Variations of Phospholipases A2 in the Geographic Venom Samples of Pitvipers

The geographic variations of phospholipases A₂ (PLAs) in the venom of four medically important pit vipers were investigated. We have studied the PLAs by HPLC‐purification, cDNA cloning and sequencing, mass characterization, and functional classification. We found that: 1) Anti‐platelet acidic PLA isoforms in the venoms of Calloselasma rhodostoma from five southeastern Asian countries, and those of the Crotalus v. viridis from seven American States are differentially expressed depending on locality. The variations could be attributed to their distinct specificities towards the platelets of different prey, and to possible adaptation for playing other functional roles. In contrast, structures of the myonecrotic and the edema‐inducing basic PLAs in both venoms were relatively conserved. 2) A special type of the acidic anti‐platelet PLA is present in the venom of some Protobothrops species. Its expression level is diminished in the snake of the southern or the tropical ranges. 3) The venom of Bamboo tree vipers (Trimeresurus stejnegeri) in Taiwan and China showed extraordinary geographic variations in their acidic and basic PLAs. The high RNA‐polymorphism of their venom proteins may have been derived from interbreeding between several ancestral pit viper species. In addition, migration, isolation of different populations and rapid evolution of the venom proteins to adapt for diversified diets may have resulted in further variations in this venom species.     

骨保护素基因修饰的大鼠骨髓基质细胞系的培养与鉴定

目的培养经骨保护素OPG修饰的大鼠骨髓基质细胞系并检测其表达能力,鉴定表达产物,为基因工程技术治疗牙周病提供物质基础。方法利用已构建的pSecTagOPG真核分泌表达穿梭载体,用脂质体法将目的基因转染至大鼠骨髓基质细胞,应用免疫组织化学和Westernblot方法证实转染的细胞表达骨保护素。结果经免疫组织化学和Westernblot方法检测,在转染OPG的大鼠骨髓基质细胞中有OPG表达。结论本实验成功培养出分泌表达OPG的大鼠骨髓基质细胞系。     

Construction of cloning‐friendly minigenes for mammalian expression of full‐length human NF1 isoforms

The neurofibromatosis type 1 (NF1) tumor suppressor gene is one of the most frequently mutated genes in human tumors. Research on the NF1 proteins has been partially hindered by the difficulties in cloning and propagating the full‐length coding cDNAs. We have now established a condition for propagating the natural open reading frames (ORFs) and have assembled the ORFs for human NF1 type 1 and 2 isoforms. Furthermore, we were able to eliminate the cDNA cloning toxicity by introducing a mini‐intron. These NF1 minigenes were expressed similarly to the intronless version and could be used to purify full‐length NF1 proteins. The NF1 isoforms expressed from the minigenes showed Ras‐GAP activity in vivo and in vitro, while the type 1 was more potent. Our constructs expand currently available full‐length NF1 constructs and should be valuable tools in expediting the understanding of NF1, particularly the isoform‐specific functions and regulation.     

幽门螺杆菌细胞空泡毒素毒性片段基因的表达及其重组蛋白免疫学鉴定

目的采用基因工程技术获得具有良好免疫反应性的重组细胞空泡毒素的毒性亚单位蛋白。方法利用PCR技术从Hp基因组中扩增VacA基因毒性片段,插入原核表达载体pQE30上,测序后,转化表达宿主菌DH5α,SDSPAGE分析表达情况,Western blot检测其免疫反应性。NiNTA^2＋树脂纯化后,用重组蛋白免疫兔,Helico Blot试剂盒检测兔血清VacA抗体以鉴定其免疫原性。结果测序表明目的序列完整,与Genbank中的多株中国菌株相比有高度的同源性。SDS—PAGE显示表达产物相对分子量为27000。表达量为30％以上。该蛋白可被Hp全菌抗血清所识别,纯化后可得到纯度为93％以上蛋白质,并证实有良好的免疫原性。结论Hp VacA基因毒性片段表达成功,获得高纯度重组蛋白并具有良好的免疫反应性与免疫原性,为研制疫苗和制备诊断VacA（＋）株感染试剂盒提供了可靠材料。     

山茱萸萜类甲羟戊酸合成途径关键酶CoHMGS基因的克隆与分析

目的克隆得到山茱萸Cornusofficinalis萜类合成途径的关键酶CoHMGS基因的cDNA序列,并进行相应的生物信息学分析,为深入研究CoHMGS基因的功能奠定基础。方法以山茱萸转录组筛选出的c102453_g2序列为参考序列设计特异引物,以山茱萸叶片总RNA,通过RT-PCR扩增获得CoHMGS基因序列,序列纯化回收后连接到pTOPO-T载体上,转化至大肠杆菌DH10B,选取阳性克隆测序。利用生物信息学软件预测CoHMGS基因及其编码蛋白质的功能。结果克隆得到长度为1645 bp的CoHMGS序列,包含1413 bp的完整开放阅读框,编码470个氨基酸。蛋白整体带负电荷,为不稳定亲水性蛋白,定位于细胞质内,不含跨膜结构。结论首次克隆得到山茱萸CoHMGS基因,对其编码蛋白质进行了初步的分析和预测,为深入揭示CoHMGS基因在山茱萸萜类物质合成途径中的功能奠定了基础。     

荆芥1-脱氧-D-木酮糖-5-磷酸合成酶基因克隆及生物信息学分析

目的克隆荆芥1-脱氧-D-木酮糖-5-磷酸合成酶(1-deoxy-D-xylulose 5-phosphate synthase,StDXS)基因,并进行生物信息学分析。方法根据荆芥转录组数据获得的StDXS基因序列设计特异性引物,通过RT-PCR技术获得StDXS基因cDNA的全长序列,并进行生物信息学分析。结果荆芥StDXS基因全长2177 bp,包含一个长度为2157 bp的开放阅读框,编码718个氨基酸,其理论相对分子质量为77240,等电点为6.32,定位于叶绿体,不存在跨膜区及信号肽,为非分泌蛋白。同源系统进化树分析表明,该序列与同科植物鼠尾草的DXS基因进化关系较近,均属于DXS1亚家族。密码子偏性分析表明,该基因偏好使用以A/T结尾的密码子,具有28个偏性密码子,烟草为该基因最适合的外源表达宿主。结论成功克隆荆芥StDXS基因并进行生物信息学分析,为从分子水平调控荆芥的生长发育和改善药材产量和品质提供理论基础。     

丙型肝炎病毒包膜蛋白基因片段的克隆与表达

应用ＲＴ－ＰＣＲ和基因重组技术，从湖南地区丙型肝炎病毒（ＨＣＶ）感染者血清中克隆了ＨＣＶ包膜蛋白基因片段（Ｅ１，Ｅ２／ＮＳ１），经与已知基因型的ＨＣＶ－１，ＨＣＶ－Ｊ，ＨＣＶ－Ｊ６和ＨＣＶ－Ｊ８进行核苷酸序列的比较分析，这些基因片段属１ｂ型ＨＣＶ基因。将克隆基因重组于表达质粒ＰＭＡＬ－ＣＲＩ中，在大肠杆菌内进行高效表达，获得了融合蛋白ＭＢＰ－Ｅ１，ＭＢＰ－Ｅ２／ＮＳ１，经ＷｅｓｔｅｒｎＢｌｏｔ分析证实这些融合蛋白具有ＨＣＶ的特异抗原活性，提示这些融合蛋白可用于ＨＣＶ感染的临床诊断和发病机理的研究。     

白血病原代骨髓基质细胞对柔红霉素作用下Jurkat细胞基因表达的影响

目的研究白血病患者骨髓基质细胞（BMSCs）抑制柔红霉素（DNR）诱导Jurkat细胞凋亡过程中Jurkat细胞相关基因表达的变化,并进一步分析差异表达基因在BMSCs对Jurkat细胞保护过程中的意义.方法采用Percoll体外分离初治急性白血病患者骨髓单个核细胞,贴壁培养BMSCs;采用抑制性消减杂交技术等分子生物学方法建立白血病BMSCs保护的Jurkat细胞差异表达基因的cDNA文库;并对差异表达的基因进行初步鉴定与分析.结果成功地建立了白血病BMSCs保护的Jurkat细胞上调和下调差异表达基因的cDNA文库;初步筛选克隆到30个上调差异表达基因和22个下调差异表达基因cDNA片段;其功能主要与细胞周期调控、细胞凋亡、细胞能量代谢相关.结论白血病患者BMSCs能诱导Jurkat细胞基因发生差异表达,为探讨白血病患者BMSCs保护白血病细胞逃避化疗药物杀伤的分子机制进行了前期研究.