梁彩凤,庞欢瑛,刘建勇

• 1:广东海洋大学水产学院

摘要(Abstract)：

【目的】克隆溶藻弧菌(Vibrio alginolyticus)HY9901 vscH基因,并分析其序列结构。【方法】参照Gen Bank上的溶藻弧菌全基因组序列设计1对特异性引物,PCR扩增溶藻弧菌HY9901株的Ⅲ型输出蛋白Yop R核心家族蛋白vscH基因全长,用生物信息学手段分析VscH序列,构建VscH系统进化树及亚基三维结构模型。【结果】vscH基因序列全长636 bp,编码211个氨基酸,推导的蛋白分子质量为24.09 ku。经预测,VscH不存在信号肽与跨膜区;含有2个ASN-糖基化位点,14个磷酸化位点,2个N-豆蔻酰化位点,2个内质网靶向序列及2个微体C-末端靶信号位点;有1个主要功能域,T3SS_needle_reg(第71-211氨基酸);α-螺旋占66.35%,无规则卷曲占23.22%,延伸链占5.69%,β-折叠占4.74%;可能的B细胞抗原优势表位,分别是第20~23,25~28,37~40,48~51和109~112区段。溶藻弧菌VscH系统进化树与弧菌(Vibrio)聚为一簇。VscH亚基三维结构模型与鼠疫耶尔森菌毒力因子Yop R蛋白的编码基因ysc H相似。

关键词(KeyWords)： 溶藻弧菌;vsc H基因;基因克隆;生物信息学分析

Abstract:

Keywords:

基金项目(Foundation): 国家科技支撑计划(2015BAD13B03-3)

作者(Author): 梁彩凤,庞欢瑛,刘建勇

参考文献(References)：

• [1]LIN J,HUANG S,ZHANG Q.Outer membrane proteins:key players for bacterial adaptation in host niches[J].Microbes and Infection,2002,4(3):325-331.
• [2]黄志坚,何建国.溶藻弧菌外膜蛋白(Va-OMP)的免疫原性及免疫保护性[J].水产学报,2006,30(4):538-543.
• [3]LEE V T,Schneewind O.Type III machines of pathogenic yersiniae secrete virulence factors into the extracellular milieu[J].Molecular Microbiology,1999,31(6):1619-1629.
• [4]ALLAOUI A,SCHULTE R,CORNELIS G R.Mutational analysis of the Yersinia enterocolitica vir C operon:characterization of ysc E,F,G,I,J,K required for Yop secretion and ysc H encoding Yop R[J].Molecular Microbiology,1995,18(2):343-355.
• [5]CORNELIS G R,WOLF-WATZ H.The Yersinia Yop virulon:a bacterial system for subverting eukaryotic cells[J].Molecular Microbiology,1997,23(5):861-867.
• [6]GALáN J E,COLLMER A.Type III secretion machines:bacterial devices for protein delivery into host cells[J].Science,1999,284(5418):1322-1328.
• [7]SCHUBOT F D,CHERRY S,AUSTIN B P,et al.Crystal structure of the protease-resistant core domain of Yersinia pestis virulence factor Yop R[J].Protein Science,2005,14(6):1679-1683.
• [8]HUECK C J.Type III protein secretion systems in bacterial pathogens of animals and plants[J].Microbiol Mol Biol Rev,1998,62(2):379-433.
• [9]王欣茹,唐小千,绳秀珍,等.基于r Gro EL和r Omp C的牙鲆迟缓爱德华氏菌血清学诊断试纸条的制备[J].中国海洋大学学报(自然科学版),2018,48(2):32-37.
• [10]刘望,杨佳杰,杨静,等.嗜水气单胞菌外膜蛋白原核表达及其免疫保护性研究[J].陕西科技大学学报,2017,35(6):125-130.
• [11]曹叶.鼠疫耶尔森菌外膜蛋白J参与宿主免疫逃逸机制的研究[D].长春:吉林大学,2017.
• [12]程海燕,庞欢瑛,鲁义善,等.溶藻弧菌acf A基因克隆与生物信息学分析[J].广东海洋大学学报,2014,34(1):9-14.
• [13]CHANG C C,YEH M S,LIN H K,et al.The effect of Vibrio alginolyticus,infection on caspase-3 expression and activity in white shrimp Litopenaeus vannamei[J].Fish&Shellfish Immunology,2008,25(5):672-678.
• [14]SADOK K,MEJDI S,NOURHEN S,et al.Phenotypic characterization and RAPD fingerprinting of Vibrio parahaemolyticus and Vibrio alginolyticus isolated during Tunisian fish farm outbreaks[J].Folia Microbiologica,2013,58(1):17-26.
• [15]SGANGA G,COZZA V,SPANU T,et al.Global climate change and wound care:case study of an off-season Vibrio alginolyticus infection in a healthy man[J].Ostomy/wound Management,2009,55(4):60-62.
• [16]刘祥,陈琛,陈春琳,等.猪重要感染病毒蛋白的二级结构?抗原表位分析及三联表位多肽疫苗的重组预测[J].生物学杂志,2017,34(3):18-23.
• [17]娄慧,张中旺,陈启伟,等.口蹄疫病毒A/WH/09株结构蛋白P1基因的克隆及其B细胞抗原表位的预测[J].中国兽医科学,2011(3):221-228.
• [18]CAI S H,WU Z H,JIAN J C,et al.Cloning and expression of the gene encoding an extracellular alkaline serine protease from Vibrio alginolyticus strain HY9901,the causative agent of vibriosis in Lutjanus erythopterus(Bloch)[J].Journal of Fish Diseases,2007,30(8):493-500.
• [19]庞欢瑛,周泽军,丁燏,等.溶藻弧菌Ⅲ型分泌系统分子伴侣护航蛋白Vsc O的基因克隆及其生物信息学分析[J].生物技术通报,2014(6):155-161.
• [20]孙金福,何利昆,耿莉,等.禽大肠埃希菌耐药谱及氨基糖苷耐药基因分子流行病学调查[J].中国兽医杂志,2009,45(6):6-8.
• [21]庞欢瑛,陈立明,蔡佳,等.溶藻弧菌二氢硫辛酰胺脱氢酶基因克隆及其生物信息学分析[J].广东海洋大学学报,2014,34(1):1-8.
• [22]SCHUBOT F D,CHERRY S,AUSTIN B P,et al.Crystal structure of the protease‐resistant core domain of Yersinia pestis virulence factor Yop R[J].Protein Science,2005,14(6):1679-1683.
• [23]BLAYLOCK B,SORG J A,SCHNEEWIND O.Yersinia enterocolitica type III secretion of Yop R requires a structure in its m RNA[J].Molecular Microbiology,2008,70(5):1210–1222.
• [24]BLAYLOCK B,BERUBE B J,SCHNEEWIND O.Yop R impacts type III needle polymerization in Yersinia species[J].Molecular Microbiology,2010,75(1):221-229.
• [25]张萃,卢文菊,李红枝,等.抗TRPC6多肽单克隆抗体的制备及其识别抗原表位分析[J].中国免疫学杂志,2013,29(7):741-744.
• [26]祁凌霞.基于HCVE2序列保守性评估及B-cell表位预测筛选基因型特异性表位多肽[D].长春:吉林大学,2015.
• [27]高孟,谢珲,姜立民,等.禽流感病毒H7N9神经氨酸酶的优势线性B细胞抗原位点的预测及确定[J].中华实验和临床病毒学杂志,2017,31(4):287-290.