可溶性55kD人肿瘤坏死因子受体(shTNFR55)在变铅青链霉菌中的分泌表达

从 Ｈｅ Ｌａ 细胞中提取总 Ｒ Ｎ Ａ,采用反转录 Ｐ Ｃ Ｒ 技术,从该总 Ｒ Ｎ Ａ 中扩增了约 ５３０ ｂｐ 的ｓｈ Ｔ Ｎ Ｆ Ｒ５５ 基因的 ｃ Ｄ Ｎ Ａ,并克隆至质粒 ｐ Ｕ Ｃ ｍ ｅｌ中酪蛋白酶 ｍ ｅｌ Ｃｌ 分泌信号肽编码序列的下游,构建成含融合基因 ｍ ｅｌ／ Ｔ Ｎ Ｆ Ｒ 的重组质粒 ｐ Ｕ Ｃ ｍ ｅｌ／ Ｔ Ｎ Ｆ Ｒ．把融合基因 ｍ ｅｌ／ Ｔ Ｎ Ｆ Ｒ 插入链霉菌表达质粒 ｐ Ｉ Ｊ４５９ 的多克隆位点,使之位于 ｅｒｍ 强启动子的下游,得到重组表达质粒 ｐ Ｉ Ｊ４５９ ｍ ｅｌ／ｓ Ｔ Ｎ Ｆ Ｒ．经 Ｓｏｕｔｈｅｒｎ 杂交证明重组质粒 ｐ Ｉ Ｊ４５９ ｍ ｅｌ／ｓ Ｔ Ｎ Ｆ Ｒ 插入了 ｓ Ｔ Ｎ Ｆ Ｒ５５ 基因片段．对重组菌株 Ｓｔｒｅｐｔｏｍ ｙｃｅｓ ｌｉｖｉｄａｎｓ（ｐ Ｉ Ｊ４５９ ｍ ｅｌ／ｓ Ｔ Ｎ Ｆ Ｒ）的发酵液进行 Ｓ Ｄ Ｓ Ｐ Ａ Ｇ Ｅ、受体配基杂交（ Ｌｉｇａｎｄ ｂｌｏｔ）分析、对 Ｔ Ｎ Ｆ 敏感的 Ｌ９２９ 细胞的细胞毒性中和试验表明,可溶性肿瘤坏死因子受体 ｓ Ｔ Ｎ Ｆ Ｒ５５ 在链霉菌中得到了分泌表达,表达产物具有生物学活性．表达产物的分子量约在 ２６～２８ ｋ Ｄ 之间．     

丙型肝炎病毒RNA多聚酶在昆虫细胞中的表达

ＨＣＶ ＮＳ５Ｂ基因片段克隆入ＢＡＣ－ＴＯ－ＢＡＣ＾ＴＭ重组杆状病毒表达系统的ｐＦＡＳＴＨＴｃ载体质粒,转化ＤＨ１０ＢＡＣ＾ＴＭ感受态细菌获得重组的Ｂａｃｍｉｄ质粒,将重组Ｂａｃｍｉｄ质粒转染Ｓｆ细胞,获得的重组杆状病毒可表达目的蛋白。免疫印迹和体外活性检测表明,所表达蛋白为ＨＣＶ ＮＳ５Ｂ蛋白,具有多聚酶活性。     

大肠杆菌表达质粒pSM43及pSM53的构建

利用已成功高表达ｅｒａ基因的质粒ｐＣＥ３１翻译起始码上游的序列,去构建大肠杆菌新的外源基因表达载体。先合成特定序列的单链脱氧寡核苷酸,以改进的实验程序插入ｐＪＬ６,其后再加上限制酶多克隆位点。所构建的ｐＳＭ４３和ｐＳＭ５３分别适合於不带翻译起码（ＡＴＧ）和带起始码的基因插入、表达非融合目的蛋白质之用。并已成功用於人肿瘤坏死因子、人骨形成蛋白、ＨＩＶ蛋白酶、Ｄｕｃｈｅｎｎｅ肌营养不良等ｃＤＮＡ基因的     

p53蛋白多肽片段在大肠杆菌中的表达

在所有研究过的人体肿瘤组织或细胞中,ｐ５３似乎是突变频率最高的一个基因,研究和检测ｐ５３基因及其编码产物的变化将具有重要的意义。我们将野生型ｐ５３基因编码区３’端７０３ｂｐ的ｃＤ－ＮＡ片段插入到大肠杆菌表达载体ｐＢＶ２２０中,得到了一个重组体表达质粒ｐＲＲ３３,经热诱导表达,用ＳＤＳ－ＰＡＧＥ和Ｗｅｓｔｅｒｎ印迹法证实ｐ５３蛋白多肽在大肠杆菌中得到了表达,它不仅可用于抗ｐ５３蛋白抗体的制备,而且也可用于对ｐ５３蛋白羧基端多肽功能的研究。     

芝田硫化叶菌麦芽寡糖基海藻糖合酶基因在大肠杆菌中的克隆和表达

用ＰＣＲ 方法从芝田硫化叶菌中扩增了编码一种新酶,即麦芽寡糖基海藻糖合酶（ ＭＴＳａｓｅ） 的基因,扩增的２－２ｋｂ ＤＮＡ 插入到原核表达载体ｐＢＶ２２０ 中,构建成重组质粒ｐＳＢＧＴ１ 。ｐＳＢＧＴ１ 中ＭＴＳａｓｅ 基因在大肠杆菌中得到表达。ＳＤＳＰＡＧＥ 分析表达产物ＭＴＳａｓｅ蛋白的分子量约为７４ｋＤａ ,同核苷酸序列测定所推导的值相符。表达产物占细胞总蛋白约４－４ ％ 。ｐＳＢＧＴ１ 产生的重组酶作用于淀粉部分水解物,使ＤＥ 值降低,得到非还原糖或低还原糖。     

大肠杆菌表达质粒pSM43及pSM53的构建

利用已成功高表达ｅｒａ基因的质粒ｐＣＥ３１翻译起始码上游的序列,去构建大肠杆菌新的外源基因表达载体。先合成特定序列的单链脱氧寡核苷酸,以改进的实验程序插入ｐＪＬ６,其后再加上限制酶多克隆位点。所构建的ｐＳＭ４３和ｐＳＭ５３分别适合於不带翻译起始码（ＡＴＧ）和带起始码的基因插入、表达非融合目的蛋白质之用。并已成功用於人肿瘤坏死因子、人骨形成蛋白、ＨＩＶ蛋白酶、Ｄｕｃｈｅｎｎｅ肌营养不良等ｃＤＮＡ基因的高表达。     

霍乱弧菌zot基因的克隆及其在大肠杆菌中的表达

从霍乱疫苗菌中抽提基因组ＤＮＡ,用ＰＣＲ的方法扩增ｚｏｔ基因。序列分析表明,ｚｏｔ基因编码３９９个氨基酸,其中４个氨基酸与文献报道有差异。将ｚｏｔ基因插入含Ｔ７启动子的质粒ｐＥＴ－２８（ａ＋）构建表达质粒ｐＥＴ－ＺＯＴ,转化大肠檑菌ＢＬ２１（ＤＥ３）筛有达菌株ＢＬＺＯＴ。表达菌株经１ｍｍｏｌ／ＬＴ ＩＰＴＧ诱导表达３－５ｈ后,表达大量ＺＯＴ蛋白,并形成包涵体,经ＳＤＳ－ＰＡＧＥ分析重组ＺＯＴ蛋白分     

霍乱毒素B亚单位基因（CtxB）的克隆及其表达

从霍乱弧菌中抽提基因组ＤＮＡ,用ＰＣＥＲ方法获取霍乱毒素Ｂ亚单位基因（ＣｔｘＢ）。序列分析结果表明,ＣｔｘＢ基因编码１２４个氨基酸,其中编码６２位Ｔｈｒ的密码子与文献报道有差异。将ＣｔｘＢ基因插入质粒ｐＧＥＸ－４Ｔ－２,构建ｐＧＥＸ－ＣＴＸＢ表达质粒,转化大肠相菌ＢＬ２１（ＤＥ３０,筛选表达菌株ＣＴＸＢ／ＢＬ２１。工程株经ＩＰＴＧ诱导表达,可产生大量的表达蛋白,经ＳＤＳ－ＰＡＧＥ分析,融合蛋白分子     

内皮型氧化氮合酶FAD间区在大肠杆菌中的表达

为了研究内皮型氧化氮合酶（ｅＮＯＳ）的功能、功能调节以及结构与功能的关系．通过ＰＣＲ技术克隆出ｅＮＯＳＦＡＤ间区８０１～９０２ＡＡ肽段的编码基因,插入ｐＥＴ２８ａ（＋）表达质粒中构建成ｐＥＴ２８ａ／ｅＮＯＳ２重组表达质粒,经转染在大肠杆菌中成功表达．表达蛋白经金属离子螯合亲和层析和ＳＤＳＰＡＧＥ回收纯化,得蛋白质纯品．为ｅＮＯＳ特异性抑制肽的筛选和ｅＮＯＳ特异性抗体的制备提供了必要的准备     

环状芽孢杆菌C-2几丁酶基因的克隆

环状芽孢杆菌（Ｂａｃｉｌｕｓｃｉｒｃｕｌａｎｓ）Ｃ２总ＤＮＡ经ＰｓｔＩ部分酶切后分离２～１０ｋｂ的片段,插入质粒ｐＵＣ１９的ＰｓｔＩ位点,转化大肠杆菌（Ｅｓｃｈｅｒｉｃｈｉａｃｏｌｉ）,利用几丁质平板从约８０００个重组子中筛选到一个几丁酶基因阳性克隆（命名为ｐＣＨＴ１）。用１２种限制酶对重组质粒进行的酶切分析表明,重组质粒中的插入片段长３０ｋｂ,其中各有一个ＫｐｎＩ,ＳａｃＩ和ＳｓｐＩ位点。把该克隆片段反向插入ｐＵＣ１９的ＰｓｔＩ位点所得到的重组子同样具有几丁酶基因表达活性,说明此片段含有一个完整的几丁酶基因,其自身的启动子能被大肠杆菌转录系统所识别。Ｓｏｕｔｈｅｒｎ杂交证实了该片段来自于Ｂ．ｃｉｒｃｕｌａｎｓＣ２基因组,且以单拷贝形式存在,它不能与来自于其它７株几丁酶产生菌的总ＤＮＡ杂交。     

Increased Nitrogenase-Dependent H(2) Photoproduction by hup Mutants of Rhodospirillum rubrum

Transposon Tn5 mutagenesis was used to isolate mutants of Rhodospirillum rubrum which lack uptake hydrogenase (Hup) activity. Three Tn5 insertions mapped at different positions within the same 13-kb EcoRI fragment (fragment E1). Hybridization experiments revealed homology to the structural hydrogenase genes hupSLM from Rhodobacter capsulatus and hupSL from Bradyrhizobium japonicum in a 3.8-kb EcoRI-ClaI subfragment of fragment E1. It is suggested that this region contains at least some of the structural genes encoding the nickel-dependent uptake hydrogenase of R. rubrum. At a distance of about 4.5 kb from the fragment homologous to hupSLM, a region with homology to a DNA fragment carrying hypDE and hoxXA from B. japonicum was identified. Stable insertion and deletion mutations were generated in vitro and introduced into R. rubrum by homogenotization. In comparison with the wild type, the resulting hup mutants showed increased nitrogenase-dependent H(2) photoproduction. However, a mutation in a structural hup gene did not result in maximum H(2) production rates, indicating that the capacity to recycle H(2) was not completely lost. Highest H(2) production rates were obtained with a mutant carrying an insertion in a nonstructural hup-specific sequence and with a deletion mutant affected in both structural and nonstructural hup genes. Thus, besides the known Hup activity, a second, previously unknown Hup activity seems to be involved in H(2) recycling. A single regulatory or accessory gene might be responsible for both enzymes. In contrast to the nickel-dependent uptake hydrogenase, the second Hup activity seems to be resistant to the metal chelator EDTA.     

Isolation of genes (nif/hup cosmids) involved in hydrogenase and nitrogenase activities in Rhizobium japonicum.

Recombinant cosmids containing a Rhizobium japonicum gene involved in both hydrogenase (Hup) and nitrogenase (Nif) activities were isolated. An R. japonicum gene bank utilizing broad-host-range cosmid pLAFR1 was conjugated into Hup- Nif- R. japonicum strain SR139. Transconjugants containing the nif/hup cosmid were identified by their resistance to tetracycline (Tcr) and ability to grow chemoautotrophically (Aut+) with hydrogen. All Tcr Aut+ transconjugants possessed high levels of H2 uptake activity, as determined amperometrically. Moreover, all Hup+ transconjugants tested possessed the ability to reduce acetylene (Nif+) in soybean nodules. Cosmid DNAs from 19 Hup+ transconjugants were transferred to Escherichia coli by transformation. When the cosmids were restricted with EcoRI, 15 of the 19 cosmids had a restriction pattern with 13.2-, 4.0-, 3.0-, and 2.5-kilobase DNA fragments. Six E. coli transformants containing the nif/hup cosmids were conjugated with strain SR139. All strain SR139 transconjugants were Hup+ Nif+. Moreover, one nif/hup cosmid was transferred to 15 other R. japonicum Hup- mutants. Hup+ transconjugants of six of the Hup- mutants appeared at a frequency of 1.0, whereas the transconjugants of the other nine mutants remained Hup-. These results indicate that the nif/hup gene cosmids contain a gene involved in both nitrogenase and hydrogenase activities and at least one and perhaps other hup genes which are exclusively involved in H2 uptake activity.     

Role of hydrogenases 1 and 2 in the hydrogen dependent nitrate respiration by Escherichia coli

The study of Escherichia coli mutants synthesizing either hydrogenase 1 (HDK203) or hydrogenase 2 (HDK103) showed that the nitrate-dependent uptake of hydrogen by E. coli cells can be accomplished through the action of either of these hydrogenases. The capability of the cells for hydrogen-dependent nitrate respiration was found to be dependent on the growth conditions. E. coli cells grown anaerobically without nitrate in the presence of glucose were potentially capable of nitrate-dependent hydrogen consumption. The cells grown anaerobically in the presence of nitrate exhibited a much lower capability for nitrate-dependent hydrogen consumption. The inhibitory effect of nitrate on this capability of bacterial cells was either weak (the mutant HDK203) or almost absent (the mutant HDK103) when the cells were grown in the presence of peptone and hydrogen. Hydrogen stimulated the growth of the wild-type strain and the mutant HDK103 (but not the mutant HDK203) cultivated in the medium with nitrate and peptone. These data suggest that hydrogenase 2 is much more active in catalyzing the nitrate-dependent hydrogen consumption than is hydrogenase 1.     

Sensitivity of Saccharomyces cerevisiae to tannic acid is due to iron deprivation

Tannic acid inhibited the growth of the yeast Saccharomyces cerevisiae. Growth medium supplementation with more nitrogen or metal ions showed that only iron ions could restore the maximal growth rate of S. cerevisiae. Tannic acid resistant mutants were previously isolated by screening for tannic acid resistance and were all cytoplasmic petite mutants. While the wild type was very sensitive to iron deprivation conditions when grown in aerobic conditions, the mutants, whether grown aerobically or anaerobically, showed the same growth rate under iron-limited conditions as under iron-repleted conditions. Also, the wild type grown anaerobically was not affected by iron-limited conditions. Cytoplasmic petite mutants obtained by ethidium bromide mutagenesis behaved like the other mutants. During iron limitation, the wild type showed a reduced oxygen uptake rate. Maximal growth rate of the wild type in iron-limited conditions could be restored by the addition to the media of unsaturated fatty acids and sterol. Iron deprivation caused by tannic acid may thus affect the synthesis of a functional respiratory chain as well as the synthesis of unsaturated fatty acids and (or) sterol.     

The nature of H+-K+-exchange in anaerobically and aerobically grown S. typhimurium

H+-K+-exchange via the Trk-like system of K+ accumulation takes place in anaerobically grown S. typhimurium LT-2 with stable ratio of DCC-sensitive ionic fluxes, equal to 2H+ of a cell for one K+ of the medium. This exchange is now observed in the mutant S. typhimurium TH-31 with unfunctional H+-ATPase. H+-K+-exchange in aerobically grown S. typhimurium LT-2 has unstable ratio of ionic fluxes. The rate of K+ uptake in anaerobically grown bacteria is higher than that in the aerobically grown ones. Q10 is about 1.8 both for H+ transfer and K+ uptake in anaerobically grown bacteria, but it is 1.7 and 0.9 respectively in the aerobically grown ones. Delta psi is not changed by different temperatures both in anaerobically and aerobically grown bacteria. The distribution of K+ in anaerobically grown bacteria is higher than 10(3) and the potassium equilibrium potential is much higher than the measured delta psi. In aerobically grown bacteria the distribution of K+ is in good conformity with the measured delta psi. H+ and K+ transport in anaerobically grown cells is likely to proceed by the same mechanism, which includes H+-ATPase and the Trk-like system. In aerobically grown bacteria these transport systems work separately, and the Trk-like system as K+-ionophore serving for K+ uptake across the electrical field on the membrane.     

The Involvement of Hydrogenases 1 and 2 in the Hydrogen-Dependent Nitrate Respiration of Escherichia coli

The study of Escherichia coli mutants synthesizing either hydrogenase 1 (HDK203) or hydrogenase 2 (HDK103) showed that the nitrate-dependent uptake of hydrogen by E. coli cells can be accomplished through the action of either of these hydrogenases. The capability of the cells for hydrogen-dependent nitrate respiration was found to depend on the growth conditions. E. coli cells grown anaerobically without nitrate in the presence of glucose were potentially capable of nitrate-dependent hydrogen consumption. The cells grown anaerobically in the presence of nitrate exhibited a much lower capability for nitrate-dependent hydrogen consumption. The inhibitory effect of nitrate on this capability of bacterial cells was either weak (the mutant HDK203) or almost absent (the mutant HDK103) when the cells were grown in the presence of peptone and hydrogen. Hydrogen stimulated the growth of the wild-type strain and the mutant HDK103 (but not the mutant HDK203) cultivated in the medium with nitrate and peptone. These data suggest that hydrogenase 2 is much more active in catalyzing nitrate-dependent hydrogen consumption than hydrogenase 1.     

Mutants of Escherichia coli with altered hydrogenase activity

Mutant strains of Escherichia coli which expressed different levels of hydrogenase activity when grown anaerobically under a variety of conditions were obtained by mutagenesis and selective growth and screening procedures. Four classes of mutants were isolated, ranging from those devoid of enzyme activity to those expressing maximal activity under all growth conditions. One class of mutants (A) could not grow on fumarate plus H2 in the presence of active fumarate reductase. Since hydrogenase is essential for growth under these conditions some of these strains may be hydrogenase-negative. Three other classes of mutants were isolated which were all hydrogenase-positive and fully expressed this activity when grown on fumarate plus H2. They differed in the level of expression of hydrogenase activity when grown anaerobically on glucose, conditions which do not require hydrogenase for growth. Class B mutants expressed less activity, while class C mutants expressed more activity than the parental strain. Class D mutants fully expressed hydrogenase activity and were dependent on the enzyme for growth. The different strains were also assayed for reduction of dyes by hydrogen and for evolution of hydrogen from reduced methyl viologen. Some of the hydrogenase-positive strains showed altered activities in these assays suggesting that mutations may have occurred either in enzymes or proteins required for reaction with dyes or in the hydrogenase enzyme itself.     

Mitochondrial function in cell wall glycoprotein synthesis in Saccharomyces cerevisiae NCYC 625 (Wild type) and [rho(0)] mutants

We studied phosphopeptidomannans (PPMs) of two Saccharomyces cerevisiae NCYC 625 strains (S. diastaticus): a wild type strain grown aerobically, anaerobically, and in the presence of antimycin and a [rho(0)] mutant grown aerobically and anaerobically. The aerobic wild-type cultures were highly flocculent, but all others were weakly flocculent. Ligands implicated in flocculation of mutants or antimycin-treated cells were not aggregated as much by concanavalin A as were those of the wild type. The [rho(0)] mutants and antimycin-treated cells differ from the wild type in PPM composition and invertase, acid phosphatase, and glucoamylase activities. PPMs extracted from different cells differ in the protein but not in the glycosidic moiety. The PPMs were less stable in mitochondrion-deficient cells than in wild-type cells grown aerobically, and this difference may be attributable to defective mitochondrial function during cell wall synthesis. The reduced flocculation of cells grown in the presence of antimycin, under anaerobiosis, or carrying a [rho(0)] mutation may be the consequence of alterations of PPM structures which are the ligands of lectins, both involved in this cell-cell recognition phenomenon. These respiratory chain alterations also affect peripheral, biologically active glycoproteins such as extracellular enzymes and peripheral PPMs.     

Coordinate expression of hydrogenase and ribulose bisphosphate carboxylase in Rhizobium japonicum Hupc mutants.

In contrast to the wild type, H2 uptake-constitutive mutants of Rhizobium japonicum expressed both hydrogenase and ribulose bisphosphate carboxylase activities when grown heterotrophically. However, as bacteroids from soybean root nodules, the H2 uptake-constitutive mutants, like the wild type, did not express ribulose bisphosphate carboxylase activity.