宋巧珍;邹枘峰;张亦陈;耿绪云;刘逸尘;

• 1:天津市动植物抗性重点实验室/天津师范大学生命科学学院
• 2:天津市水生动物疫病预防控制中心

摘要(Abstract)：

[目的]研究丝氨酸蛋白酶基因(Lv-SP)在凡纳滨对虾天然免疫应答过程中的作用。[方法]利用PCR技术克隆凡纳滨对虾丝氨酸蛋白酶基因(Lv-SP),并进行生物信息学分析,进一步通过实时荧光定量PCR(qRT-PCR)方法对其进行组织表达分析以及白斑杆状病毒(WSSV)刺激后的表达变化分析。[结果]成功从凡纳滨对虾体内克隆了Lv-SP基因的开放阅读框ORF,碱基数为1 104 bp,共编码367个氨基酸,在线分析软件预测其蛋白分子量为41 k D,等电点PI为6.93;在线分析软件预测显示:Lv-SP基因预测的氨基酸序列保守性很高,含有1个clip结构域和1个高度保守的SP结构域(Tryp-SPc);系统进化发生分析表明:Lv-SP氨基酸序列与中国明对虾以及中华绒螯蟹的丝氨酸蛋白酶(SP)同源性较高,而与按蚊、小蜂窝甲虫的SP同源性较低;qRT-PCR分析结果显示:Lv-SP基因在凡纳滨对虾不同组织中均有表达,并且在鳃和血中表达量较高,而在心和肝中表达量较低,病毒WSSV刺激后,Lv-SP基因在48.0 h和72.0 h有显著的上调表达趋势。[结论]Lv-SP基因保守性比较高,并且其在一定程度上参与了对虾应答病毒侵染的免疫反应,推测它有可能作为重要的免疫调控基因在对虾的防御应答过程中发挥作用。

关键词(KeyWords)： 凡纳滨对虾;丝氨酸蛋白酶;基因克隆;免疫应答;WSSV刺激

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金项目(31472299);; 天津市应用基础与前沿技术研究计划(15JCZDJC33800);; 天津市水产生态及养殖重点实验室开放基金项目(TJAE2015005)

作者(Author): 宋巧珍;邹枘峰;张亦陈;耿绪云;刘逸尘;

Email:

DOI:

参考文献(References)：

• [1]王兴强,马甡,董双林.凡纳滨对虾生物学及养殖生态学研究进展[J].海洋湖沼通报,2004(4):94-100.
• [2]MURUGASU-OEI B,RODRIGUES V,YANG X,et al.Masquerade:A novel secreted serine protease-like molecule is required for somatic muscle attachment in the Drosophila embryo[J].Genes&development,1995,9(2):139-154.
• [3]IWANAGA S,KAWABATA S,MUTA T.New types of clotting factors and defense molecules found in horseshoe crab hemolymph:Their structures and functions[J].Journal of biochemistry,1998,123(1):1-15.
• [4]郭慧芝.中华绒鳌蟹c DNA文库的构建及免疫相关基因的克隆与表达[D].武汉:华中农业大学,2010.
• [5]张智明.凝血酶的研究进展[J].海峡药学,2006,18(6):1-3.
• [6]陈晓翔,杨程德,顾越英.纤溶酶功能研究进展[J].诊断学理论与实践,2005,4(5):430-432.
• [7]LEE K Y,ZHANG R,KIM M S,et al.A zymogen form of masquerade-like serine proteinase Homologue is cleaved during pro-phenoloxidase activation by Ca2+in coleopteran and Tenebrio molitor larvae[J].European journal of biochemistry,2002,269(17):4375-4383.
• [8]YU X Q,JIANG H B,WANG Y,et al.Nonproteolytic serine proteinase homologs are involved in prophenoloxidase activation in the tobacco hornworm,Manduca sexta[J].Insect biochemistry&molecular biology,2003,33(2):197-208.
• [9]ASGARI S,ZHANG G,ZAREIE R,et al.A serine proteinase homolog venom protein from an endoparasitoid wasp inhibits melanization of the host hemolymph[J].Insect biochemistry and molecular biology,2003,33(10):1017-1024.
• [10]KAWABATA S,TOKUNAGA F,KUGI Y,et al.Limulus factor D,a 43-k Da protein isolated from horseshoe crab hemocytes,is a serine protease homologue with antimicrobial activity[J].FEBS Letters,1996,398(2/3):146-150.
• [11]JOHANSSON M W,KEYSER P,SRITUNYALUCKSANA K,et al.Crustacean haemocytes and haematopoiesis[J].Aquaculture,2000,191(1):45-52.
• [12]LEE S Y,SDERHLL K.Characterization of a pattern recognition protein,a masquerade-like protein,in the freshwater crayfish Pacifastacus leniusculus[J].Journal of immunology,2001,166(12):7319-7326.
• [13]KWON T H,KIM M S,CHOI H W,et al.A masquerade-like serine proteinase homologue is necessary for phenoloxidase activity in the coleopteran insect,Holotrichia diomphalia larvae[J].FEBS Journal,2000,267(20):6188-6196.
• [14]HOLMSKOV U,THIEL S,JENSENIUS J C.Collectins and ficolins:Humoral lectins of the innate immune defense[J].Annual review of immunology,2003,21(1):547-578.
• [15]PRESANIS J S,KOJIMA M,SIM R B.Biochemistry and genetics of mannan-binding lectin(MBL)[J].Biochemical society transactions,2003,31(4):748-752.
• [16]PENG M X,NIU D H,WANG F,et al.Complement C3 gene:Expression characterization and innate immune response in razor clam Sinonovacula constricta[J].Fish&shellfish immunology,2016,55:223-232.
• [17]MEGYERI M,JANI P K,KAJDCSI E,et al.Serum MASP-1 in complex with MBL activates endothelial cells[J].Molecular immunology,2014,59(1):39-45.
• [18]SRIPHAIJIT T,FLEGEL T W,SENAPIN S.Characterization of a shrimp serine protease homolog,a binding protein of yellow head virus[J].Developmental&comparative immunology,2007,31(11):1145-1158.
• [19]NICHOLL S M,ROZTOCIL E,DAVIES M G.Urokinase-induced smooth muscle cell responses require distinct signaling pathways:A role for the epidermal growth factor receptor[J].Journal of vascular surgery,2005,41(4):672-681.
• [20]LI X C,ZHU L,LI L G,et al.A novel myeloid differentiation factor 88homolog,Sp My D88,exhibiting Sp Toll-binding activity in the mud crab Scylla paramamosain[J].Developmental&comparative immunology,2013,39(4):