 bp的序列，顯示此選殖分子應為較長轉錄型的草蝦蝦激素。根據推衍，此分子編碼區含有124個胺基酸，包括一段訊息胜肽，並具有一個prokineticin功能區塊與五對雙硫鍵鍵結。推算其成熟的蛋白質分子量為11,295 Da，pI值為5.2。分析此分子的親緣關係，得知與節肢動物astakine分子最為相似，與脊椎動物的兩種分子－prokineticin及dickkopf之親緣關係遙遠，唯其序列C端皆富含半胱胺酸區塊。巢狀反轉錄聚合酶連鎖反應結果顯示，此分子的mRNA在眼柄、殼下皮膜、鰓、心臟、肝胰臟、淋巴器官、腸、肌肉、神經及血球中皆可偵測到，而定量反轉錄聚合酶連鎖反應顯示此分子在血球中的表現量最高。然而，草蝦經注射LPS後的24小時內，皆無法誘發血球中的蝦激素mRMA大量產生。利用昆蟲細胞-桿狀病毒表現系統合成草蝦蝦激素重組蛋白，並以MALDI-MS/MS spectrometry分析重組蛋白之真確性。另以ESI-MS分析C端帶有6×His tag的重組蛋白之分子量，發現其較預期分子量少10 Da，顯示已形成五對雙硫鍵鍵結。利用BrdU攝入法分析經注射重組蛋白的草蝦，發現此蝦激素蛋白具有能夠促使其造血組織細胞進行增生的能力，由此推論草蝦蝦激素可作為一種細胞激素。

Hemocytes play important roles in crustacean immune responses. Generation of new hemocytes (hematopoiesis) is thus necessary to maintain homeostasis which is vital to crustaceans. In vertebrates, certain cytokines have been demonstrated to regulate hematopoiesis and immune responses. In invertebrates, however, little is known about cytokines related to hematopoiesis. In the present study, we cloned an astakine molecule from hemocytic cDNA of tiger shrimp (Penaeus monodon) (PmAst) which was 1,509 bp in length with a 5'-UTR of 143 bp, a coding region of 375 bp and a 3'-UTR of 991 bp. The present clone showed to be a longer form of astakine transcript with an extra insert of 671 bp in the 3'-UTR than the NCBI-recorded shrimp astakine cDNA sequence (GenBank accession no. AY787657). The deduced protein had 124 amino acid residues, including a signal peptide, one prokineticin domain and five pairs of disulfide bonds. The calculated molecular weight (MW) of the mature peptide was 11,295 Da and pI was 5.2. Phylogenetically, this molecule is most similar to astakine-related molecules of arthropod and the distant was found from vertebrate prokineticin and dickkopf molecules, the common among these sequences was that all contained cysteine-rich domain in C-terminal region. Nested RT-PCR showed that PmAst is expressed in many tissues and organs of the shrimp such as eyestalk, subcuticular epithelium, gills, heart, hepatopancreas, lymphoid organ, intestine, muscle, nerve and hemocytes. Real-time PCR further revealed that PmAst is expressed mainly in the hemocytes. The PmAst transcript is however not inducible in the hemocytes until 24 hrs post LPS injection of shrimp. The recombinant protein of PmAst (rPmAst) was synthesized using insect cell-baculovirus expression system. The authenticity of rPmAst protein was examined by MALDI-MS/MS spectrometry. Using ESI-MS it was determined that the MW of C-terminally histidine-tagged recombinant protein is 10 Da less than the expected MW, allowing the formation of five pairs of disulfide bonds. Using BrdU incorporation assay it was demonstrated that the injection of rPmAst to the shrimp promoted cell proliferation in hematopoietic tissues. Therefore, we conclude that PmAst functions as a cytokine that influences cell proliferation in the hematopoietic tissues.