Cloning of DNA fragments of the genetic transfer factor pAP39

Large HindIII digested fragments of the plasmid pAP39 have been cloned on the cosmid vector pHC79. The study of the structure of HindIII fragments of plasmid pAP39 in the recombinant plasmids has shown that these fragments are represented by f1 + f2 fragments from the plasmid pAP1055, by f1 + f6 fragments from the plasmid pAP1056, by f2 + f3 fragments from the plasmid pAP1057 and by two f3 fragment from the plasmid pAP1058. Physical maps of the recombinant plasmids have been constructed. The plasmid pAP39 is shown to contain two functionally active tra regions.     

Replication of a plasmid containing two origins of bacteriophage f1

A chimeric plasmid was constructed that contains a tandem duplication of the bacteriophage f1 origin of DNA replication. This plasmid replicates stably in the absence of phage. When cells carrying this plasmid are infected with f1, two new plasmid-derived DNA species are generated: a smaller, chimeric plasmid containing only one f1 origin of replication, and a miniphage, the genome of which consists of the f1 fragment that was located between the two f1 origins of the original plasmid. These data support the hypothesis (Horiuchi, 1980) that the nucleotide sequence recognized for initiation of plus strand synthesis in f1 DNA replication is also the signal for its termination.     

Genetic organization of transfer region of F-like plasmid pAP18-1

The results of complementation analysis of nitrosoguanidine-induced mutants of F-like drd-plasmid pAP18-1 (Tc, ColV) testified to the existence of at least 3 tra regions (tra1, tra2, tra3) and regulating locus fin V in the genome of this plasmid. By means of molecular cloning of tra2 region and locus fin V of plasmid pAP18-1drd were located in Sall-fragment f5 (3.9 MD).     

Second EcoRI fragment of F capable of self-replication.

The cloning of fragments of F' plasmid deoxyribonucleic acid produced by restriction endonuclease EcoRI has revealed that fragment f7, not previously suspected to have replicative properties, is able to replicate autonomously. The ability of f7 to replicate was observed when it was cloned with fragments coding for resistance to either kanamycin or streptomycin and sulfonamide. Such f7 miniplasmids have been obtained from an F'lac+ and two F'gal+ temperature-sensitive mutant plasmids and from the unmutated F plasmid. Plasmids containing both f5 and f7 fragments were also obtained. Expression of resistance to "female-specific" bacteriophages requires that f5 and f7 be present in the same plasmid since cells containing separate f5 and f7 plasmids are not resistant to bacteriophage phi II. f7 plasmids were less stable than miniplasmids containing f5, particularly at fast growth rates. The bearing of these results on the isolation and behavior of temperature-sensitive F mutants is discussed.     

The genome localization of the plasmid pAP27 fin N system]

Plasmid transfer genetic regulatory system fin N of F-like plasmid pAP27 have been localized on BamHI-restriction fragment f3 (length 8.7 kb). Plasmid pUC19 Fin-activity have been cleared up and characterized from the point of view of its specificity of action on some plasmid transfer functions.     

上调hMLH1基因表达对卵巢癌药物敏感性的影响

构建携带错配修复基因hMLH1编码序列全长的真核表达质粒pCAN—hMLHl,并探讨其对卵巢癌细胞顺铂耐药的逆转作用。应用基因重组技术将pET28-hMLHl中的目的基因hMLHl定向克隆到真核表达载体pCAN,经酶切及测序鉴定：分别将pCAN—hMLHl和空质粒pCAN转染进卵巢癌耐药细胞SKOV3／DDP,同时以对顺铂敏感的sKOV3细胞和未转染的SKOV3／DDP细胞作为对照：应用RT-PCR和Westemblo凇测转染前后细胞内hMLHlmRNA和蛋白的表达变4Jc;四甲基偶氮唑蓝（MTT）比色法检测转染前后sKOv3／DDP细胞对顺铂敏感性的变化;Hoechst染色检测转染前后细胞的凋亡。结果提示：pCAN—hMLHl重组质粒经酶切及测序鉴定,表明真核表达质粒构建正确;采用脂质体法转染sKOv3／DDP细胞后,RT-PCR和Westernblot检测到耐药细胞内hMLHl的表达增强：MTT结果显示转染重组质粒后sKOv3／DDP细胞对顺铂的敏感性显著增加;Hoechst染色观察到转染后耐药细胞的凋亡明显增强。该研究成功构建了pCAN．hMLHl重组质粒,在sKOV3／DDP细胞中进行表达,并能增强耐药细胞对顺铂的敏感性,促进耐药细胞的凋亡。     

Recombinant plasmid associated cell aggregation and high-frequency conjugation of Streptococcus lactis ML3

Lactose-positive (Lac+) transconjugants resulting from matings between Streptococcus lactic ML3 and S. lactis LM2301 possess a single plasmid of approximately 60 megadaltons (Mdal) which is nearly twice the size of the lactose plasmid of the donor. The majority of these Lac+ transconjugants aggregated in broth and were able to transfer lactose-fermenting ability at a frequency higher than 10(-1) per donor on milk agar plates or in broth. Lac+ transconjugants which did not clump conjugated at a much lower frequency. Lactose-negative derivatives of Lac+ clumping transconjugants did not aggregate in broth and were missing the 60-Mdal plasmid. The ability to aggregates in broth was very unstable. Strains could lose the ability to clump but retain lactose-fermenting ability. The majority of these Lac+ nonclumping derivatives of clumping transconjugants contained a plasmid of approximately 33 Mdal, the size of the lactose plasmid of the original donor ML3. These strains transferred lactose-fermenting ability at a frequency of approximately 10(-6) per donor, resulting in both Lac+ clumping transconjugants which contained a 60-Mdal plasmid and Lac+ nonclumping transconjugants which possessed a 33-Mdal plasmid. Our results suggest that the genes responsible for cell aggregation and high-frequency conjugation are on the segment of deoxyribonucleic acid which recombined with the 33-Mdal lactose plasmid in S. lactis ML3.     

Block Copolymer/DNA Vaccination Induces a Strong Allergen-Specific Local Response in a Mouse Model of House Dust Mite Asthma

Background

Allergic asthma is caused by abnormal immunoreactivity against allergens such as house dust mites among which Dermatophagoides farinae (Der f) is a common species. Currently, immunotherapy is based on allergen administration, which has variable effect from patient to patient and may cause serious side effects, principally the sustained risk of anaphylaxis. DNA vaccination is a promising approach by triggering a specific immune response with reduced allergenicity.

Objective

The aim of the study is to evaluate the effects of DNA immunization with Der f1 allergen specific DNA on allergic sensitization, inflammation and respiratory function in mice.

Methods

Mice were vaccinated 28 and 7 days before allergen exposure with a Der f1-encoding plasmid formulated with a block copolymer. Asthma was induced by skin sensitization followed by intra-nasal challenges with Der f extract. Total lung, broncho-alveolar lavage (BAL) and spleen cells were analyzed by flow cytometry for their surface antigen and cytokine expression. Splenocytes and lung cell IFN-γ production by CD8+ cells in response to Der f CMH1-restricted peptides was assessed by ELISPOT. IgE, IgG1 and IgG2a were measured in serum by ELISA. Specific bronchial hyperresponsiveness was assessed by direct resistance measurements.

Results

Compared to animals vaccinated with an irrelevant plasmid, pVAX-Der f1 vaccination induced an increase of B cells in BAL, and an elevation of IL-10 and IFN-γ but also of IL-4, IL-13 and IL-17 producing CD4+ lymphocytes in lungs and of IL-4 and IL-5 in spleen. In response to CD8-restricted peptides an increase of IFN-γ was observed among lung cells. IgG2a levels non-specifically increased following block copolymer/DNA vaccination although IgE, IgG1 levels and airways resistances were not impacted.

Conclusions & Clinical Relevance

DNA vaccination using a plasmid coding for Der f1 formulated with the block copolymer 704 induces a specific immune response in the model of asthma used herein.     

人源抗狂犬病毒单链抗体基因原核表达质粒的构建及高效表达

在原核系统中高效表达抗狂犬病毒单链抗体scFv41,以便进一步研究其生物学功能,预测临床应用前景。以重组质粒pCANTABscFv41为模板,PCR扩增带NcoI和NotI位点的scFv41基因,克隆入原核表达载体pET-22b( ),酶切鉴定重组表达质粒,转化大肠杆菌BL21(DE3),IPTG诱导表达。竞争ELISA检测表达蛋白的特异结合活性。酶切鉴定证实scFv41基因已插入原核表达载体pETl-22b( ),重组表达质粒pET-scFv41在大肠杆菌BL21(DE3)中获得了高效表达,表达量约占菌体蛋白总量的30％。竞争ELISA检测结果表明scFv41表达蛋白可特异抑制抗狂犬病毒IGY与狂犬病毒的特异性结合。该实验为进一步研究scFv41的生物学特性和免疫保护作用,及基因工程抗体的制备奠定了基础。     

Development of high-level streptomycin resistance affected by a plasmid in lactic streptococci

Some lactose-negative (Lac-) mutants of Streptococcus lactis C2 and ML3 exhibited development of very high level streptomycin resistance after incubation with subinhibitory concentrations of the drug for 18 to 22 h. These drug-resistant mutants showed no loss of resistance even after 6 months of subculturing in broth without any drug. The parental Lac+ strains did not show mutation to high-level streptomycin resistance. The Lac+ characteristic of the parental strain was conjugally transferred to Lac- derivatives of C2 and ML3, showing the ability to mutate to high-level resistance. When transconjugants were analyzed for this characteristic, they showed both mutable and nonmutable Lac+ types. The results suggested that genetic information for mutation to high-level streptomycin resistance in lactic streptococci resides on the chromosome, and its expression is affected by a plasmid. The plasmid profiles of strains C2, ML3, C2 Lac-, ML3 Lac-, and two kinds of transconjugants confirmed the presence of a plasmid of approximately 5.5 megadaltons in strains showing no mutation to high-level streptomycin resistance, while strains missing such a plasmid exhibited high-level streptomycin resistance after incubation with subinhibitory concentrations of the drug.     

Development of high-level streptomycin resistance affected by a plasmid in lactic streptococci.

Some lactose-negative (Lac-) mutants of Streptococcus lactis C2 and ML3 exhibited development of very high level streptomycin resistance after incubation with subinhibitory concentrations of the drug for 18 to 22 h. These drug-resistant mutants showed no loss of resistance even after 6 months of subculturing in broth without any drug. The parental Lac+ strains did not show mutation to high-level streptomycin resistance. The Lac+ characteristic of the parental strain was conjugally transferred to Lac- derivatives of C2 and ML3, showing the ability to mutate to high-level resistance. When transconjugants were analyzed for this characteristic, they showed both mutable and nonmutable Lac+ types. The results suggested that genetic information for mutation to high-level streptomycin resistance in lactic streptococci resides on the chromosome, and its expression is affected by a plasmid. The plasmid profiles of strains C2, ML3, C2 Lac-, ML3 Lac-, and two kinds of transconjugants confirmed the presence of a plasmid of approximately 5.5 megadaltons in strains showing no mutation to high-level streptomycin resistance, while strains missing such a plasmid exhibited high-level streptomycin resistance after incubation with subinhibitory concentrations of the drug.     

紫色非硫细菌质粒的制备与性质

介绍一种适于紫色非硫细菌质粒的制备方法,该法简单,易操作,质粒ＤＮＡ纯度好,收率高,通过在加富培养基上连续传代数次,质粒ＤＮＡ可自行消除。     

Conjugal transfer but not quorum-dependent tra gene induction of pTiC58 requires a solid surface.

Donors of Agrobacterium tumefaciens harboring a transfer-constitutive derivative of the nopaline-type Ti plasmid pTiC58 transferred this element at frequencies 3 to 4 orders of magnitude higher in matings conducted on solid surfaces than in those conducted in liquid medium. However, as measured with a lacZ reporter fusion, the tra genes of the wild-type Ti plasmid were inducible by opines to indistinguishable levels on solid and in liquid medium. Donors induced in liquid transferred the Ti plasmid at high frequency when mated with recipients on solid medium. We conclude that while formation of stable mating pairs and subsequent transfer of the Ti plasmid is dependent on a solid stratum, the regulatory system can activate tra gene expression to equivalent levels in liquid and on solid surfaces.     

Plasmids containing many tandem copies of a synthetic lactose operator

Up to 12 tandem copies of the lactose operator sequence AATTCCACATGTGGAATTGTGAGCGGATAACAATTTGTGG (3') GGTGTACACCTTAACACTCGCCTATTGTTAAACACCTTAA (5') have been cloned in the EcoRI site of plasmid pMB9. A 12-operator plasmid is about 8% operator by weight and represents a rich source of this DNA segment. A procedure for the rapid and convenient isolation of operator in mg quantities is presented. The lifetimes of complexes formed between repressor and oligo-operator plasmids increased with increasing numbers of tandem operators per plasmid. Evidence is presented indicating that only one tetrameric repressor molecule binds strongly to a segment of four (or fewer) tandem operators, but that two repressor molecules can be accommodated on segments containing at least six tandem operators.     

Construction of recombinant plasmid carrying the lambda DNA fragment responsible for prophage integration.

The recombinant DNA molecules were constructed from plasmid RSF2124 and the EcoRI fragment of lambda DNA containing the genes responsible for prophage integration. The presence of these genes in recombinant plasmids was detected genetically. lambda int-gene was shown to be expressed in either orientation of insertion in the plasmid. We found that recombinant plasmid was able to integrate into chromosome of lambda lysogens. The integration of plasmid into host chromosome was demonstrated by contransduction of chromosome and plasmid markers using generalized transducer P1 and by specialized transduction with lambda phages.     

Presence of a family of plasmids (29 to 65 kilobases) with a 26-kilobase common region in different strains of the sulfur-oxidizing bacterium Acidithiobacillus caldus

Three large cryptic plasmids from different isolates of Acidithiobacillus caldus were rescued by using an in vitro transposition system that delivers a kanamycin-selectable marker and an Escherichia coli plasmid origin of replication. The largest of the plasmids, the 65-kb plasmid pTcM1, was isolated from a South African A. caldus strain, MNG. This plasmid was sequenced and compared to that of pTcF1 (39 kb, from strain "f," South Africa) and pC-SH12 (29 kb, from strain C-SH12, Australia). With the exception of a 2.7-kb insertion sequence, pC-SH12 appears to represent the DNA common to all three plasmids and includes a number of accessory genes plus the plasmid "backbone" containing the replication region. The two larger plasmids carry, in addition, a number of insertion sequences of the ISL3 family and a composite transposon related to the Tn21 subfamily containing a highly mosaic region within the borders of the inverted repeats. Genes coding for arsenic resistance, plasmid mobilization, plasmid stability, and a putative restriction-modification system occur within these mosaic regions.     

Restriction endonuclease mapping and mutagenesis of the F sex factor replication region.

The plasmids pSC138 and pML31 each contain the EcoRI-generated f5 replicator fragment of the conjugative plasmid F in addition to an EcoRI fragment encoding antibiotic resistance: ampicillin resistance derived from Staphylococcus aureus in pSC138 and kanamycin resistance from Escherichia coli in pML31. We have mapped one HindIII and two BamHI restriction sites in the f5 region of these plasmids and one HindIII site in the antibiotic resistance region of each plasmid. The HindIII site in the Km region of pML31 occurs in the kan gene whereas the HindIII site in the Ap region of pSC138 appears to occur in an area important for the regulation of beta-lactamase production. By means of in vitro recombinant DNA manipulation of plasmids pML31 and pSC138, we have shown that approximately 1.9 X 10(6) daltons of the 6.0 X 10(6) dalton f5 fragment can be deleted without disrupting plasmid stability. In addition, we have used these same techniques to isolate a novel F-controlled Ap plasmid cloning vehicle which contains a single restriction site for each of the enzymes EcoRI, HindIII, and BamHI. This cloning vehicle has been linked via either its EcoRI or HindIII site to a ColE1 plasmid replicon to yield stable recombinants.     

Cloned DNA fragment specifying major outer membrane protein a in Escherichia coli K-12.

Plasmid pMC44 is a recombinant plasmid that contains a 2-megadalton EcoRI fragment of Escherichia coli K-12 DNA joined to the cloning vehicle, pSC101. The polypeptides specified by plasmid pMC44 were identified and compared with those specified by pSC101 to determine those that are unique to pMC44. Three polypeptides specified by plasmid pMC44 were localized in the cell envelope fraction of minicells: a Sarkosyl-insoluble outer membrane polypeptide (designated M2), specified by the cloned 2-megadalton DNA fragment, and two Sarkosyl-soluble membrane polypeptides specified by the cloning plasmid pSC101. Bacteria containing plasmid pMC44 synthesized quantities of M2 approximately equal to the most abundant E. coli K-12 outer membrane protein. Evidence is presented that outer membrane polypeptide M2, specified by the recombinant plasmid pMC44, is the normal E. coli outer membrane protein designated protein a by Lugtenberg and 3b by Schnaitman.     

Production of lentiviral vectors in suspension cells using low proportion of supercoiled circular plasmid DNA

The supercoiled circular (SC) topology form of plasmid DNA has been regarded to be advantageous over open circular or linearized analogue in transfection and expression efficiency, and therefore are largely demanded in the biopharmaceutical manufacturing. However, production of high-purity SC plasmid DNA would result in high manufacturing cost. The effect of SC proportion in plasmid DNA on the quality of packaged lentiviral vectors has never been reported. In this study, we established an efficient system for production of high-titer lentiviral vectors using suspension HEK293SF cells in serum-free media, and the lentiviral titer was not associated with the proportion of SC plasmid DNA. Plasmids DNA with different proportion of SC, open-circular, and linearized forms were prepared using the thermal denaturation method, and were transfected to adherent HEK293T or suspension HEK293SF cells for packaging of lentiviral vectors. The titer of lentiviral vectors from HEK293T cells, but not from HEK293SF cells, was significantly impaired when the proportion of SC plasmid DNA decreased from 60–80% to 30–40%. Further decrease of SC plasmid proportion to 3% led to a dramatic reduction of lentiviral titer no matter the packaging cell line was. However, lentiviral vectors from HEK293SF cells still showed a high titer even when the proportion of SC plasmid DNA was 3%. This study demonstrated that extremely high proportion of SC plasmid DNA was not required for packaging of high-titer lentiviral vector in HEK293SF cells, at least under our manufacturing process.