霍乱毒素A、B亚基基因比例表达的精确调节

目的;研究乱毒素Ａ亚基基因内部翻译起始序列的翻译起始效率与Ａ亚基基因翻译起始效率的关系。方法：将基因内翻译起始序列合成后克隆到上游含有起始密码和无起始密码的报告质粒中,研究表达水平的差异。结果：在上流起始区（ｔｒａｎｓｌａｔｉｏｎ ｉｎｉｔｉａｔｉｏｎ ｒｅｇｉｏｎ,ＴＩＲ）与报告基因间插入该序列,基因的表达水平提高１倍,当上上游的翻译起始密码由ＡＴＧ突变为ＡＴＣ时,内部翻译起始序列几乎失去起始功     

霍乱毒素B亚基融合蛋白表达载体的构建

霍乱毒素Ｂ亚基（ＣＴ－Ｂ）具有很强的免疫原性。口服时，在宿主小肠内ＣＴ—Ｂ与神经节苷脂ＧＭ１结合，能够激发机体产生粘膜免疫球蛋白Ａ（ＳＩｇＡ）和全身免疫球蛋白Ｇ（ＩｇＧ）。近几年的研究表明，ＣＴ－Ｂ还能辅佐其他抗原刺激机体产生ＳＩｇＡ，具有免疫佐剂作用。ＣＴ－Ｂ亚基被认为是亚单位疫苗和肽苗的良好载体。在研制预防霍乱和大肠杆菌腹泻的新型口服多价疫苗中，已经注意利用ＣＴ－Ｂ的Ｌ述特性。本研究通过ＰＣＲ技术定点突变，在ＣＴ－Ｂ基因３’端终止密码子处进行两个碱基的突变，消除了转译终止密码子并引入了限制性…     

用抗体捕获法从噬菌体肽库中筛选志贺毒素B亚基结？…

避免生物毒化对志贺毒素Ｂ亚基结构的功能和生物学活性的影响，从呼菌体肽库中筛选与志贺毒素Ｂ亚基结合的短肽分子。方法采用抗体捕获法。结果：经过四轮筛选，ＥＬＩＳＡ结果表明，与ＳｔｘＢ 噬菌体得到了有效富集在８６个随机挑选的克隆中，４９个与ＳｔｘＢ     

乳酸和乙酸对重组霍乱毒素B亚单位工程菌表达的影响

霍乱毒素Ｂ亚单位(ＣＴＢ)是口服霍乱疫苗的重要抗原之一。工程菌ＭＭ2是一株表达ＣＴＢ的大肠杆菌,产物ＣＴＢ能分泌到培养液中[1]。重组质粒ｐＭＭＣＴＢ来源于ｐＵＣ19,ｃｔｘＢ基因位于ｌａｃ启动子的下游。ｌａｃ启动子是一个化学诱导型启动子,无诱导物ＩＰＴＧ(异丙基βＤ硫代半乳糖苷)存在时,其下游基因则不能表达。但是我们的研究结果[2,3]表明,在不用ＩＰＴＧ诱导的情况下ＣＴＢ仍可得到表达,而加入ＩＰＴＧ对ＣＴＢ的表达几乎没有影响。此外,在培养基中加入乳酸或乙酸还可促进ＣＴＢ表达,这一点与多数工程菌的表达特点不同。为此,…     

多重PCR同时检测粪便中的志贺菌、侵袭性大肠埃希菌和O－1群霍乱弧菌的致病基因

本研究建立一种快速标本处理和多重聚合酶链反应（ＰＣＲ）诊断方法，在一次ＰＣＲ反应中同时扩增志贺菌、侵袭性大肠埃希菌的侵袭性质粒（ｉａｌ）基因和Ｏ－１群霍乱弧菌的霍乱毒素Ａ亚单位（ｃｔｘＡ）基因。扩增产物经电泳，出现与已知阳性对照细菌条带大小一致的条带即判断为该标本阳性。对一组经常规培养生化鉴定法诊断为霍乱的２６份腹泻病人粪便标本检测后，ＰＣＲ法有２４例检测到ｃｔｘＡ基因，ＰＣＲ阴性的２例为非Ｏ－１群；对另一组５６例粪标本亦进行了检测。本方法具有简便、快速、特异、经济和不须培养等特点，宜于临床应用。     

用抗体捕获法从噬菌体肽库中筛选志贺毒素B亚基结合分子

目的：避免生物素化对志贺毒素Ｂ亚基结构功能和生物学活性的影响，从噬菌体肽库中筛选与志贺毒素Ｂ亚基结合的短肽分子。方法：采用抗体捕获法。结果：经过四轮筛选，ＥＬＩＳＡ结果表明，与ＳｔｘＢ结合的噬菌体得到了有效富集，在８６个随机挑选的克隆中，４９个与ＳｔｘＢ结合，阳性克隆的序列也表现出一定的偏向性，在结构明确的２９个克隆中，有１２个克隆（４１％）所编码的短肽序列完全一致。结论：抗体捕获法是有效的。运用噬菌体表面显示技术筛选目的分子时，对于生物素化或其他固定化策略影响到结构功能和生物学活性的某些配体分子而言，抗体捕获法是一种较好的选择，可能具有广泛的适用性。     

弗氏志贺菌2aT32株asd基因缺失突变体的构建

目的：构建痢疾弗氏志贺菌２ａＴ３２的ａｓｄ基因全缺失突变体。方法；采用全菌Ｐ座痢疾弗氏２ａ Ｔ３２株染色体扩增了ａｓｄ基因及其上下游各约５００ｂｐ的染色体例 ＤＮＡ序列，并将其克隆至ｐＵＣ１８，测定其核苷酸序列，在体外用ｃｔｘＢ基因置换了ａｓｄ基因，然后将其克隆至自杀载体ｐＸＬ２７５。通过细菌酱和人同源重组，用ｃｔｘＢ基因完全取代痢疾弗氏２ａ Ｔ３２株染色体上的ａｓｄ基因。结果与结论：构建成ａｓｄ     

普卡霉素抑制静脉移植物C-myc在mRNA水平的表达

本实验旨在研究普卡霉素 (光辉霉素 ,ｍｉｔｈｒａｍｙｃｉｎ)抑制Ｃ ｍｙｃ基因在ｍＲＮＡ水平的过量表达对静脉移植物内膜增生的作用。1　材　料1.1　主要试剂　普卡霉素 ( 1ｍｇ ,Ｓｉｇｍａ)、Ｃ ｍｙｃｃＤ ＮＡ探针 (长度 1.4ｋｂ ,酶切点ＥｃｏＲＩ/ＣｌａＩ ,北医大癌基因诊断研究室 )、Ｄｉｇ标记及检测试剂盒(ＢｅｏｈｒｉｎｇｅｒＭｅｎｎｈｅｉｍ ,Ｇｅｒｍａｎ)、蛋白酶Ｋ、ＤＥＰＣ、ＳＳ ＤＮＡ(Ｓｉｇｍａ)。1.2　主要仪器　手术显微镜 (ＳＸＢ 1型 ,10× ,上海)、显微手术器械 (上海 )、低温切片机 (Ｌｅｉｃａ ,Ｊａｐａ…     

甲亢性低血钾型周期性麻痹家系基因突变的初步研究

目的筛查甲亢性低血钾型周期性麻痹家系的钙离子通道α1亚基和电压门控钠离子通道α亚基基因是否存在突变。方法总结甲亢性低血钾型周期性家系Ⅵ代患者的临床特点,并应用酶联免疫测定和测序技术筛查编码钙离子通道α1亚基的528位及1239位精氨酸和钠离子通道α亚基669位及672位精氨酸基因的突变点。结果钠离子通道α亚基基因2012位碱基序列发生错义突变（T→C）,671位苯丙氨酸变为丝氨酸,而其他3个已知突变点的碱基序列完全正常。结论华人甲亢性低血钾型周期性麻痹家系患者中存在突变,致使钠离子通道α亚基基因671位苯丙氨酸为丝氨酸替代,该位点国内外未见报道,是一个新的突变位点。     

脑损伤早期神经细胞c－fos基因表达变化与脑水肿

采用免疫细胞化学方法（ＡＢＣ）研究大鼠脑损伤早期神经细胞ｃ－ｆｏｓ基因表达变化，探讨脑损伤时神经细胞ｃ－ｆｏｓ基因表达变化与脑水肿的关系。结果发现，脑损伤后仅０．５ｈ，脑损伤区周围神经细胞中ｃ－ｆｏｓ基因表达已明显，伤后３～６ｈ更为显著。同时伴有相应部位脑灰质及脑白质水含量增加。初步研究结果提示，脑损伤早期神经细胞ｃ－ｆｏｓ基因表达变化与脑水肿的发生有密切关系。     

含CpG和CTB的HIV多表位基因真核表达载体的构建及表达

目的构建含非甲基化的CpG ODN序列及霍乱毒素B亚单位（CTB）的HIV复合多表位基因的真核表达载体,并在体外进行表达。方法设计并合成含CpG ODN序列和linker序列的CTB基因引物,用PCR方法扩增CpG-CTB基因（CC）,定向连接到真核表达载体pVL上,然后在CC基因下游插入HIV复合多表位基因MEGNp24,构建重组质粒pVL-CC-MEGNp24,酶切鉴定。纯化重组质粒,转染293T细胞,RT-PCR和细胞免疫染色方法分别检测CTB和p24基因的转录及表达,同时设质粒pVL及空白对照。结果构建了重组质粒pVL-CC-MEGNp24,该质粒转染293T细胞后,CTB和复合多表位基因在293T细胞中得到稳定表达。结论成功构建了含免疫佐剂的HIV复合多表位真核表达载体,为后期疫苗的研究奠定了基础。     

Preparation of a DNA probe for thermolabile toxin and its use in the identification of enterotoxic Escherichia coli

Enterotoxic Escherichia coli (ETEC) having a gene for thermolabile toxin were identified by the hybridization technique. HindIII-SmaI fragment with 423 basic pairs (dp) including subunit portions A and B of the gene for thermolabile toxin (elt) was used as the DNA probe.     

Strong static magnetic field and the induction of mutations through elevated production of reactive oxygen species in Escherichia coli soxR

Purpose : Although strong static magnetic fields (SMF) are supposed to have the potential to affect biological systems, the effects have not been evaluated sufficiently. Experiments should be performed with a powerful SMF-generating apparatus to evaluate the biological effects of SMF. Materials and methods : An Escherichia coli mutation assay was used to assess the mutagenic effects of strong SMF. Various mutant strains of E. coli were exposed to up to 9 Tesla (T) for 24 h and the frequencies of rifampicin-resistant mutations were then determined. The expression of the soxS::lacZ fusion gene was assessed by measurement of β-galactosidase activity. Results : The results for survival or mutation were obtained with wild-type E. coli strain GC4468 and its derivatives defective in DNA repair enzymes or redox-regulating enzymes were all negative. On the other hand, the mutation frequency was significantly increased by the SMF exposure in soxR and sodAsodB mutants, which are defective in defence mechanisms against oxidative stress. Furthermore, the expression of superoxide-inducible soxS::lacZ fusion gene was stimulated 1.4- and 1.8-fold in E. coli when exposed to 5 and 9 T, respectively. Conclusions : These results indicate that strong SMF induce mutations through elevated production of intracellular superoxide radicals in E. coli.     

Strong static magnetic field and the induction of mutations through elevated production of reactive oxygen species in Escherichia coli soxR

PURPOSE: Although strong static magnetic fields (SMF) are supposed to have the potential to affect biological systems, the effects have not been evaluated sufficiently. Experiments should be performed with a powerful SMF-generating apparatus to evaluate the biological effects of SMF. MATERIALS AND METHODS: An Escherichia coli mutation assay was used to assess the mutagenic effects of strong SMF. Various mutant strains of E. coli were exposed to up to 9 Tesla (T) for 24 h and the frequencies of rifampicin-resistant mutations were then determined. The expression of the soxS::lacZ fusion gene was assessed by measurement of beta-galactosidase activity. RESULTS: The results for survival or mutation were obtained with wild-type E. coli strain GC4468 and its derivatives defective in DNA repair enzymes or redox-regulating enzymes were all negative. On the other hand, the mutation frequency was significantly increased by the SMF exposure in soxR and sodAsodB mutants, which are defective in defence mechanisms against oxidative stress. Furthermore, the expression of superoxide-inducible soxS::lacZ fusion gene was stimulated 1.4- and 1.8-fold in E. coli when exposed to 5 and 9 T, respectively. CONCLUSIONS: These results indicate that strong SMF induce mutations through elevated production of intracellular superoxide radicals in E. coli.     

肠毒素性大肠杆菌菌毛抗原CS6和霍乱毒素B亚基在人伤寒沙门菌中的共表达及其免疫学效果观察

目的：构建表达肠毒素性大肠杆菌菌毛抗原基因CS6和霍乱毒素B亚基（CTB）基因的细菌性腹泻基因工程多价疫苗。方法：以基因工程减毒的人伤寒沙门菌为抗原载体，利用基于asd基因功能互补的宿主染色体－质粒平衡致死表达系统，实现了在没有抗生素选择压力的条件下，CS6和CTB在人伤寒沙门菌中的稳定表达。结果与结论：检测结果表达，CS6和CTB的表达虽对宿主菌的生长有一定影响。但经过一段时间的培养，重组菌株仍可达到高密度生长，动物免疫结果显示，重组菌株在家兔体内均可诱生相应抗体，但从诱生的抗体效价，抗体持续时间方面评价，单独表达CS6与CTB的重组菌株的免疫学效果均好于两者共表达的重组菌株，提示在以后的疫苗研究实践中，可以考虑将分别表达CS6和CTB的重组菌株按一定比例混合，组成联合疫苗，用于细菌性腹泻的的免疫预防，本研究结果对于细菌性腹泻基因工程多价疫苗的构建有指导意义。     

培养基和溶氧对重组霍乱毒素B亚单位产量的影响

研究了培养基和溶氧对ＭＭ２工程菌产霍乱毒素Ｂ亚单位的影响。用正交试验获得了高产且廉价的ＹＣＰ培养基，０．０２５－０．１ｍｏｌ／Ｌ磷酸盐是霍乱素Ｂ亚单位高产所必需的。提高溶氧以提高产率和缩短培养时间，在５Ｌ发酵罐中培养平均产量达９３．３ｍｇ／Ｌ。分析并讨论了ＭＭ２工程菌的培养动力学过程。     

Radiation-induced mutations in the spleen and brain of lacZ transgenic mice

PURPOSE: To study the dose-response and molecular nature of radiation-induced mutations in the spleen and brain of lacZ transgenic mice. MATERIALS AND METHODS: Line 60 transgenic mice containing the bacterial lacZ gene in a plasmid background were used. Mutants were selected using phenyl-beta-D-galactoside. The nature of mutants was determined by sequencing DNAs of mutant lacZ genes found in control and irradiated tissues. RESULTS: X-ray irradiation at 50 and 100 Gy showed linear dose-responses for mutation induction in both tissues. The slope, however, was about twice as steep in spleen than in brain. DNA sequence analyses showed that the predominant type of mutation induced by radiation in both tissues were large deletions. CONCLUSIONS: Radiation induces mutations in spleen and brain at different efficiencies but the molecular nature of the induced mutations are similar in the two issues.     

Radiation-induced mutations in the spleen and brain of lacZ transgenic mice

Purpose : To study the dose-response and molecular nature of radiation-induced mutations in the spleen and brain of lacZ transgenic mice. Materials and methods : Line 60 transgenic mice containing the bacterial lacZ gene in a plasmid background were used. Mutants were selected using phenyl-beta-D-galactoside. The nature of mutants was determined by sequencing DNAs of mutant lacZ genes found in control and irradiated tissues. Results : X-ray irradiation at 50 and 100 Gy showed linear dose-responses for mutation induction in both tissues. The slope, however, was about twice as steep in spleen than in brain. DNA sequence analyses showed that the predominant type of mutation induced by radiation in both tissues were large deletions. Conclusions : Radiation induces mutations in spleen and brain at different efficiencies but the molecular nature of the induced mutations are similar in the two issues.