重组酶法定量分析吡咯喹啉醌

用ＤＥＡＥ－ｓｅｐｈａｃｅｌ和ＣＭ－ｃｅｌｕｌｏｓｅ柱层析的方法从Ｃｏｍａｍｏｎａｓｔｅｓｔｏｓｔｅｒｏｎｉ菌体中纯化得到一定纯度的脱辅基的乙醇脱氢酶。在含３ｍＭＣａＣｌ２的Ｔｒｉｓ／ＨＣｌ缓冲液（２０ｍＭ,ｐＨ７．０）中,该酶能与ＰＱＱ重组成有活性的全酶,测出的全酶活性大小与外加ＰＱＱ的量成正比,从而定量分析ＰＱＱ。该法专一、灵敏、可靠。     

丙型肝炎病毒C33_c单克隆抗体的研制和鉴定

用丙型肝炎病毒重组蛋白Ｃ３３_ｃ抗原免疫ＢＡＬＢ／ｃ小鼠,运用杂交瘤技术成功地建立了７株能稳定分泌抗Ｃ３３_ｃ单克隆抗体的杂交瘤细胞１Ｈ６Ｄ２、２Ｇ１Ａ６、３Ａ４Ａ８、３Ｅ３Ｅ７、４Ｇ１２Ｃ１０、４Ａ１０Ｃ２、５Ｆ４Ｂ６．试验结果表明,７株ＭｃＡｂｓ具有良好的ＨＣＶ特异性,间接ＥＬＩＳＡ法测得小鼠腹水ＭｃＡｂ效价为１：１０￣４－１：４×１０￣４;竞争抑制实验和相加指数测定证实７株ＭｃＡｂｓ识别相关的抗原表位;７株ＭｃＡｂｓ中１株为ＩｇＭ（５Ｆ４Ｂ６）,其它６株为ＩｇＧ（２ａ）。     

王瓜的组织培养和快速繁殖

１植物名称王瓜（Ｔｒｉｃｈｏｓａｎｔｈｅｓｃｕｃ－ｕｍｅｒｏｉｄｅｓ），采自浙江天目山。２材料类别顶芽、带腋芽的茎段。３培养条件诱导分化培养基：（１）ＭＳ＋６－ＢＡ０．５ｍｇ·Ｌ－１（单位下同）；（２）ＭＳ＋６－ＢＡ１；（３）ＭＳ＋６．ＢＡ２＋２，４－Ｄ１。诱导生根与生长培养基：（４）ＭＳ；（５）ＭＳ＋ＩＡＡ２；（６）ＭＳ＋６－ＢＡ１＋２，４－Ｄ１。每种培养基均附加３％蔗糖，０，７％琼脂，ｐＨ５．８。培养温度（２５±１）℃，光照每天１２ｈ，光照度２０００１ｘ左右。４生长与分化情况４．１无菌材料的…     

簇毛麦低拷贝cDNA序列的克隆和鉴定

从簇毛麦叶片中提取总ＲＮＡ,进一步分离ｍＲＮＡ。以ｍＲＮＡ为模板反转录合成ｃＤＮＡ,两端经Ｔ４ＤＮＡ多聚酶修平后加ＥｃｏＲ１接头分子,连接于质粒ｐＧＥＭ－７Ｚｆ（＋）的ＥｃｏＲ１克隆位点,转化大肠杆菌ＪＭ１０３菌株建立了ｃＤＮＡ文库。用ＰＣＲ扩增重组质粒的ｃＤＮＡ插入序列,用＾３２Ｐ标记后分别与ＨｉｎｄⅢ或ＸｂａⅠ酶切的小麦－黑麦附加系ＤＮＡ进行Ｓｏｕｔｈｅｒｎ杂交。根据其杂交结果,目前已鉴定出４     

巨大芽孢杆菌青霉素酰化酶在枯草杆菌中的表达及其分离纯化

青霉素酰化酶（ＰＧＡ）在医药工业起着重要的作用,它能够水解青霉素Ｇ产生６－氨基青霉烷酸（６－ＡＰＡ）和苯乙酸,６－ＡＰＡ是半合成青霉素的关键中间体．该酶广泛存在于各种微生物中如真菌和细菌中．国际上对Ｅ．ｃｏｌｉ、Ａｒｔｈｒｏｂａｃｔｅｒｖｉｓｃｏｓｕ...     

大肠杆菌苯丙氨酸生物合成关键酶的串联表达

ａｒｏＧ基因编码的 ３－脱氧－２－阿拉伯庚酮糖－７－磷酸合成酶（ＤＡＨＰ　Ｓｙｎｔｈｅｔａｓｅ　ＤＳ）和 ｐｈｅＡ基因编码的分支酸变位酶／预苯酸脱水酶（Ｃｈｏｒｉｍａｔｅ ｍｕｔａｓｅ／ Ｐｒｅｐｈｅｎａｔｅ　ｄｅｈｙｄｒａｔａｓｅ,ＣＷ／ＰＤ）都是本丙氨酸合成途径中的关键酶,为了通过基因工程手段来增加本丙氨酸生物的产量,在利用高效的原核表达载体ｐＢＶ２２ ０对ｐｈｅＡ基因编码的ＣＭ／ ＰＤ 酶进行了表达的基础上,采用ＰＣＲ方法扩增了抗反馈抑制的ａｒｃＧ基因,进行克隆表达,并与ｐｈｅＡ基因串联,以ＰＲＰＬ－ａｒｏＧ－ＰＬ－ｐｈｅＡ的形式,实现了２种酶基因在大肠杆菌中的表达, ＳＤＳＰＡＧＥ 图谱显示了新增的４３ｋｕ及３５ｋｕ蛋白带,经酶活性测定ＤＳ、ＣＭ／ＰＤ酶的比活分别提高了 ４．６７倍、８０５／１０．７１倍。     

宫粉郁金的组织培养和快速繁殖

１植物名称宫粉郁金（Ｃｕｒｃｕｍａ　ｋｗａｎｇｓｉｅｎｓｉｓ）。２材料类别　块茎萌动芽。３培养条件　萌动芽生长培养基：（１）ＭＳ＋６－ＢＡ１ｍｇ·Ｌ－１（单位下同）＋ＮＡＡ０．２。不定芽增殖与愈伤组织诱导培养基：（２）ＭＳ＋６－ＢＡ１０＋ＫＴ５;（３）ＭＳ＋６．ＢＡ１０;（４）ＭＳ＋６－ＢＡ５＋ＫＴ２．５;（５）ＭＳ＋６－ＢＡ５;（６）ＭＳ＋６－ＢＡ２＋ＫＴ１。生根培养基：（７）ＭＳ＋ＮＡＡ０．５;（８）ＭＳ＋６－ＢＡ０．５＋ＮＡＡ０．５;（９）ＭＳ。以上培养基均加０．７％琼脂,３％蔗糖,ｐＨ５…     

地高辛精标记酪氨酸羟化酶RNA探针的制备研究

为制备用地高辛精（Ｄｉｇｏｘｉｇｅｎｉｎ,Ｄｉｇ）标记的酶氨酸羟化酶（ＴｙｒｏｓｉｎｅＨｙｄｒｏｘｙｌａｓｅ,ＴＨ）ＲＮＡ探针,本研究用分子生物学技术重组质粒ＰＧＥＭＴＨＩ,即分别将携带Ｔ７和Ｓｐ６启动子的ＰＧＥＭ－３ｚｆ质粒和携带ＴＨ基因的ＰＫＳＴＨ质粒用限制性内切酶消化并行分离纯化,得到带Ｔ７和ｓｐ６启动子的ＤＮＡ片段和ＴＨ基因片段;经Ｔ４ＤＮＡ连接酶连接后转入大肠杆菌,得到ｐＧＥＭＴＨ１重组克隆。经小量提取质粒井用酶切分析检测,证实其确有ＴＨ基因且方向正确。将此质粒再经限制性内切酶消化则得到线状ＤＮＡ片段,用Ｔ７ＲＮＡ聚合酶转录合成带有Ｄｉｇ标记的高比活度的单链ＲＮＡ探针;经斑点杂交试验证实该探针具有较高的可靠性。这将为基因治疗帕金森氏病动物模型的疗效检测提供有效手段。     

新型生长抑肽对成纤维细胞的DNA合成及某些癌基因表达的影响

从细胞水平和基因水平研究了新型生长抑肽（ＥＰＰ）的生物学作用,研究表明：ＥＰＰ对ＮＣ３Ｈ１０及ＴＣ３Ｈ１０细胞的ＤＮＡ合成均有抑制作用,当ＥＰＰ与ｃＡＭＰ的位点选择性类似物８－Ｂｒ－ｃＡＭＰ共同作用时,其对ＮＣ３Ｈ１０细胞的ＤＮＡ合成的抑制作用消失,而对ＴＣ３Ｈ１０仍具有抑制作用;核酸杂交分析表明,ＥＰＰ可以抑制ｃ－ｆｏｓ、ｎｅｕ、ｋｉ－ｒａｓ三类癌基因在转化细胞中的表达。证明了ＥＰＰ对转化细胞的生长具有一定的抑制效应,且与８－Ｂｒ－ｃＡＭＰ联合使用时其效果更佳。     

人骨形态发生蛋白—7全长cDNA的克隆及序列测定

从人胎儿肾中提取总ＲＮＡ,反转录得ｃＤＮＡ,ＰＣＲ扩增获得１．３ｋｂ的骨形态发生蛋白－７（ＢＭＰ－７）的全长ｃＤＮＡ。克隆的ＢＭＰ－７基因编码的氨基酸序列与献报道相同。     

Characterization and nucleotide sequence of pqqE and pqqF in Methylobacterium extorquens AM1.

Methylobacterium extorquens AM1 pqqEF are genes required for synthesis of pyrroloquinoline quinone (PQQ). The nucleotide sequence of these genes indicates PqqE belongs to an endopeptidase family, including PqqF of Klebsiella pneumoniae, and M. extorquens AM1 PqqF has low identity with the same endopeptidase family. M. extorquens AM1 pqqE complemented a K. pneumoniae pqqF mutant.     

常压室温等离子体诱变扭脱甲基杆菌AM1高产吡咯喹啉醌

吡咯喹啉醌(Pyrroloquinoline quinone,PQQ)作为一种新型的氧化还原酶辅酶,在医药和食品等领域有广阔的应用前景。为改善扭脱甲基杆菌Methylobacterium extorquens AM1 PQQ生产性能,采用常压室温等离子体(Atmospheric and room temperature plasma,ARTP)进行诱变,结合高通量快速筛选方法,得到以PQQ产量为指标的正向突变株。ARTP诱变的菌株正突变率为31.6%,筛选得到的较优正突变株M.extorquens AM1(E-F3),PQQ产量达到54.0 mg/L,是出发菌株的近3倍。系统的高通量方法筛选ARTP诱变菌为后续进一步提高M.extorquens AM1菌株PQQ的产量奠定了基础,亦为改善菌株生产性能提供了新思路。     

一株新的产吡咯喹啉醌甲基营养菌全基因组测序和比较基因组学分析

【背景】由于甲基营养菌被发现的时间较短,而且可以生产吡咯喹啉醌(pyrroloquinoline quinone,PQQ)的甲基杆菌属细菌只有少数菌株的全基因组序列被公布,增加了该类细菌基因组学和生物代谢途径研究的难度。【目的】将本实验室筛选的PQQ生产菌经多种诱变方式处理,用于提高PQQ的发酵产量。对高产突变菌株进行全基因组解析,以探究甲基杆菌PQQ合成的分子机制,为后续分子育种提供序列背景信息。【方法】将野生型PQQ生产菌株进行紫外诱变、亚硝基胍诱变、甲基磺酸乙酯诱变、硫酸二乙酯诱变和紫外-氯化锂复合诱变。将突变菌株利用PromethION三代测序平台和MGISEQ-2000二代测序平台测序,然后进行组装和功能注释。组装得到的全基因组序列与模式菌株扭脱甲基杆菌AM1 (Methylobacterium extorquens AM1)进行比较基因组学分析。【结果】经11轮诱变获得一株突变菌株NI91,其PQQ产量为19.49 mg/L,相较原始菌株提高44.91%。突变菌株NI91的基因组由一个5 409 262 bp的染色体组成,共编码4 957个蛋白,与模式菌株M. extorquens AM1比较发现其PQQ合成过程中剪切加工相关的基因pqqF和pqqG缺失,但首次在甲基营养菌中发现与基因pqqF具有相似功能的基因pqqL,且基因pqqC/D的序列存在较大差异。【结论】为甲基营养类细菌甲基杆菌的功能基因组学研究及PQQ合成机理研究提供了基础数据支持,NI91与模式菌株M. extorquens AM1的比较基因组学分析为揭示PQQ合成的不同机理提供了分子基础。     

Novel dephosphotetrahydromethanopterin biosynthesis genes discovered via mutagenesis in Methylobacterium extorquens AM1

Methylobacterium extorquens AM1 was used to explore the genetics of dephosphotetrahydromethanopterin (dH(4)MPT) biosynthesis. Strains with mutations in eight "archaeal-type" genes linked on the chromosome of M. extorquens AM1 were analyzed for the ability to synthesize dH(4)MPT, and six were found to be dH(4)MPT negative. Putative functions of these genes in dH(4)MPT biosynthesis are discussed.     

PqqC/D,which converts a biosynthetic intermediate to pyrroloquinoline quinone

PqqC/D was purified from Escherichia coli transformant. The purified enzyme converted an intermediate that accumulated in a pqqC mutant of Methylobacterium extorquens AM1 to PQQ. The reaction did not show any dependence of NAD(P)H that was observed in the crude extract before purification. PqqC/D reacted with the intermediate stoichiometrically, but not catalytically. When partially purified proteins from the crude extract of E. coli were added to the reaction mixture, the rate of PQQ production increased dependent on the amount of NADPH added and the total amount of PQQ produced increased.     

Characterization of two methanopterin biosynthesis mutants of Methylobacterium extorquens AM1 by use of a tetrahydromethanopterin bioassay

An enzymatic assay was developed to measure tetrahydromethanopterin (H(4)MPT) levels in wild-type and mutant cells of Methylobacterium extorquens AM1. H(4)MPT was detectable in wild-type cells but not in strains with a mutation of either the orf4 or the dmrA gene, suggesting a role for these two genes in H(4)MPT biosynthesis. The protein encoded by orf4 catalyzed the reaction of ribofuranosylaminobenzene 5'-phosphate synthase, the first committed step of H(4)MPT biosynthesis. These results provide the first biochemical evidence for H(4)MPT biosynthesis genes in bacteria.     

Nucleotide sequence of the Methylobacterium extorquens AM1 moxF and moxJ genes involved in methanol oxidation

The nucleotide sequence has been determined for two genes involved in methanol oxidation in the facultative methylotroph, Methylobacterium extorquens AM1. The two genes are moxF, encoding the 66-kDa subunit of the methanol dehydrogenase and moxJ, located immediately downstream from moxF, which encodes a 30-kDa protein with unknown function. This information completes the sequence of the 5.86-kb XhoI-SalI fragment containing the moxFJGI region in M. extorquens AM1, and the structure of this gene cluster is presented. Evidence is presented that moxJ is also present in Paracoccus denitrificans. The aa sequence of MoxJ has provided little information concerning its function, but it does appear to contain a signal sequence suggesting a periplasmic location.     

Novel methylotrophy genes of Methylobacterium extorquens AM1 identified by using transposon mutagenesis including a putative dihydromethanopterin reductase

Ten novel methylotrophy genes of the facultative methylotroph Methylobacterium extorquens AM1 were identified from a transposon mutagenesis screen. One of these genes encodes a product having identity with dihydrofolate reductase (DHFR). This mutant has a C(1)-defective and methanol-sensitive phenotype that has previously only been observed for strains defective in tetrahydromethanopterin (H(4)MPT)-dependent formaldehyde oxidation. These results suggest that this gene, dmrA, may encode dihydromethanopterin reductase, an activity analogous to that of DHFR that is required for the final step of H(4)MPT biosynthesis.     

Cloning, mutagenesis, and physiological effect of a hydroxypyruvate reductase gene from Methylobacterium extorquens AM1.

The gene encoding the serine cycle hydroxypyruvate reductase of Methylobacterium extorquens AM1 was isolated by using a synthetic oligonucleotide with a sequence based on a known N-terminal amino acid sequence. The cloned gene was inactivated by insertion of a kanamycin resistance gene, and recombination of this insertion derivative with the wild-type gene produced a serine cycle hydroxypyruvate reductase null mutant. This mutant had lost its ability to grow on C-1 compounds but retained the ability to grow on C-2 compounds, showing that the hydroxypyruvate reductase operating in the serine cycle is not involved in the conversion of acetyl coenzyme A to glycine as previously proposed. A second hydroxypyruvate-reducing enzyme with a low level of activity was found in M. extorquens AM1; this enzyme was able to interconvert glyoxylate and glycollate. The gene encoding hydroxypyruvate reductase was shown to be located about 3 kb upstream of two other serine cycles genes encoding phosphoenolpyruvate carboxylase and malyl coenzyme A lyase.