间日疟原虫深圳株红内期SSUrDNA基因片段的克隆与序列分析

目的 体外扩增间日疟原虫深圳株红内期小亚单位核糖体核糖核酸编码基因(SSUrDNA)片段，研究其结构与功能。方法 设计一对特异性引物，采用聚合酶链反应(PCR)从间日疟原虫患者血样中扩增出间日疟原虫SSUrDNA片段，以PUC19质粒T载体构建重组子导入大肠杆菌JM109；阳性克隆双酶切鉴定后，双脱氧末端终止法测定序列。结果 间日疟原虫SSUrDNA扩增片段大小为341bp；阳性克隆双酶切及PCR扩增均得到预期大小的片段；序列测定插入片段为341bp，与Sal I株顺序相比，仅在第151位处缺失一个碱基C。结论 成功克隆了间日疟原虫SSUrDNA片段．该序列在间日疟原虫虫株间高度保守。     

人抵抗素基因cDNA的克隆

目的克隆人抵抗素基因(hRETN)cDNA,为进一步研究RETN的结构和功能提供实验基础.方法应用RT-PCR方法从中国人网膜脂肪垫总RNA中扩增出RETN 基因cDNA,克隆入载体pMD18-T中,形成重组载体pMD18-T/hRETN.通过蓝白斑筛选出阳性克隆,限制性内切酶酶切鉴定后对其进行测序.结果从脂肪组织总RNA中扩增得到363 bp片段hRETN基因,其cDNA序列与Genbank hRETN基因序列基本相同.结论成功地克隆中国人hRETN cDNA.     

霍乱毒素B亚单位基因的克隆和表达

用DNA重组技术构建CT-B表达性质粒pXB1及进一步修饰的pXB2,使CT-B基因在大肠杆菌中得到较高水平的表达(280～350ng/ml),且有71.5％分泌率。表达动力学研究阐明,克隆子培养24h产物收率较高。一定浓度的Mg~(2+)、L-组氨酸和天冬酰胺能刺激CT-B的表达。GM1-ELISA、CHO细胞测毒结合间接免疫荧光检测和家兔肠段结扎试验证实,表达的CT-B能与游离的或活细胞膜上的GM1受体结合,但无细胞和肠段毒性。这种细胞测毒结合免疫荧光序贯试验,为客观证实CT-B的生物学活性开拓了新途径。     

连续核移植对异种山羊(Bore)克隆胚胎发育的影响

目的探讨连续核移植与异种山羊克隆胚胎发育之间的关系。方法分别以波尔(Bore)山羊耳部成纤维细胞、山羊-兔异种克隆桑椹胚卵裂球为核供体,以兔卵母细胞为受体,进行连续核移植。结果共构建145枚异种原代重构卵、73枚继Ⅰ代及20枚继II代重构卵,经电融合后,获得重构胚数分别为90、58和14枚,融合率分别为62.1%、79.5%和70%;162枚重构胚在同等条件下进行体外共培养,卵裂率分别为72.2%、75.9%和28.6%,囊胚率分别为10%、13.8%和0%;融合率方面,原代、继I及继II重构卵之间无显著差异(P>0.05);早期发育率无显著差异,但继Ⅰ代重构胚高于原代重构胚,相反,继II代远低于前两者,差异极显著(P<0.01)。结论波尔山羊体细胞核经第1次核移植过程中异种受体卵胞质作用后,比高度分化的体细胞更有利于重构胚的发育,但异种山羊克隆胚卵裂球反复多次暴露在异种卵胞质中,可能不利于细胞核的发育。     

食管癌3p24等位基因LOH及其扩增产物克隆的研究

目的；研究3p24EAβMD位点与食管癌的关系。为寻找该位点附近可能存在的抑癌基因奠定基础。方法：采用PCR—RFLP法检测45例食管癌患者3p24位点杂合缺失情况。并对该片段进行克隆。结果：在22例食管癌信息个体中共检出9例杂合缺失。并通过序列分析可知变化为点突变。结论：3p24EAβMD较高的杂合缺失率显示该位点附近可能存在潜在的抑癌基因。     

人白细胞介素12双亚基共表达真核载体的构建

目的 :为满足基因治疗的需要构建人白细胞介素 12 (hIL 12 )双亚基共表达质粒。方法 :先从人胚肾组织的逆转录产物中用PCR方法扩增出它的两个亚基P4 0 和P3 5的cDNA全长 ,分别克隆入真核表达载体pcDNA3.1(+/ )中构建单亚基质粒———P(+) /P4 0 和P( ) /P3 5,然后再将二者串联克隆入真核表达载体 pcDNA3.1(+)中得到P(+) /IL 12质粒。脂质体转染HepG2后 2 4,48hELISA检测细胞培养上清内hIL 12蛋白质表达。结果 :P(+) /IL 12质粒经酶切和测序证明两亚基连接方向正确 ,序列无突变 ;ELISA检测细胞上清结果证实可表达hIL 12蛋白质。结论 :成功构建P4 0 和P3 5双亚基真核共表达质粒———P(+) /IL 12 ,为模拟hIL 12生理表达方式 ,简化hIL 12基因治疗操作奠定了基础。     

Two novel gastric cancer-associated genes identified by differential display

AIM To clone novel gastric cancer-associated genes and investigate their roles in gastric cancer occurrence.METHODS A method called differential display was used which allows the identification of differentially expressed genes by using PAGE to display PCR-amplified cDNA fragments between gastric cancer cells and normal gastric mucosa cells. These fragments were cloned into plasmid vector pUC18. Homology analysis was made after sequencing these fragments.RESULTS Two novel genes were identified compared with sequences from GenBank. One was registered with the AD number AF 051783. In situ hybridization showed that these two novel genes expressed specifically in gastric cancer tissues.CONCLUSION The two novel genes obtained by differential display were confirmed to be gastric cancer-associated genes using in situ hybridization.     

Identification and characterization of a DR4-restricted T cell epitope within chlamydia heat shock protein 60

An epitope within the 60 kD Chlamydia trachomatis heat shock protein (hsp) 60, recognized by a HLA-DRB1*0401-restricted T cell clone from a reactive arthritis patient, has been characterized. Stimulatory peptides contained a nine amino acid sequence (residues 38–46) predicted by algorithm to confer strong binding to DRB1*0401, with valine in the P1 position. The overall length of the peptide was critical for efficient recognition; peptides with at least one residue N-terminal to the putative P1 position were markedly more stimulatory than a peptide whose N-terminal is the P1 valine. Optimal responses were seen with 14mer peptides having two to three amino acids N- and C-terminal to the core 9mer. The sequence of the defined epitope is identical in hsp60 from both C. trachomatis and C. pneumoniae. Since the latter is a common respiratory pathogen, patients infected with C. trachomatis may already be primed for responses to hsp60 by prior infection with C. pneumoniae. Such secondary responses are important in the pathogenesis of chlamydia-induced inflammatory diseases such as trachoma. Priming by infection with enteric organisms was considered because of the similarity of the epitope sequence in Escherichia coli hsp60. However, although an E. coli-related peptide was recognized, intact E. coli hsp60 was not, suggesting that the epitope is cryptic in E. coli hsp60. Human hsp60 has six amino acid differences from chlamydial hsp60 in the epitope sequence and was not recognized. Thus cross-reactive recognition of self hsp60 could not be implicated in the pathogenesis of chlamydia-induced reactive arthritis in this patient.     

艰难梭茵毒素基因3''''末端重复区域基因片段的PCR克隆

目的探寻一种简单、快速、灵敏的检测艰难梭菌的方法.方法以艰难梭菌毒素A和毒素B基因3'末端高度保守重复区分别设计一对引物,进行PCR反应,同时在同一体系相同反应条件下使用相同引物分别以大肠杆菌和乳杆菌DNA为模板进行扩增,比较二者结果.结果从艰难梭菌成功克隆了毒素A基因3'端重复区域960 bp的基因片段及毒素B基因3'端重复区域1851 bp的基因片段,不同来源的菌株出现了相同的2条电泳带,并通过测序鉴定;而对照的大肠杆菌和乳杆菌株无特异电泳带出现.结论从艰难梭菌毒素A、毒素B基因3'末端重复区域克隆的基因片段具有保守性,可以作为基因探针在临床上进行艰难梭菌检测,该方法简便、灵敏,可直接用粪便标本进行检测.此外,这些基因编码的多肽具有较高的疏水性并存在着膜受体结合区域,可以此为基础进行基因工程蛋白质疫苗的研究.     

一种直接高效克隆PCR产物的载体制备

Ｂｌｕｅｓｃｒｉｐｔ经ＥｃｏＲ Ｖ酶切后，在Ｔａｑ酶作用下加入ｄＴＴＰ使其３’端加上碱基“Ｔ”而成为３’端突出的粘性末端载体。用此载体直接与ＰＣＲ产物连接进行克隆。克隆效率比采用平端载体克隆的效率提高约５０倍。