乙肝病毒核衣壳启动子内三链DNA的形成

用电泳迁移分析方法研究了２１ｎｔ脱氧寡核苷酸Ｇ３ＴＧ２ＴＧＴ２Ｇ５ＴＧ２ＴＧＴ（ＣＰ１）与１２９ｂｐ的乙肝病毒（ＨＢＶ）核衣壳启动子（Ｃｐ）片段内一位点结合形成的三链ＤＮＡ的特异性及稳定性．在克隆有ＨＢＶ基因组的质粒ｐＣＰ１０的酶切产物中,ＣＰ１仅与含Ｃｐ的１２９ｂｐ片段结合．在２０ｍｍｏｌ／ＬＭｇ２＋溶液中其解离常数（Ｋｄ）为１．４×１０－７ｍｏｌ／Ｌ．不同离子稳定三链ＤＮＡ的效果依次为ｓｐ４＋（精胺）＞Ｍｇ２＋＞Ｚｎ２＋＞Ｎａ＋＞Ｋ＋,离子之间存在相互竞争作用．比ＣＰ１多一误配碱基的脱氧寡核苷酸Ｇ２ＴＧ２ＴＧＴＧ３ＴＧ２ＴＧ２ＴＧ２Ｔ（ＣＰ２）在２０ｍｍｏｌ／ＬＭｇ２＋溶液中与Ｃｐ结合的Ｋｄ值约为ＣＰ１的１／７,而在６０ｍｍｏｌ／ＬＫ＋或５ｍｍｏｌ／ＬＺｎ２＋溶液中检测不到它与Ｃｐ的结合,这进一步显示了三链ＤＮＡ形成的特异性．细胞的生理离子浓度被认为是：Ｓｐ４＋１ｍｍｏｌ／Ｌ,Ｍｇ２＋１０ｍｍｏｌ／Ｌ,Ｋ＋１４０ｍｍｏｌ／Ｌ,因此,ＣＰ１在细胞内将能特异地与Ｃｐ结合并具有较好的稳定性．     

金属硫蛋白 - 潜伏膜蛋白融合基因的体外转录调控

ＥＢ病毒（ＥＢＶ）是一种与地区性伯基特氏淋巴瘤、鼻咽癌、何杰金氏病等多种人体肿瘤有关的疱疹病毒．已往的研究表明,潜伏膜蛋白（ＬＭＰ）基因是ＥＢＶ最可能的致瘤基因．为制备ＬＭＰ基因转基因小鼠,探讨ＬＭＰ的体内致瘤作用,首先构建了含鼠金属硫蛋白－１（ＭＴ－１）基因调控区和ＬＭＰ基因编码区的ｐＢＲ３２２－ＭＴ－ＬＭＰ质粒,并用电击法将该质粒与ｐＫＪ１－Ｎｅｏ质粒共转染人胃癌细胞株ＭＧＣ,对ＭＴ－ＬＭＰ基因在转染细胞中的整合、转录情况及重金属镉和镍对该融合基因的转录调控进行了研究．结果表明：（１）两质粒共转染效率为８６．７％;（２）ＰＣＲ和Ｓｏｕｔｈｅｒｎ杂交分析显示,完整的ＭＴ－ＬＭＰ基因已整合入转染的ＭＧＣ细胞基因组,且在不同的转染细胞克隆中,ＭＴ－ＬＭＰ基因整合的方式及拷贝数不同,拷贝数从１到１９不等;（３）ＲＴ－ＰＣＲ和Ｎｏｒｔｈｅｒｎ杂交分析证实,ＭＴ－ＬＭＰ基因不仅在转染的ＭＧＣ中能够转录,而且在１０μｍｏｌ／Ｌ镉诱导下,ＭＴ－ＬＭＰ基因转录增强,平均增高约１．４倍．结果说明,在ＭＴ－１基因调控区指导下,ＬＭＰ基因不但有ｍＲＮＡ水平的表达,而且其表达受重金属镉的调控,上述结果为制备ＭＴ－ＬＭＰ转基因小鼠     

白腐菌产锰过氧化物酶条件的研究

白腐菌ＰｈａｎｅｒｏｃｈａｅｔｅｃｈｒｙｓｏｓｐｏｒｉｕｍＭＩＧ．３８３降解桉木时具有显著的选择性,３０天内降解３７．２３％Ｋｌａｓｏｎ木素,７．２９％综纤维素。该菌株产胞外锰过氧化物酶,并在高碳低氧培养基中显示较高酶活,静置液体培养的优化培养条件是（Ｌ＾－１）：１０ｇ葡萄糖,２ｍｍｏｌ酒石酸铵,１０ｍｍｏｌｐＨ４．５醋酸钠缓冲液;１ｇ吐温８０,２ｇＫＨ２ＰＯ４,０．５ｇＭｇＳＯ４．７Ｈ２Ｏ,０．     

环状芽孢杆菌C—2几丁酶基因在大肠杆菌中的表达

对已克隆的环状芽孢杆菌Ｃ－２的几丁酶基因片段所作的亚克隆分析表明,该几丁酶基因位于１．７ｋｂ Ｐｓｔ Ｉ－Ｓｔｙ Ｉ片段 。ＣｈｉＩ基因在大肠杆菌ＪＭ１０７,ＤＨ５α,ＸＬ１－ｂｌｕｅ,ＴＧ－１等菌株中均能表达,但表达水平不同,其中ＪＭ１０７的表达活性最高,其胞外几丁酶活性与供体菌Ｃ－２菌株的胞外酶活性几乎相当。     

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     

肾动脉内注射内皮素—1对麻醉大鼠肾神经传入放电的影响

本实验旨在观察肾动脉内注射ＥＴ－１对麻醉大鼠血压（ＢＰ）、心率（ＨＲ）和肾神经传入放电（ＡＲＮＡ）的影响,以及ＥＴＡ受体阻断剂ＢＱ－１２３和硝苯吡啶（Ｎｉｆ）对ＥＴ生物学效应的拮抗作用。结果如下：（１）肾动脉注射ＥＴ－１（１μｇ／ｋｇ）后,平均动脉压（ＭＡＰ）先有短暂的降低（由１３．７７±０．１３ｋＰａ降至１０．２±１．１２ｋＰａ）,随后为较显著的持久增高,增值达３．４４±１．６０ｋＰａＰ＜０．００１）,ＨＲ无明显变化,ＡＲＮＡ增加１０８．３３±１６．６７％（Ｐ＜０．００１）。（２）肾动脉内注射ＥＴＡ受体选择性拮抗剂ＢＱ－１２３（１５０μｇ／ｋｇ）,ＥＴ－１的上述效应即被拮抗。与ＥＴ组相比差异非常显著（Ｐ＜０．００１）。（３）肾动脉内注射二氢吡啶敏感性Ｌ－型钙通道阻断剂Ｎｉｆ（０．１ｍｇ／ｋｇ）,也可明显抑制ＥＴ－１的上述效应,与ＥＴ组相比差异十分显著（Ｐ＜０．００１）。以上结果表明：肾动脉内注射ＥＴ－１引起的麻醉大鼠ＭＡＰ增高和ＡＲＮＡ积分增加的作用,可能是由ＥＴＡ受体介导的,其作用的细胞机制可能在于胞内钙超载     

Epstein—Barr病毒反义LMP1基因对鼻咽癌细胞CNE2株生长的抑制

将０．６ｋｂＬＭＰ１前段基因反向插入逆转录病毒载体（ｐＺＩＰ）中,构建成ｐＺＩＰ－反义ＬＭＰ１载体,再转入ＰＡ３１７包装细胞中,用Ｇ４１８筛选性克隆,获得产生１．７×１０＾５（ＣＦＵ／ｍｌ）反杂交实验证实ｐＺＩＰ－反义ＬＭＰ１载体基车整合到ＣＮＥ２细胞的ＤＮＡ中,并且有载体基因ＲＮＡ的表达,观察反义ＬＭＰ１基罟对ＣＮ＃２细胞生长的影响,发现反义ＬＭＰ１基因可降低细胞生长速度,减弱ＣＮＥ２细胞在这果     

人突变appE基因在转基因鼠体内的表达及血清脂质变化

为了研究人突变ａｐｏＥ７基因在血脂代谢中的作用．采用微注射的方法建立了人ａｐｏＥ７转基因鼠,三个首建鼠（ｔｇ１,ｔｇ２,ｔｇ３）整合目的基因的拷贝数相差２倍左右,其血中表达的人ａｐｏＥ７的水平也不相同,低水平表达的ｔｇ１为１．２６ｍｇ／ｄｌ,高水平表达的首建鼠ｔｇ３血清中ａｐｏＥ７浓度可高达２１．１ｍｇ／ｄｌ．异常ａｐｏＥ基因的表达导致了转基因鼠血清甘油三酯和胆固醇明显升高,为对照的１．５～３倍．高密度脂蛋白ＨＤＬ降低,低密度脂蛋白ＬＤＬ和极低密度脂蛋白ＶＬＤＬ升高．经２０ｍｍｏｌ／ＬＺｎＳＯ４诱导后,Ｆ１代Ｔｇ３鼠系血清甘油三酯（ＴＧ）水平高达４４４ｍｇ／ｄｌ,胆固醇（ＴＣ）高达２３４ｍｇ／ｄ１．ＨＤＬ升高和ＬＤＬ／ＶＬＤＬ降低十分明显,表现了高脂血症的指征．     

重组干扰素γ的中间试制

用带有表达质粒ＰＢＶ２２０（含有γ干扰素基因）的大肠杆菌ＤＨ５α株进行发酵培养,３批中试的菌产量平均为１４．１克／Ｌ,γ干扰素的表达量平均为１．０２±１０＾９ＩＵ／Ｌ,收集的菌体经高压匀质破菌后收集包涵体,用７ｍｏｌ／Ｌ盐酸胍提取干扰素,此过程中去除７８．４％菌体蛋白,而干扰素活性仅损失１０．４１％,粗制干扰素经复性可使干扰素活性提高４０５％,比活也有明显提高,３批平均为１．７６×１０＾７ＩＵ／ｍ     

SAPGTP和PG为接头的5'IL6-TNFα衍生物融合蛋白

通过计算机模拟比较十种理论上柔性较好的接头在 ５′ Ｉ Ｌ６ Ｔ Ｎ ＦΔ融合蛋白中对 Ｉ Ｌ ６ 和 Ｔ Ｎ ＦΔ空间结构的影响情况,从中选择了 Ｓ Ａ Ｐ Ｇ Ｔ Ｐ接头．以 Ｓ Ａ Ｐ Ｇ Ｔ Ｐ 作为接头的 ５′ Ｉ Ｌ６ Ｓ Ａ Ｐ Ｇ Ｔ Ｐ Ｔ Ｎ ＦΔ和以 Ｐ Ｇ 为接头的５′ Ｉ Ｌ６ Ｐ Ｇ Ｔ Ｎ ＦΔ空间结构预测结果相似． Ｄ Ｎ Ａ 序列分析两种蛋白的接头序列均与设计的一致．５′ Ｉ Ｌ６ Ｓ Ａ Ｐ Ｇ Ｔ Ｐ Ｔ Ｎ ＦΔ和 ５′ Ｉ Ｌ６ Ｐ Ｇ Ｔ Ｎ ＦΔ蛋白的大肠杆菌表达产物经初步分离、纯化及鉴定后,生物学活性及对高表达 Ｉ Ｌ ６ 受体肿瘤细胞的杀伤作用比较结果显示：在 Ｌ９２９细胞上,前者的生物学活性是后者的 ２７ 倍;在 Ｕ９３７ 细胞上,前者对肿瘤细胞的抑制率是后者的１３ 倍．它们对高表达 Ｉ Ｌ ６ 受体的 Ｕ９３７ 细胞杀伤作用分别是同样突变位点的人 Ｔ Ｎ Ｆα衍生物的３７ 和 ２９ 倍．实验表明, Ｓ Ａ Ｐ Ｇ Ｔ Ｐ作为接头构建的 ５′ Ｉ Ｌ６ Ｓ Ａ Ｐ Ｇ Ｔ Ｐ Ｔ Ｎ ＦΔ融合蛋白优于以 Ｐ Ｇ 作为接头构建的 ５′ Ｉ Ｌ６ Ｐ Ｇ Ｔ Ｎ ＦΔ融合蛋白．     

A 'phase-shift' fusion system for the regulation of foreign gene expression by lambda repressor in gram-negative bacteria

A 'phase-shift' translation fusion vector was constructed in which mutually compatible restriction sites BamHI, BclI and BglII are positioned in such a manner that the cut point is in a different reading frame, immediately following the ATG start codon and ribosome-binding site of the lambda cro gene. The lambda cro gene is expressed from promoter pR and controlled by a thermosensitive (cI857) lambda repressor. The usefulness of the expression vector was demonstrated using a galK gene lacking the ATG start codon and fusing this to the pR promoter and ATG start codon of the lambda cro gene, resulting in cI857-regulated expression of galactokinase. The vector is of general use for foreign gene expression in Escherichia coli when the target gene has a compatible cohesive end (5'-GATC-3') at the N terminus (provided, for example, by a BamHI linker). The lambda cI857-pR-cro-galK cassette was cloned into pJRD215, a wide-host-range plasmid and transferred by conjugation to a variety of Gram-negative bacteria. In all cases, thermosensitive regulation of galactokinase could be demonstrated, though the levels of induction varied considerably. These results show that the powerful lambda pR promoter and the efficient lambda repressor can be used to regulate expression of foreign genes in Gram-negative organisms other than E. coli.     

Translational initiation frequency of atp genes from Escherichia coli: identification of an intercistronic sequence that enhances translation.

The c, b and delta subunit genes of the Escherichia coli atp operon were cloned individually in an expression vector between the tac fusion promoter and the galK gene. The relative rates of subunit synthesis directed by the cloned genes were similar in vitro and in vivo and compared favourably with the subunit stoichiometry of the assembled proton-translocating ATP synthase of E. coli in vivo. The rate of synthesis of subunit c was at least six times that of subunit b and 18 times that of subunit delta. Progressive shortening of the long intercistronic sequence lying upstream of the subunit c gene showed that maximal expression of this gene is dependent upon the presence of a sequence stretching greater than 20 bp upstream of the Shine-Dalgarno site. This sequence thus acts to enhance the rate of translational initiation. The possibility that similar sequences might perform the same function in other operons of E. coli and bacteriophage lambda is also discussed. Translation of the subunit b cistron is partially coupled to translation of the preceding subunit c cistron. In conclusion, the expression of all the atp operon genes could be adjusted to accommodate the subunit requirements of ATP synthase assembly primarily by means of mechanisms which control the efficiency of translational initiation and re-initiation at the respective cistron start codons.     

TGATG vector: a new expression system for cloned foreign genes in Escherichia coli cells

A TGATG vector system was developed that allows for the construction of hybrid operons with partially overlapping genes, employing the effects of translational coupling to optimize expression of cloned cistrons in Escherichia coli. In this vector system (plasmid pPR-TGATG-1), the coding region of a foreign gene is attached to the ATG codon situated on the vector, to form the hybrid operon transcribed from the phage lambda PR promoter. The cloned gene is the distal cistron of this hybrid operon ('overlappon'). The efficiently translated cro'-cat'-'trpE hybrid cistron is proximal to the promoter. The coding region of this artificial fused cistron [the length of the corresponding open reading frame is about 120 amino acids (aa)] includes the following: the N-terminal portions of phage lambda Cro protein (20 aa), the CAT protein of E. coli (72 aa) and 3' C-terminal codons of the E. coli trpE gene product. At the 3'-end of the cro'-cat'-'trpE fused cistron there is a region for efficient translation reinitiation: a Shine-Dalgarno sequence of the E. coli trpD gene and the overlapping stop and start codons (TGATG). In this sequence, the last G is the first nucleotide of the unique SacI-recognition site (GAGCT decreases C) and so integration of the structural part of the foreign gene into the vector plasmid may be performed using blunt-end DNA linking after the treatment of pPR-TGATG-1 with SacI and E. coli DNA polymerase I or its Klenow fragment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression and secretion of Mirabilis antiviral protein in Escherichia coli and its inhibition of in vitro eukaryotic and prokaryotic protein synthesis

Mirabilis antiviral protein (MAP), a ribosome-inactivating protein, exhibits inhibitory effects on both plant virus infection and protein synthesis. To study these functions by site-specific mutagenesis, the total synthetic gene of MAP was constructed and expressed in Escherichia coli. However, the growth of the host was inhibited by the products, and the yield of MAP was very low. To improve the system for expressing MAP, an expression vector, pSH7, was constructed. This vector is based on the high copy number plasmid pUC19 and includes PL promoter and temperature-sensitive cI857 repressor. The plasmid also contains the ompA signal sequence and the total synthetic MAP gene. The MAP gene was expressed and its product was secreted into the culture medium after E. coli transformants were cultivated at 30 degrees C and the temperature was raised to 42 degrees C. The secreted MAP was then purified and characterized. This protein was identical to native MAP as determined by its mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the amino acid sequence at the NH2 terminus, and its inhibitory effect on in vitro protein synthesis. MAP was found to inhibit the in vitro protein synthesis of rabbit reticulocyte and wheat germ. It further showed an IC50 concentration of approximately 200 nM in an E. coli in vitro translation system in contrast to ricin A-chain, a well known ribosome-inactivating protein.     

Heat-inducible autolytic vector for high-throughput screening

In directed evolution, a high-throughput screening system is often a prerequisite for sampling the enzyme variants. When the target enzyme is expressed intracellularly, for example when Escherichia coli is used as the host, chemical or enzymatic disruption of cell membrane is often required in many cases, which can be tedious, time-consuming, and costly. In this study, a set of heat-inducible autolytic vectors were constructed to solve this problem, in which the SRRz lysis gene cassette from bacteriophage lambda was placed downstream of heat-inducible promoters, lambda cI857/pR promoter and its mutant, c1857/pR(M). The artificial autolytic units were inserted into the backbone of pUC18 (away from the multiple cloning sites). For the wild promoter; cI857/pR, the SRRz lysis cassette was expressed by temperature up-shift from 28 degrees to 38 degrees C, and the lysis efficiency of transformed bacterial cells was found to be consistent and could reach 96.3% as measured by the reporter beta3-galactosidase assay. In order to obtain a higher cell growth rate, the mutant promoter cI857/pR(M) was utilized to allow bacteria growth at 35 degrees C and lysis at 42 degrees C. However; this heat-inducible system showed significant inconsistency in terms of lysis efficiency. Bacillus subtilis 168 lipase A gene was further inserted into the multiple cloning sites of the autolytic vector containing cI857/pR, and 93.7% of the expressed lipase activity was found in the culture medium upon heat induction, demonstrating the utility of the vector for expression and rapid extracellular assay of heterologous enzymes.