马尔尼菲青霉菌溶血磷脂酶基因的识别和序列分析及功能预测

目的 从马尔尼菲青霉菌(PM)酵母相全长cDNA文库中识别溶血磷脂酶基因,预测其结构和功能,为进一步实验研究提供理论依据.方法 利用NCBI在线分析工具对PM酵母相全长cDNA文库进行筛选,识别溶血磷脂酶基因;通过Vector NTI suite 8.0软件包,对其编码蛋白质的各种结构与功能特征进行预测,并构建种系分子进化树.结果 筛选得到的基因长1014 bp,Blastx分析该基因可能是编码PM溶血磷脂酶的全长基因,完整的开放读码框(ORF)共含729 bp,编码243个氨基酸;其编码蛋白的相对分子质量为26 800,预测等电点为5.49,疏水氨基酸占47.7%,亲水氨基酸占26.8%,酸性氨基酸占12.7%,碱性氨基酸占12.8%;该蛋白有潜在的2个酪蛋白激酶Ⅱ磷酸化位点、3个蛋白激酶C磷酸化位点和7个N-肉豆蔻酰位点.结论 该氨基酸序列属于溶血磷脂酶样功能域特征蛋白酶;与球孢子菌亲源关系最近.通过研究,一个PM溶血磷脂酶基因被成功发现,这为进一步探讨该基因的功能提供了条件.

Abstract：

Objective To identify the gene encoding lysophospholipase from the full length cDNA library of Penicllium marneffei (PM) in yeast phase and predict the structure and function of its deduced protein, to provide with theoretical evidences for further experiments. Methods The PM full-length cDNA library in yeast phase was screened to identify the gene encoding lysophospholipase with the help of NCBI on-line analytical tool. Then, by utilizing the software package of Vector NTI suite 8.0, the protein deduced by the gene was analyzed to predict its corresponding structure and functions, and its molecular cladogram was constructed. Results The gene was composed of 1014 base pairs in the length and was presumed to be the full-length gene encoding lysophospholipase by Blastx, with a complete open reading frame (ORF) comprised of 729 base pairs encoding 243 amino acids with relative molecular weight being 26 800 and the predicted isoelectric point being S.49. The deduced protein included 47.7% hydrophobic amino acids, 26.8% hydrophilic amino acids, 12.7% acid amino acids and 12.8% basic amino acids. The protein had 2 potential casein kinase Ⅱ phosphorylation site, 3 potential protein kinase C phosphorylation site and 7 N-myristoyl site. Conclusions The amino acid sequence belongs to this kind of protease with lysophospholipase-like domain. The protein is most close to Coccidioides posadasii in genetic relationship. A novel gene encoding lysophospholipase is successfully found and the work has made necessary preparations for further research on the gene's function.     

粤西某市406例病毒抑制失败艾滋病患者基因型耐药分析

目的了解病毒抑制失败的艾滋病患者基因型耐药流行特点。方法收集2014-2018年粤西某市病毒抑制失败患者血浆共406例,扩增HIV-1蛋白酶全长及逆转录酶部分基因,测序拼接后基因亚型,提交斯坦福大学HIV-1耐药数据库获得耐药突变位点,分析比较耐药突变位点及耐药情况。结果 406例病毒抑制失败患者中,共检出696个耐药相关突变位点。PI区耐药位点L10V为3.7%和L10I为3.4%;NRTI主要突变位点为M184V(22.7%);NNRTI主要突变位点为K103N(20.2%)、Y181C(16.2%)和G190A(10.6%);耐药率为48.0%,在发生不同程度耐药的195例患者中,3.1%(6例)对PI、NRTI和NNRTI 3类药物都耐药,1.5%(3例)对PI和NNRTI两类药物都耐药,54.4%(106例)对NRTI和NNRTI两类药物都耐药,41.0%(80例)对单一药物耐药。结论粤西某市病毒抑制失败者M184V和K103N突变位点检出率高,耐药发生率偏高,其中以NNRTIs的耐药为主。应加强监测防止耐药株传播。     

2010年广州登革热患者临床及实验室特征分析

目的:分析2010年广州地区登革热患者的临床及实验室特征。方法:统计2010年8-12月我院收治47例登革热患者的临床及实验室检查结果,采用巢式PCR方法扩增并鉴定感染患者的登革病毒血清型别。结果:患者临床特征为发热(100%)、头痛(44.7%)、肌肉痛(55.3%)、骨关节痛(19.1%)以及皮疹(59.6%),实验室特征为白细胞、血小板计数减少(80.9%、63.8%)以及肝脏转氨酶升高(丙氨酸转氨酶和天冬氨酸转氨酶升高分别为23.4%和36.2%);巢式PCR结果提示14例患者为DENV-4感染,6例为DENV-2感染。结论:2010年广州地区登革热患者症状典型、病情较轻,流行型别是DENV-4和DENV-2,DENV-4均为本土病例有输入及本土病例     

艾滋病患者马尔尼菲青霉菌优势株酵母相全长cDNA文库的构建和初步分析

Objective To construct a full length cDNA library of the dominant strain of Penicillium marneffei (PM) in yeast phase isolated from AIDS patients in Guangdong province and screen UniGenes as well as full-length genes, so as to establish the foundation for the study of PM's functional genes and pathogenic mechanisms. Methods CloneMiner cDNA construction kit was utilized to extract mRNA of the dominant PM strain isolated from AIDS patients in Guangdong province. The mRNA was reversed into cDNA, then cloned into a pDONR222 vector by BP recombination to obtain an Uncut cDNA library, which was homogenized later to construct a normalized cDNA library with the principal of saturation hybridization for DNA genome. 2000 clones were chosen randomly to make a bi-directional sequencing and analyzed with bioinformatics for screening UniGenes and full-length genes. Results The total clone number of the Uncut cDNA library was 1.16 × 107 cfu/mL, with a recombination rate of 95% and an average insertion element being over 1 kb. The total clone number of the normalized cDNA library was 1.18 × 106 cfu/mL, with a recombination rate of 95% and an average insertion element being over 1 kb as well. 1945 genes which DNA length were longer than 1 kb were obtained by sequencing and merged into 1360 UniG enes, of which 632 genes were full-length ones. Conclusions The full-length cDNA library of the dominant strain of PM from AIDS patients in Guangdong province possesses good quality.Meanwhile, the technical routine presents high efficiency in obtaining full-length genes and establishing a gene expression spectrum, which can contentedly meet the needs of future experiments.     

马尔尼菲青霉菌溶血磷脂酶基因的识别和序列分析及功能预测

Objective To identify the gene encoding lysophospholipase from the full length cDNA library of Penicllium marneffei (PM) in yeast phase and predict the structure and function of its deduced protein, to provide with theoretical evidences for further experiments. Methods The PM full-length cDNA library in yeast phase was screened to identify the gene encoding lysophospholipase with the help of NCBI on-line analytical tool. Then, by utilizing the software package of Vector NTI suite 8.0, the protein deduced by the gene was analyzed to predict its corresponding structure and functions, and its molecular cladogram was constructed. Results The gene was composed of 1014 base pairs in the length and was presumed to be the full-length gene encoding lysophospholipase by Blastx, with a complete open reading frame (ORF) comprised of 729 base pairs encoding 243 amino acids with relative molecular weight being 26 800 and the predicted isoelectric point being S.49. The deduced protein included 47.7% hydrophobic amino acids, 26.8% hydrophilic amino acids, 12.7% acid amino acids and 12.8% basic amino acids. The protein had 2 potential casein kinase Ⅱ phosphorylation site, 3 potential protein kinase C phosphorylation site and 7 N-myristoyl site. Conclusions The amino acid sequence belongs to this kind of protease with lysophospholipase-like domain. The protein is most close to Coccidioides posadasii in genetic relationship. A novel gene encoding lysophospholipase is successfully found and the work has made necessary preparations for further research on the gene's function.     

枯草杆菌芽孢抵抗胃肠道环境的耐性评估

目的 对益生菌一枯草杆菌芽孢进行耐性评估,为研制口服枯草杆菌芽孢载体疫苗奠定基础.方法 采用耗竭法,DSM培养基长时间振荡培养(24 h)获得芽孢,使用pH 2.0盐酸胃蛋白酶液模拟胃液,胆酸盐、胰液素混合液模拟肠液,对枯草杆菌芽孢进行体外耐受实验.结果 在DSM培养基中,80%～90%的枯草杆菌WB600可形成芽孢,每升DSM液所生成芽孢约1×1011.在模拟胃液中1 h后,仅0.0024%枯草杆菌WB600繁殖体细菌仍成活,大肠杆菌JM109活力完全丧失,而枯草杆菌芽孢基本不受影响,93.3%仍成活.在模拟小肠环境中3 h后,枯草杆菌WB600繁殖体细菌活力显著降低(仅0.0013%存活),枯草杆菌芽孢基本不受影响(92%仍存活),而大肠杆菌JM109有一定量的增殖.结论 枯草杆菌芽孢能耐受模拟胃肠道环境,有望成为新型的口服疫苗载体.     

华支睾吸虫CsSCCRO基因的克隆表达和蛋白纯化

目的原核表达华支睾吸虫鳞状上皮癌相关肿瘤基因(SCCRO)全长编码区序列,获得纯化的重组蛋白,为进一步功能研究做好准备。方法用生物信息学方法从华支睾吸虫成虫全长cDNA文库中识别出与人鳞状上皮癌相关基因的同源序列及其全长编码区,将其编码区序列克隆到原核表达载体PET-30a(+),测序鉴定重组质粒,在大肠杆菌BL-21/DE3中用IPTG诱导表达,重组产物用His-镍蛋白纯化柱纯化。结果华支睾吸虫鳞状上皮癌相关基因长度为1 218bp,其全长编码序列长度为780bp,编码259个氨基酸,1-22位为分泌信号肽,在羧基端有一个高度保守的区域。该基因在大肠杆菌中得到了高效的可溶性表达,重组蛋白达总蛋白的39%,纯化后的重组蛋白纯度可达98%。结论鳞状上皮癌相关基因的PET-30a(+)原核重组质粒在大肠杆菌BL-21/DE3中可以稳定高效地表达可溶性蛋白。     

204例登革热患者病原学特征分析

目的 分析204例登革病毒感染者的病毒血清型别分布及抗体产生特征,为研究登革热流行动态特征及临床诊治策略提供实验依据.方法 采集登革热患者急性期血清,应用实时荧光PCR检测登革病毒核酸及分型,ELISA法检测登革病毒特异性抗体IgG和IgM.结果 共检测204例标本,其中179例登革病毒核酸阳性,DENV-1型、DENV-2型及DENV-3型分别占97.2％ (174/179)、1.1％(2/174)及0.6％(1/174);DENV-1合并DENV-2型感染和DENV-1合并DENV-3型感染各1例.ELISA法结果显示IgM、IgG抗体阳性者分别为193例和47例,且IgG抗体阳性者的IgM抗体同为阳性,表明初次感染和二次感染分别为146例(75.6％,146/193)和47例(24.4％,47/193).结论 广州存在多种血清型流行及二次感染患者,提示今后广东省重症登革热的发生几率可能会有所增加,应引起重视.     

中国首例甲型H1N1流感二代病例的临床特征

目的了解2009年我国首例甲型H1N1流感二代病例的流行病学、临床、病原学检查特点及预后转归。方法对患者流行病学及临床资料进行回顾性分析,并采用实时荧光聚合酶链反应测定甲型H1N1流感病毒核酸。结果患者与甲型H1N1流感输入病例接触1天后发病。以发热、咽痛、咳嗽起病,白细胞及CD4＋T淋巴细胞计数降低,无肺炎等并发症。多级机构检测咽拭子甲型H1N1流感病毒核酸阳性确诊甲型H1N1流感。RT-PCR测序证实其病毒核苷酸序列与一代输入病例的一致,同源性为100%。经奥司他韦抗病毒及对症治疗痊愈出院。结论本病例的传染源明确,为我国首例报告的甲型H1N1流感二代确诊病例,其临床表现轻,病情恢复快。未发生院内感染,早隔离早诊断等防控措施有效。     

广州市医疗机构HIV抗体不确定的蛋白印迹试验结果及与转归情况的关系

目的:了解HIV抗体不确定结果的特征及其与HIV感染转归的关系。方法:收集广州医科大学附属市八医院HIV确证实验室2015—2019年报告HIV抗体不确定的蛋白印迹试验（WB）结果,分析带型特征及其与转归的关系。结果:共3 365份样本进行了WB试验,其中HIV抗体不确定199份,有确定转归结果185例,其中转归为阳性...