xyLE基因在嗜热脂肪芽孢杆菌中的表达

以嗜热脂肪芽孢杆菌CU21的表达型质粒pFDC11为载体,插入来自恶臭假单孢菌的编码邻苯二酚2,3-双加氧酶(CatO_2ase)的xylE基因,构建成重组质粒pFDX1转化入CU21受体,在48℃培养时得到了表达产物,表明常温细菌的xylE基因可以在高温菌启动子的带动下在高温菌宿主中表达。采用提高培养温度后选择卡那毒素抗性突变的方法,得到了在55℃及6O℃的CatO_2ase表达量明显提高的变异菌株CU21-161。本文同时报道一种用完整细胞悬浮液测定CatO_2ase活力的方法。     

Asia 1型口蹄疫病毒抗原表位突变株的构建及其生物学特性分析

为了研制口蹄疫抗原表位突变标记疫苗,本研究以含有Asia 1型口蹄疫病毒(FMDV)c DNA全长的感染性克隆p Asia 1-FMDV作为骨架,将3D蛋白中第27位氨基酸的H和31位的氨基酸N分别突变成Y和R,从而突变3D蛋白的一个抗原表位,将构建的带有突变表位的重组质粒转染BHK-21细胞,成功拯救出一株突变FMDV。经比较后发现,重组病毒的生物学特性与亲本毒株相似。病毒中和试验结果显示,抗重组病毒的血清与亲本病毒有良好的反应性。Western blotting结果表明重组病毒诱导的抗体能与突变的表位合成肽反应而不与野生型病毒的表位合成肽发生反应,从而区分重组病毒与亲本病毒。综上所述,这株抗原表位突变FMDV有望作为口蹄疫标记疫苗候株进一步评估。     

ERCC1对H520细胞Mdr1及SP细胞的影响分析

目的:分析ERCC1对H520细胞中Mdr1及SP细胞比例的影响。方法:利用RNA干扰技术构建ERCC1-shRNA重组质粒表达载体,转染并筛选出阳性稳定转染的H520细胞;采用RT-PCR和WesternBlot的方法检测ERCC1对H520细胞多药耐药1(Mdr1)的影响;采用流式细胞仪检测ERCC1对H520细胞SP细胞比例的影响。结果:RT-PCR检测出Mdr1 mRNA在含有ERCC1-shRNA的H520细胞和含有空载体的细胞中和内参的A比值分别为0.423±0.147和0.468±0.02(n=5),两者有显著性差异;Western-Blot检测出Mdr1蛋白浓度在含有ERCC1-shRNA的H520细胞较含有空载体的细胞中明显降低。含有ERCC1-shRNA的H520细胞SP细胞比例达到0.8%,而含有空载体的H520细胞的SP细胞比例则达到2.2%。结论:RNA干扰技术可以构建重组质粒,稳定转染的H520细胞能被筛选出,当ERCC1基因被抑制时,Mdr1基因也会收到抑制,同时使H520细胞SP细胞比例明显降低。     

电转染哺乳类动物细胞DG44-CHO的方法探讨

目的：建立并验证DG44-CHO细胞快速、无血清培养体系的转染方法。方法：将绿色荧光蛋白表达质粒pEGFP-N1电转入DG44-CHO细胞,用流式细胞仪检测绿色荧光蛋白的表达量;将不同重组蛋白表达质粒C1—28／GL1／pCMV163、C1-28／GL2／pCMV163和TmHL／pCMV163分别电转入DGd4-CHO细胞,ELISA检测其培养上清中相应重组蛋白的表达。结果：280V电压电击20ms、质粒用量为20pg时,转染细胞中绿色荧光蛋白的表达量最高;同样在该条件下,培养上清中重组蛋白浓度最高。结论：上述电转染条件具有一定的适用性。     

酿酒酵母(Saccharomyces cerevisiae)半乳糖可诱导表达系统特性的研究

把大肠杆菌β-半乳糖苷酶基因克隆到带有酵母半乳糖可诱导启动子GAL1的穿梭表达质粒pYESZ中,并把得到的重组质粒分别转化到两种不同遗传性状的宿主菌中,其中一株菌为蛋白酶活性缺失90％以上的pep4－3突变菌株。通过比较两株重组菌产生的β-半乳糖苷酶活性水平发现在所述实验条件下,蛋白酶缺失突变菌株中产生的β-卜半乳糖苷酶活水平不仅均要高于另一对照菌株,并且pep4-3突变菌株表现出受葡萄糖阻遏的严紧程度高及对诱导反应迅速等特点。此外,带有重组质粒的pep4－3突变菌株在葡萄糖阻遏培养基中最大生长量和重组对照菌株基本相同,但β-半乳糖苷酶在pep4－3突变菌株中的表达对细胞生长的影响明显小于对照菌株。     

同源重组法制备口蹄疫病毒多基因重组腺病毒

通过细菌内同源重组的方法成功构建了含有O型口蹄疫病毒P1-2A和3C蛋白酶基因和3D基因的重组腺病毒表达载体.首先将P1-2A、3C和3D基因亚克隆连接到穿梭质粒pShuttle-CMV上,再将重组穿梭质粒用PmeI线性化后电转化携带有腺病毒骨架载体pAdeasy-1的大肠杆菌BJ5183感受态菌,经细菌内同源重组产生pAdcmv-p12x3c和pAdcmv-p12x3cd重组腺病毒质粒,经序列测定证实目的基因已正确的插入到腺病毒骨架载体中.重组腺病毒质粒经PacI线性化后转染HEK293细胞,转染1w内细胞出现典型病变.取转染细胞裂解液上清连续传代至第4代时,细胞于24~48h内即病变完全,收取接毒后24h细胞进行PCR和RT-PCR检测,表明目的基因已整合到腺病毒基因组内,且在mRNA水平上有表达.取第4、6、8和10代病毒,用蛋白酶K处理后可扩增出目的基因,证明此重组病毒可稳定存在.本研究为FMDV腺病毒活载体疫苗的研究奠定了基础.     

人胰岛素原基因B10位定点突变对增强胰外表达效率的实验研究

目的构建在肝细胞内增强表达成熟人胰岛素的生理调控性人胰岛素原基因重组质粒。方法利用PCR定点突变技术在B-C及C-A连接处已两点突变的人胰岛素原基因上进行B10位突变,并在其上游连接肝细胞特异的3倍体葡萄糖反映元件(GLRE3)和胰岛素样生长因子结合蛋白-1启动子(IGFBP-1P)。经酶切后将含有调控元件的3点突变人胰岛素原基因(GLRE3-IGFBP-1P-3mINS)插入逆转录病毒载体(pLXSN),脂质体介导转染大鼠肝癌细胞CBRH7919后检测成熟胰岛素表达情况及与含有调控元件的2点突变人胰岛素原基因(GLRE3-IGFBP-1P-3mINS)表达体的表达差异。结果人胰岛素原基因的B10位组氨酸编码序列CAC突变为门冬氨酸编码序列GAC。构建了含调控元件的B10位门冬氨酸人胰岛素原基因逆转录病毒载体质粒(pLXSN-GLRE3-IGFBP-1P-3mINS),酶切、PCR及测序鉴定各段基因碱基序列及连接方向正确。pLXSN-GLRE3-IGFBP-1P-3mINS经脂质体包裹转染大鼠肝癌细胞,细胞外液含5.0、25.0mmol/L的葡萄糖浓度下胰岛素含量分别为5.03±0.72、43.90±2.30mU/L。未进行B10位突变的重组体转染组在同样葡萄糖浓度下胰岛素含量分别为<2.00、2.10±0.23mU/L。结论成功构建了含调控元件的B10位门冬氨酸人胰岛素原基因逆转录病毒载体重组质粒,该重组体已整合入鼠肝癌细胞基因组,其表达效率显著提高并受葡萄糖生理性调控。     

异形胞分化蓝细菌dnaA温度敏感型突变体的构建

以能分化异形胞的蓝细菌(Anabaenasp.PCC7120)为材料,采用重组PCR在体外对控制DNA复制起始的dnaA基因进行定点突变后克隆到整合质粒中,再通过三亲本杂交将整合质粒转移到Anabaena PCC7120中,以分离和筛选温度敏感型突变体。结果成功获得Anabaena PCC 7120 dnaA高温敏感性突变体。研究表明,利用重组PCR技术可在体外实现对Anabaena PCC 7120的dnaA的定点突变,并可通过同源重组双交换成功实行整合质粒中突变基因对野生型基因的置换,使突变基因插入到细胞染色体中,进而成功构建温度敏感型突变菌株。     

H5N1禽流感病毒HA和NA基因重组腺病毒共表达载体的构建

利用口蹄疫病毒(FMDV)2A蛋白具有自我裂解的功能,将其作为连接肽构建携带有H5N1亚型AIVHA和NA基因的重组腺病毒表达载体,进而为AIV基因工程疫苗的开发以及相关诊断试剂的开发提供依据。采用融合PCR的方法扩增出含有H5N1 AIV HA-2A-NA的基因,定向插入pAdtrack-CMV腺病毒穿梭质粒中,含有目的基因的腺病毒穿梭质粒pAdtrack-HA-2A-NA与腺病毒骨架质粒pAdeasy-1在基因工程菌BJ5183中进行同源重组,获得腺病毒质粒pAdeasy-HA-2A-NA,将pAdeasyd-H5经PacI线性化后转染HEK293细胞株包装出含有HA-2A-NA基因的腺病毒pAd-HA-2A-NA。结果表明,构建的含有目的基因的腺病毒穿梭质粒pAdtrack-HA-2A-NA和含有目的基因的腺病毒质粒pAdeasy-HA-2A-NA经PCR、双酶切及核苷酸测序测定无误。线性化后的pAdeasy-HA-2A-NA转染HEK293细胞包装成功获得腺病毒pAd-HA-2A-NA载体,经绿色荧光蛋白和RT-PCR分析证实,目的基因在该细胞中成功表达。本试验构建的含有AIV H5N1亚型HA-2A-NA基因的重组腺病毒表达载体,将为进一步研究开发基因工程疫苗提供病毒模型。     

大豆胚胎致死黄化突变沈农2015(y_(21))的遗传研究

在以地理远缘材料为亲本组配的一个单交后代中发现一个胚胎致死黄化突变。通过F_2至F_4表现型可知,这种胚胎致死黄化突变是受一对隐性基因控制的。由于它与迄今为止发现的延迟性黄化突变有所不同,故应命名为y_(21)。y_2~1和ms_1及其相对应的Y_(21)和Ms_1为两对独立遗传的基因。这一单交后代群体(沈农2015)含有)y_(21)和ms_1两种隐性基因。对它作进一步的研究,将有助于解决生产杂交大豆的一些实际问题。     

Appearance in Japan of highly macrolide-resistant Escherichia coli producing macrolide 2'-phosphotransferase II.

Escherichia coli CU1, a clinical isolate recovered in Japan in 1997, was found to be highly-resistant to both 14-membered and 16-membered ring macrolide antibiotics. A crude extract prepared from strain CU1 inactivated 14-, 15- and 16-membered ring macrolides in the presence of ATP and the Rf value of inactivated oleandomycin was identical to that of oleandomycin 2'-phosphate. This suggested that strain CU1 produced the enzyme macrolide 2'-phosphotransferase [MPH(2')]. Substrate specificity of the crude enzyme from strain CU1 against 14-, 15- and 16-membered ring macrolides was basically similar to that of MPH(2')II from strain BM2506, differing in that the former more effectively inactivated roxithromycin and tylosin. Subsequent attempts were made to clone the novel mph gene encoding for MPH(2') in strain CU1. The mph gene carried by strain CU1 was located on nontransmissible plasmid DNA, designated pCU001. Its molecular weight, estimated by agarose electrophoresis, was approximately 57 kD. The DNA sequence of the cloned mph gene from the Japanese isolate CU1 was identical to that of mphB, which until now had only been recovered in France. The variance in the substrate specificity of MPH(2')II from each strain led us to speculate that other factors in the reaction affect the enzymatic inactivation activity.     

嗜热脂肪芽孢杆菌启动子克隆载体pFDC4和表达型载体pFDC11的构建

以pPL703的衍生质粒pPGV5为载体,从嗜热脂肪芽孢杆菌CU21总DNA的Sau 3A酶切产物中得到1个0.54kb的启动子片段,它能促进载体上的无启动子的cat-86基因在嗜热脂肪芽孢杆菌及枯草芽孢杆菌中表达。这一片段以正、反向插入pPGV5载体,都能使重组质粒转化CU21原生质体的效率提高10~(?)至10(?)倍。Southern杂交实验表明,这一启动子片段与Imanaka等报道的来自CU21中的隐蔽性质粒pBS02的能提高转化效率的1.6kb Eco RI片段是同源的。利用所得到的0.54kb Sau 3A片段构建了新的启动子克隆载体pFDC4和表达型载体pFDC11,二者都能以很高的效率转化CU21原生质体。     

Phenotypic expression of PCR-generated random mutations in a Pseudomonas putida gene after its introduction into an Acinetobacter chromosome by natural transformation

Localized sets of random point mutations generated by PCR amplification can be transferred efficiently to the chromosome of Acinetobacter ADP1 (also known as strain BD413) by natural transformation. The technique does not require cloning of PCR fragments in plasmids: PCR-amplified DNA fragments are internalized by cells and directly incorporated into their genomes by homologous recombination. Previously such procedures for random mutagenesis could be applied only to Acinetobacter genes affording the selection of mutant phenotypes. Here we describe the construction of a vector and recipient that allow for mutagenesis, recovery, and expression of heterologous genes that may lack a positive selection. The plasmid carries an Acinetobacter chromosomal segment interrupted by a multiple cloning site next to a kanamycin resistance marker. The insertion of heterologous DNA into the multiple cloning site prepares the insert as a target for PCR mutagenesis. PCR amplifies the kanamycin resistance marker and a flanking region of Acinetobacter DNA along with the insert of heterologous DNA. Nucleotide sequence identity between the flanking regions and corresponding chromosomal segments in an engineered Acinetobacter recipient allows homologous recombination of the PCR-amplified DNA fragments into a specific chromosomal docking site from which they can be expressed. The recipient strain contains only a portion of the kanamycin resistance gene, so donor DNA containing both this gene and the mutagenized insert can be selected by demanding growth of recombinants in the presence of kanamycin. The effectiveness of the technique was demonstrated with the relatively GC-rich Pseudomonas putida xylE gene. After only one round of PCR amplification (35 cycles), donor DNA produced transformants of which up to 30% carried a defective xylE gene after growth at 37 degrees C. Of recombinant clones that failed to express xylE at 37 degrees C, about 10% expressed the gene when grown at 22 degrees C. The techniques described here could be adapted to prepare colonies with an altered function in any gene for which either a selection or a suitable phenotypic screen exists.     

Excision process of integrated plasmid pBD12 from the Bacillus subtilis chromosome

We studied the behavior of pBD12 plasmid integrated into Bacillus subtilis chromosome via homologous recombination. One copy of the plasmid was integrated into the chromosome, it conferred resistance to low concentrations of antibiotics. Clones with enhanced resistance bearing autonomous plasmid DNAs appeared with a frequency 10(-6) in rec+ but not in recE strain with the integrated plasmid. By restriction and hybridization analysis of some excised plasmids, the sites of excision were determined, chromosomal location of pBD12 plasmid was found to be at the terminal fragment of prophage DNA, so that the att site of phi 105 phage is supposed to be situated on the EcoRI fragment of phage DNA.     

Survival of nonculturable Aeromonas salmonicida in lake water.

The survival of Aeromonas salmonicida subsp. salmonicida was investigated in sterile and untreated lake water. In sterile lake water (filtered and autoclaved), it was found that cells of A. salmonicida entered a nonculturable but viable condition. Viability was determined by flow cytometry with the dye rhodamine 123, which is taken up and maintained within cells with a membrane potential. For survival studies in untreated lake water, A. salmonicida was marked with the xylE gene by using the plasmid pLV1013. Marked cells were detected by growth on tryptone soy agar and tryptone soy agar supplemented with kanamycin. Cells were also detected by polymerase chain reaction DNA amplification of the xylE gene and a chromosomal DNA fragment specific for A. salmonicida (pLV1013). The results indicated that A. salmonicida entered a nonculturable condition in untreated lake water over a 21-day study. The viability of nonculturable cells could not be determined in mixed samples; however, the presence of nonculturable cells containing both chromosomal and plasmid DNA was confirmed.     

Complete nucleotide sequence of the metapyrocatechase gene on the TOI plasmid of Pseudomonas putida mt-2

Metapyrocatechase which catalyzes the oxygenative ring cleavage of catechol to form alpha-hydroxymuconic epsilon-semialdehyde is encoded by the xylE gene on the TOL plasmid of Pseudomonas putida mt-2. We have cloned the xylE region in Escherichia coli and determined the nucleotide sequence of the DNA fragment of 985 base pairs around the gene. The fragment included only one open translational frame of sufficient length to accommodate the enzyme. The predicted amino acid sequence consisted of 307 residues, and its NH2- and COOH-terminal sequences were in perfect agreement with those of the enzyme recently determined (Nakai, C., Hori, K., Kagamiyama, H., Nakazawa, T., and Nozaki, M. (1983) J. Biol. Chem. 258, 2916-2922). A mutant plasmid was isolated which did not direct the synthesis of the active enzyme. This plasmid had a DNA region corresponding to the NH2-terminal two-thirds of the polypeptide. From the deduced amino acid sequence, the secondary structure was predicted. Around 10 base pairs upstream from the initiator codon for metapyrocatechase, there was a base sequence which was complementary to the 3'-end of 16 S rRNAs from both E.coli and Pseudomonas aeruginosa. A preferential usage of C- and G-terminated codons was found in the coding region xylE, which contributed to the relatively high G + C content (57%) of this gene.     

Isolation of an autonomously replicating DNA fragment from the region of defective bacteriophage PBSX of Bacillus subtilis

We have isolated a 5.4-kilobase fragment of Bacillus subtilis DNA that confers the ability to replicate upon a nonreplicative plasmid. The B. subtilis 168 EcoRI fragment was ligated into the chimeric plasmid pCs540, which contains a chloramphenicol resistance determinant from the Staphylococcus aureus plasmid pC194 and an HpaII fragment from the Escherichia coli plasmid, pSC101. A recE B. subtilis derivative, strain BD224, is capable of maintaining this DNA as an autonomously replicating plasmid. In rec+ recipients, chloramphenicol-resistant transformants do not contain free plasmid. The plasmid is integrated as demonstrated by alterations in the pattern of chromosomal restriction enzyme fragments to which the plasmid hybridizes. The site of plasmid integration was mapped by PBS1-mediated transduction to the metC-PBSX region. A strain was a deletion in the region of defective bacteriophage PBSX differs in the hybridization profile obtained by probing EcoRI digests with this cloned fragment. This same deletion mutant, though proficient in normal recombinational pathways, permits autonomous replication of the plasmid apparently owing to the lack of an homologous chromosomal region with which to recombine. We believe that, like E. coli. B. subtilis contains at least one DNA fragment capable of autonomous replication when liberated from its normally integrated chromosomal site and that this cloned DNA fragment comes from the region of defective bacteriophage PBSX.     

Streptomyces marker plasmids for monitoring survival and spread of streptomycetes in soil.

Plasmid constructs pNW1 through pNW6 containing a controllable xylE gene (for catechol 2,3-dioxygenase) were introduced into Streptomyces lividans strains to provide a selectable marker system. xylE functions in S. lividans under the control of bacteriophage lambda promoters lambda pL and lambda pR. Thermoregulated expression of xylE is provided through the lambda repressor cI857. Catechol 2,3-dioxygenase activity was increased 2.8-fold from plasmid construct pNW2 (lambda pL, xylE, cI857) and 9.5- and 7.4-fold from constructs pNW3 (lambda pR, xylE, cI857) and pNW5 (lambda pR, xylE, cI857), respectively, when the temperature was shifted from 28 degrees C to 37 degrees C. The stability of the constructs varied from 4.7% for pNW2 to 99.4% for pNW4 (lambda pL, xylE) over two rounds of sporulation. Marked S. lividans strains released into soil systems retained the XylE phenotype for more than 80 days, depending on the marker plasmid, when examined by a selective plating method. Furthermore, S. lividans harboring plasmid pNW5 was detectable by nucleic acid hybridization at less than 10 CFU g-1 (dry weight) of soil as mycelium and 10(3) CFU g-1 (dry weight) of soil as spores with the xylE marker DNA extracted from soil and amplified by using the polymerase chain reaction.     

Streptomyces marker plasmids for monitoring survival and spread of streptomycetes in soil.

Plasmid constructs pNW1 through pNW6 containing a controllable xylE gene (for catechol 2,3-dioxygenase) were introduced into Streptomyces lividans strains to provide a selectable marker system. xylE functions in S. lividans under the control of bacteriophage lambda promoters lambda pL and lambda pR. Thermoregulated expression of xylE is provided through the lambda repressor cI857. Catechol 2,3-dioxygenase activity was increased 2.8-fold from plasmid construct pNW2 (lambda pL, xylE, cI857) and 9.5- and 7.4-fold from constructs pNW3 (lambda pR, xylE, cI857) and pNW5 (lambda pR, xylE, cI857), respectively, when the temperature was shifted from 28 degrees C to 37 degrees C. The stability of the constructs varied from 4.7% for pNW2 to 99.4% for pNW4 (lambda pL, xylE) over two rounds of sporulation. Marked S. lividans strains released into soil systems retained the XylE phenotype for more than 80 days, depending on the marker plasmid, when examined by a selective plating method. Furthermore, S. lividans harboring plasmid pNW5 was detectable by nucleic acid hybridization at less than 10 CFU g-1 (dry weight) of soil as mycelium and 10(3) CFU g-1 (dry weight) of soil as spores with the xylE marker DNA extracted from soil and amplified by using the polymerase chain reaction.