Plasmid DNA Analysis of Pathogenic Escherichia coli in Musk Deer

[Objective] The pathogenic Escherichia coli in musk deer was classified at molecular level to provide basic materials for molecular epidemiology of pathogenic Escherichia coli in musk deer. [Method] Plasmids from 24 pathogenic Escherichia coli in musk deer were extracted by the Lysis Triton method, and then identified by single enzyme digestion with three endonucleases of Hind Ⅲ, EcoR Ⅰ and BamH Ⅰ. [Result] The yield rate of plasmids was 91.6%, and 24 pathogenic Escherichia coli in musk deer had the identical or similar plasmid profiles. [Conclusion] Plasmid DNA analysis offers scientific basis for molecular epidemiology of pathogenic Escherichia coli in musk deer in Sichuan Institute of Musk Deer Breeding.

Researches of Agrobacterium rhizogenes Ri Plasmid rol Genes

Expression of rol genes from Ri plasmid of Agrobacterium rhizogenes not only leads to the excessive formation of adventitious roots, but also exhibits various genetically modified characteristics that have broad prospects for the application of plant genetic improvement. Since the 1980s of the last century, much progress has been made in the studies of A. rhizogenes, in particular the agropine type Ri plasmid rol genes and their applications for plant genetic improvement, which involves the structure and function of Ri plasmid, the characters of rol genes, the influence of rol genes expression on plants growth and development, and the applications of rol genes for genetic improvement of forest tree. In this paper, the advances in this field are reviewed and the existing problems about the application of rol genes for genetic improvement of forest tree are also discussed.

Curing of the Bacillus subtilis Plasmid Using Sodium Dodecyl Sulfate

Curing of Bacillus subtilis plasmid using sodium dodecyl sulfate (SDS)was studied in order to obtain a host strain. An overnight culture of Bacillus subtilis 24/pMX45 was used to inoculate fresh LB containing SDS (0-0.008%). No growth of 24/pMX45 was observed when LB contained an SDS concentration of 0.006% or greater, and the sublethal concentration (w/v) of SDS was 0.005% with a killing rate of 99%. Samples were diluted and plated on LB agar, individual colonies were randomly picked to a selective agar medium by tooth to screen for loss of plasmid-encoded erythomycin resistance. CsCl-EtBr gradient centrifugation and plasmid DNA profile demonstrated that plasmid-cured derivative A7 has completely lost its plasmid. A7 had a shorter lag, and its cell concentration was consistently higher than that of the 24/pMX45. Elimination of the plasmid was first observed after 24/pMX45 had been treated with SDS for 8 h. The percent elimination then continued to increase until about 22 h, after which the fraction of cured cell in the population remained constant. Plasmid cured cell numbers were measured in a separate control culture of 24/pMX45 untreated by SDS. No spontaneous loss of pMX45 was observed after 24/pMX45 were incubated for 24 h and 48 h with shaking at 37 ℃.These results suggested that SDS can be used as curing agent to eliminate the plasmid of Bacillus subtilis.

Construction of the Plasmid Reference Molecule for Detection of Transgenic Soybean MON89788

[Objective] The aim was to construct a plasmid reference molecule (PRM) for detection of transgenic soybean MON89788. [Method] the lectin gene sequence,3'-junction and 5'-junction sequence between host plant DNA integrated DNA of MON89788 soybean were amplified independently,and the three fragments were cloned into the cloning vector pMD18-T in order through molecular manipulation method to construct pMD-LM3M5,the applicability of the constructed novel PRM was tested. [Result] Sequencing confirmation result showed that the PRM was 3 700 bp in length,containing 1 029 bp of recombined DNA fragment. The limits of qualitative detection of the PRM were 10 copies. [Conclusion] The PRM constructed in this study was suitable for the identification of MON89788 event.

Construction and Application of Plasmid pUC19-CM-D

[Objective] The aims were to construct a new suicide plasmid of Lactobacillus and gene deletion engineering bacteria of Lactobacillus with pUC19 vector. [Methods] pUC19-CM was constructed by inserting a chloramphenicol resistant gene into the multi-cloning site of pUC19,and then two homologous fragments were cloned into each side of the pUC19-CM to construct suicide plasmid pUC19-CM-D. [Results] A replacement mutant strain,whose target gene was replaced by resistant gene,could be obtained by transforming the suicide plasmid pUC19-CM-D into Lactobacillus for resistance screening. [Conclusion] The construction and application of pUC19-CM-D provided a fast and efficient means of construction of gene deletion engineering bacteria of Lactobacillus,and laid a foundation for study of gene function of Lactobacillus.

Transfection of bone marrow mesenchymal stem cells using green fluorescence protein labeled hVEGF165 recombinant plasmid mediated by liposome

Objective:To study the role of bone marrow mesenchymal stem cells(BMSCs)in construction of vascularized engineered tissue.Methods:hVEGF165 was amplified via RT-PCR before recombinant with pShuttle-green fluorescence protein;green fluorescent protein(GFP)-CMV.Then the recombinant shuttle plasmid was transfected into BMSCs with Lipofectamine^(TM)2000 for packaging and amplifying.hVTGF165 mRNA expression in BMSCs cells was tested.Results:The sequence of hVEGFI65 in pShutlle-GFP-hVFGF165 plasmid was confirimed by double-enzyme cleavage method and sequencing.hVECF165 was highly expressed in BMSCs.Conclusions:The GFP/hVECF165 recombinant plasmid vector was constructed successfully and expressed effectively in host cells,which may be helpful for discussing the possibility of the application of VEGF165-BMSCs in tissue engineering and ischemic disease cure.

ISOLATION OF PLASMID FROM THE BLUE-GREEN ALGA SPIRULINA PLATENSIS

CCC plasmid was isolated from an economically important blue-green alga- Spirulina platensis (1.7×106 dalton from the S6 strain and 1.2×106 dalton from the F, strain) using a rapid method based on ultrasonic disruption of algal cells and alkaline removal of chromosomal DNA. The difference in the molecular weight of the OOC DNAs from the two strains differing in form suggests that plasmid may be related with the differentiation of algal form. This modified method, which does not use any lysozyme, is a quick and effective method of plasmid isolation, especially for filamentous blue-green algae.

Catalytic hydrolysis of phosphate diester (BNPP) and plasmid DNA by mononuclear macrocyclic polyamine metal complexes

The activities of the catalytic hydrolysis of phosphate diester （BNPP） [bis（p-nitrophenyl）phosphate diester] and plasmid DNA （pUC 18） by mononuclear macrocyclic polyamine metal complexes have been investigated in this paper. The results showed that the highest activity in hydrolysis of BNPP was obtained with le--Zn（II） complex （composed of lipophilic group） as catalyst. The hydrolysis rate enhancement is up to 3.64 × 10^4 fold. These metal complexes could effectively promote the cleavage of plasmid DNA （pUC18） at physiological conditions.

CABYR RNAi plasmid construction and NF-κB signal transduction pathway

AIM:To construct the CABYR RNAi plasmid and study its relation with the nuclear factor(NF)-κB signal transduction pathway.METHODS:Human CABYR mRNA sequence was obtained from GenBank.The structure of cDNA sequence for the short hairpin RNA was BbsⅠ+sense+loop+ antisense+transcription terminator+KpnⅠ+Bam HⅠ.A CABYR silencing plasmid was constructed and transfected into the human embryo cell line 293T.Quantitative real-time polymerase chain reaction was used to analyze CABYR and NF-κB gene expression.RESULTS:The CABYR and NF-κB expressions were detected in 293T cells.The oligonucleotide(5'-GCT-CAGATGTTAGGTAAAG-3')efficiently silenced the expression of CABYR.The expression of NF-κB was not significantly affected by silencing CABYR(P=0.743).CONCLUSION:CABYR can be found in the human embryo cell line 293T.Cabyrmid 2 can efficiently silence its target,CABYR,indicating that CABYR is not related with the NF-κB signal transduction pathway.

Construction of human eukaryotic expression plasmid vascular endothelial growth factor 165 and its expression in transfected vascular smooth muscles

BACKGROUND: The highly specific vascular endothelialgrowth factor (VEGF) induces the growth of vascular en-dothelial cell. This study was to construct the eukaryoticexpression plasmid of vascular endothelial growth factorl65(VEGF165) and observe its expression in vascular smoothmuscles (VSMCs).METHODS: The primers were designed and synthesizedaccording to the gene sequences of human VEGF165. TheVEGF165 gene was obtained from umbilic artery tissue bythe method of RT-PCR, then it was cloned to eukaryoticexpression plasmid pBudCE4.1 by recombination strategy.The eukaryotic expression plasmid named pBudCE4.1/VEGF165 was identified by restriction enzyme digestion,and was sequenced. The pBudCE4.1/VEGF165 was trans-fected into VSMCs by using lipofection. The VEGF165 ex-pression of mRNA and protein was detected by RT-PCRand Western blot respectively.RESULTS: VEGF165 was shown about 576bp by RT-PCR.Sequencing revealed the amplified VEGF165 gene was iden-tical with that in the GeneBank. Restrictive enzyme (HindBam HI) digestion analysis showed that recombinantexpression plasmid pBudCE4. l/tVEGF165 had been con-structed successfully. The expression of VEGF165 at mRNAand protein levels in the transformed VSMCs had beendemonstrated by RT-PCR and Western blot.CONCLUSIONS: The recombinant eukaryotic expressionplasmid pBudCE4.1/VEGF165 has been successfully con-structed and expressed in transformed VSMCs. The presentstudy has laid a foundation for VEGF165 gene therapy ofvascular stenosis in the transplant organ.

Construction of Shuttle Expression Plasmid and Stable Expression of Foreign Gene in Mycobacteria and E．Coli

By employing the pUC19 as a backbone,the regulatory and signal sequences which encode kanamycin resistance, and mycobacterial plasmid origin of replication (oriM) were cloned into the pUC19. The recombinant E. Coli-mycobacteria shuttle expression plasmid PBCG-8000 was constructed. The PBCG-8000 was able to replicate in both E. Coli and mycobacteria (including BCG) systems, and to confer stable kanamycin resistance upon transformants. The study should facilitate the development of BCG and other mycobacteria into multivalent vaccine vectors.

Elimination of Multidrug Resistant Plasmid pEIB202 in Fish Pathogenic Edwardsiella tarda

[Objective] The aim of the research was to investigate the function of large plasmid pEIB202 in the pathogenesis of Edwardsiella tarda EIB202 and to eliminate the plasmid pEIB202,so as to lay the foundation for developing safe and live attenu- ated vaccine against E, tarda. [Method] sacB was used as reverse screening marker to eliminate the plasmid by using homologous recombination technique. [Result] The plasmid pEIB202 was sequenced and it was found that the plasmid encoded multiple resistant genes and some components in type IV secretion system（T4SS）,which sug- gested that the plasmid might be related with the multiple drug-resistance and pathogenicity of E. tarda. The plasmid-eliminated strain EIB202Ap lost the resistance to chloramphenicol and tetracycline,but its growth,virulence and secretion of extra- cellular proteins had no significant difference with wild-type plants. [Conclusion] pEIB202 plasmid is the main reason that caused the multi-drug resistance of EIB202 and might have indirect effects in the pathogenesis of EIB202.

Construction of human VEGF165 gene eukaryotic expression plasmid and its effect on proliferation of vascular endothelial cells

After organ transplantation, rapid repair of injured vascular endothelial cell (VEC) is a key to prevent graft chronic dysfunction besides control of immunological rejection. Many studies have confirmed that vascular endothelial growth factor 165 (VEGF165) could accelerate the repair of VEC injury, decrease thrombosis and thrombotic occlusion, and inhibit hyperplasia of the intima. This study was designed to construct eukaryotic expression plasmid pBudCE4.1/VEGF165, and observe its effect on the prolife ration of VEC. METHODS:The VEGF165 gene cloned from human heart tissue by RT-PCR was cloned into eukaryotic expression plasmid pBudCE4.1. The recombinant expression plasmid pBudCE4.1/VEGF165 was identified by restriction enzyme (Hind III and BamH I) digestion analysis, and was sequenced. The pBudCE4.1/VEGF165 was introduced into VEC through lipofection transfection. The VEGF165 mRNA expression by Northern blot and VEGF165 protein expression was detected by immunocytochemical staining. The effect of expression protein on VEC proliferation was detected by flow cytometry. RESULTS:The RT-PCR product of the VEGF165 gene was about 576bp. Sequencing analysis revealed that the sequence of the amplified VEGF165 gene was identical with that in GenBank. Restrictive enzyme digestion analysis showed that recombinant expression plasmid pBudCE4.1/ tVEGF165 had been constructed successfully. The expression of VEGF165 at mRNA and protein levels in the transformed VSMCs had been demonstrated by Northern blot and immunocytochemical staining respectively. The expressed product of VEGF165 could notably accelerate the proliferation of VECs. CONCLUSIONS:pBudCE4.1/VEGF165 is successfully cons- tructed and is expressed in VECs. Expressed VEGF165 can accelerate the VEC proliferation. The present study has laid a foundation for potential use of VEGF165 gene transfection to prevent and treat vascular stenosis in the transplanted organ.

Construction of Eukaryotic Expression Plasmid of hTGF-β3 and Its Inducing Effect on Differentiation of Precartilaginous Stem Cells into Chondroblasts

This study examined the construction of eukaryotic expression plasmid of human transforming growth factor-β3（hTGF-β3） and its inducing effect on the differentiation of precartilaginous stem cells（PSCs） into chondroblasts.hTGF-β3 gene was amplified by using polymerase chain reaction（PCR） and then inserted into the eukaryotic expression plasmid pcDNA3.1 to construct the eukaryotic expression plasmid pcDNA3.1（＋）-hTGF-β3.Rat PSCs were isolated and purified by employing an immunomagnetic cell sorting system.pcDNA3.1（＋）-hTGF-β3 was transfected into purified PSCs with the use of linear polyamines.The expression of TGF-β3 and cartilage-specific extracellular matrix（ECM） components was detected after transfection by real-time quantitative PCR,ELISA,immunochemistry and Western blotting,respectively.The results showed that the eukaryotic expression plasmid pcDNA3.1（＋）-hTGF-β3 was successfully established as identified by enzyme digestion and DNA sequencing.Real-time quantitative PCR and ELISA revealed that hTGF-β3 was strongly expressed in pcDNA3.1（＋）-hTGF-β3-transfected PSCs.Real-time quantitative PCR,immunochemistry and Western blotting showed that the cartilage-specific ECM markers,i.e.,cartilage oligomeric matrix protein（COMP）,Aggrecan,collagen type Ⅹ and Ⅱ were intensely expressed in the pcDNA3.1（＋）-hTGF-β3transfected cells.It was concluded that hTGF-β3 could be stably expressed in pcDNA3.1（＋）-hTGFβ3-transfected PSCs and induce the differentiation of PSCs into chondroblasts.

Extraction of plasmid-like DNA and high-quality total DNA from Porphyra yezoensis

Somatic cells were prepared from sea snail enzyme digests of Porphyra yezoensis thalli. Us ing SDS - Proteinase K as extraction solution, total DNA was isolated from the somatic cells. The crude extracts of total DNA were purified with glassmilk, and the resulting DNA was of sufficient quality for digestion of restriction endonuclease. DNA bands were clearly observed in the restriction patterns of EcoRI, PstI and HaeIII respectively. The presence of DNA hands in the restriction pattern of total DNA indicated that the genome of Porphyra yezoensis may be small. Unexpectedly, using guanidinium isoth iocyanate and sarcosyl as extraction solution, a plasmid-like DNA band (2.3 Kb) was directly found in the isolated total DNA of Porphyra yezoensis. A very simple and convenient method for plasmid-like DNA isolation has been established.

Knocking-down of Nogo-A Gene Expression in PC12 Cell Line by Plasmid-based RNAi

To study the inhibitory effect of Nogo-A shRNA on cell line PC12, the Nogo-A shRNA （short hairpin RNA, or shRNA） was designed and synthesized. The annealed shRNA template was inserted into plasmid pGenesil-1 containing enhanced green fluorescent protein （EGFP） gene by gene cloning technique to generate eukaryotic expression vector. The recombinant plasmid was transfected into PC12 cells by lipofecamine2000 and the mRNA and protein expression level of Nogo-A gene was detected by RT-PCR and Western blotting 48 h after the transfection. Gene sequencing showed that that the Nogo-A shRNA eukaryotic expression vector was successfully constructed. No significant change was found in the Nogo-A mRNA and protein expression level in empty vector-transfected group as compared with controls （P〉0.05）, while the expression level in shRNA-transfected group decreased significantly （P〈0.05）. It is concluded that the pGenesil-1/Nogo-AshRNA recombinant plasmid can effectively suppress the expression of Nogo-A gene in PC12 cells.

Transfection Efficiency Comparison of Oligonucleotide and Plasmid to the HL-60 Cell Line with Liposomes

The transfection efficiency of oligonucleotide and plasmid to the HL-60 cell line with lipofectaminePLUS was compared through observing the transfection rate and the expression duration of exogenous gene in the target cells. The results showed that the transfection rate of oligonucleotide to the HL-60 was about 90 %—95 % and it had no obvious attenuation within 84 h. However, the plasmid transfection rate was only 5 %—25 % and it was decreased significantly within 60 h. It was suggested that the transfection of oligonucleotide with liposomes was better than that of plasmid.

Antimicrobial Resistance and Plasmid Profiles of <i>Salmonella enterica</i>Serovars from Different Sources in Lagos, Nigeria

Introduction: Salmonella enterica Serovars remains one of the leading pathogens that cause diarrhoea and bloodstream infections in developing countries. The emergence of multidrug resistant (MDR) Salmonella has become a serious problem globally. This study investigated the antibiotic resistance and plasmid profiles of Salmonella isolates from different sources. Methods: Seventy-three samples comprised of clinical (30), hand swab (15), food (10) and water (18) were analyzed bacteriologically. Salmonella isolates were identified and characterized by standard procedures. Isolates were subjected to antimicrobial susceptibility testing and were further screened for plasmid DNA by standard methods. Results: A total of 27 Salmonella isolates made up of 5 (18.5%) S. typhi, 6 (22.2%) S. enteritidis, 9 (33.3) S. typhimurium, 5 (18.5%) S. cholerasuis, and 1 (3.7%) each of S.arizonae and S. vichow were obtained in this study. All the isolates developed resistance to three or more antibiotics evaluated. Four distinct resistance profiles: TetAmpCol, TetAmpColCot, TetAmpColCip and TetAmpColCotCip were recorded with 63% of the isolates exhibiting resistance profile TetAmpColCot. Specifically 23 of 27 (85.2%) of the isolates harboured plasmid DNA comprised of 12 distinct plasmid profiles of different sizes ranging from 3.2 kb to 30.2kb. Salmonella isolates of the same species from different sources differed in plasmid profile. Plasmid profile was found to show good discriminatory capability compared to antibiotics resistance profile. Conclusion: This study revealed that both resistance antibiogram and plasmid profile are still viable epidemiological tools for tracing the source of Salmonella isolates. A need for prudent use of antibiotics is suggested.

The Bacterial Isolates and Plasmid Profile of Extended Spectrum Beta-Lactamases Producers Causing Urinary Tract Infection among Pregnant Women in Uyo, Nigeria

Background: Extended spectrum beta-lactamases (ESBLs) are enzymes that compromise the efficacy of all beta-lactams and are spread by plasmids. They are of public health importance the world over;however, in Nigeria in general and Uyo in particular, tests for their detection are not routinely done in hospital laboratories despite increase in treatment failures observed for common clinical conditions like urinary tract infection. Objective: To isolate ESBLs producing uropathogens and the plasmid underlying their resistance to antibiotics. Materials and Methods: Three hundred urine specimens (n = 300) were collected from pregnant women attending antenatal clinics at St. Lukes Hospital, Anua, cultured and incubated according to accepted standard. Identification of isolates was done using Microbact 24E (Oxoid, UK) system. The predominant bacterial pathogens were Escherichia coli (42%) followed by Klebsiella pneumonia (21%), Klebsiella oxytoca (12%), Citrobacter spp. (5%), Proteus mirabilis (7%), Enterobacter spp. (12%) and Acinetobacter baumanii (1%). The isolated bacteria were tested for their antibiotic susceptibility using Clinical Laboratory Standard Institute (CLSI) recommended disc diffusion method. A Double Disk Synergy Test (DDST) and Phenotypic Disk Confirmatory Test (PDCT) were performed to determine ESBL production. Chromagar ESBL was also used to test for the presence of ESBL producing isolates. The plasmid content of ESBL producing isolates and their participation in drug resistance were investigated. Results: Of the 80 bacterial isolates causing urinary tract infection in these women, the ESBL producers were found to be 16 (20%). Out of these 16 ESBL producing urogenital isolates Klebsiella pneumonia (8, 50%) was the most prevalent. Others include Escherichia coli (38%), Klebsiella oxytoca (6%) and Enterobacter cloacae (6). Plasmid content of ESBL producing isolates was found to be 87.5%. Conclusion: The Extended Spectrum Beta-lactamase producing uropathogens mainly of plasmid origin are increasingly responsible for the cause of community acquired urinary tract infections in pregnant women in Uyo.

Characterization of NDM-5-producing Enterobacteriaceae isolates from retail grass carp(Ctenopharyngodon idella) and evidence of blaNDM-5-bearing IncHI2 plasmid transfer between ducks and fish

We aimed to characterize NDM-5-producing Enterobacteriaceae from aquatic products in Guangzhou,China.A total of 196 intestinal samples of grass carp collected in 2019 were screened for carbapenemase genes.Characterization of bla_(NDM-5) positive isolates and plasmids was determined by antimicrobial susceptibility testing,conjugation experiments,Illumina HiSeq,and Nanopore sequencing.One Citrobacter freundii and six Escherichia coli strains recovered from seven intestinal samples were verified as bla_(NDM-5) carriers(3.57%,7/196).The bla_(NDM-5) genes were located on the lncX3(n=5),lncHI2(n=1),or lncHI2-lncF(n=1)plasmids.All bla_(NDM-5)-bearing plasmids were transferred by conjugation at frequencies of~10^(-4)-10^(-6).Based on sequence analysis,the lncHI2 plasmid pHNBYF33-1 was similar to other bla_(NDM-5)-carrying lncHI2 plasmids deposited in GenBank from Guangdong ducks.In all lncHI2 plasmids,bla_(NDM-5)was embedded in a novel transposon,Tn7057(IS3000-△ISAba125-IS5-△ISAba125-bla_(NDM-5)-bleMBL-trpF-tat-△dct-IS26-△umuD-△ISKox3-IS3000),which was identical to the genetic structure surrounding bla_(NDM-5)found in some IncX3 plasmids.The lncHI2-lncF hybrid plasmid pHNTH9F11-1 was formed by homologous recombination of the bla_(NDM-5)-carrying lncHI2 plasmid and a heavy-metal-resistant IncF plasmid through△Tn1721 To the best of our knowledge,this is the first report on the characterization of bla_(NDM-5)-bearing plasmids in fish in China.The lncHI2 plasmid pHNBYF33-1 may be transmitted from ducks,considering the common duck-fish freshwater aquaculture system in Guangdong.Tn7051 is likely responsible for the transfer of bla_(NDM-5) from lncX3 to lncHI2 plasmids in Enterobacteriaceae,resulting in the expansion of transmission vectors of bla_(NDM-5).