CAJ | 학술논문

前酚氧化酶是一种黑色素合成酶,是节肢动物体内的一种重要免疫蛋白.为了克隆中华按蚊Anopheles sinensis前酚氧化酶(Prophenoloxidase,PPO) 基因部分序列,根据已报道的多种昆虫 PPO 氨基酸的保守序列设计简并引物,以中华按蚊总 RNA 为模板进行RT-PCR 扩增,目的片段经 TA 克隆后测序.所得中华按蚊 PPO 基因(ASPPO) 部分序列长597 bp,编码 199 个氨基酸;与几种近缘蚊种的PPO基因序列同源性分别达57%~100%不等.经在线比对,该序列在基因组中分属两个外显子区域,中间含有一个内含子.推导的氨基酸序列含有两个铜离子结合位点,与目标序列相符,表明成功克隆出ASPPO部分序列.所得序列登录GenBank,登录号:JX295575,JX295576.推导的氨基酸序列提交瑞士蛋白质空间构象模拟平台Swiss-model,以冈比亚按蚊免疫相关前酚氧化酶为模板进行空间3D构象模拟,SPDview软件分析拉氏构象图.结果显示拉氏构象得分较高而QMEAN得分较低,近似跨膜蛋白值,提示该该蛋白是否为中华按蚊中肠和血淋巴疟原虫免疫蛋白尚不确定,需进一步研究.
전분양화매시일충흑색소합성매,시절지동물체내적일충중요면역단백.위료극륭중화안문Anopheles sinensis전분양화매(Prophenoloxidase,PPO) 기인부분서렬,근거이보도적다충곤충 PPO 안기산적보수서렬설계간병인물,이중화안문총 RNA 위모판진행RT-PCR 확증,목적편단경 TA 극륭후측서.소득중화안문 PPO 기인(ASPPO) 부분서렬장597 bp,편마 199 개안기산;여궤충근연문충적PPO기인서렬동원성분별체57%~100%불등.경재선비대,해서렬재기인조중분속량개외현자구역,중간함유일개내함자.추도적안기산서렬함유량개동리자결합위점,여목표서렬상부,표명성공극륭출ASPPO부분서렬.소득서렬등록GenBank,등록호:JX295575,JX295576.추도적안기산서렬제교서사단백질공간구상모의평태Swiss-model,이강비아안문면역상관전분양화매위모판진행공간3D구상모의,SPDview연건분석랍씨구상도.결과현시랍씨구상득분교고이QMEAN득분교저,근사과막단백치,제시해해단백시부위중화안문중장화혈림파학원충면역단백상불학정,수진일보연구.
Prophenoloxidase, a melanin-synthesizing enzyme, is considered to be an important arthropod immune protein. In order to clone the prophenoloxidase gene of Anopheles sinensis, degenerative primers were designed according to the conserved protein fragments of prophenoloxidase gene from sibling species deposited on GenBank. RNA sequence of adult An. sinensis was reverse-transcripted into cDNA strand which was amplified by PCR to get the 600 bp target fragment, the partial prophenoloxidase cDNA sequence. The target fragment was cloned into PMD18-T vector and sequenced. The partial cDNA sequences of prophenoloxidase of An. sinensis was identified and named ASPPO1, ASPPO2 respectively. The homology blasted with An. gambiae ranged from 57.58% to 100%. The two sequences were submitted to NCBI with the accessing number: JX295575, JX295576. The rooted phylogenetic tree showed the homology with PPO2 of An. gambiae. The putative amino acid fragments were submitted to Swiss-model blasting with An. gambiae to simulate the 3D spatial structure. The QMEAN score of the model showed a further study was needed to ensure whether the two PPO genes were from the midgut and hemolymph against plasmodium or not.