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.

Methylome and transcriptome data integration reveals potential roles of DNA methylation and candidate biomarkers of cow Streptococcus uberis subclinical mastitis

Background:Mastitis caused by different pathogens including Streptococcus uberis(S.uberis)is responsible for huge economic losses to the dairy industry.In order to investigate the potential genetic and epigenetic regulatory mecha‑nisms of subclinical mastitis due to S.uberis,the DNA methylome(whole genome DNA methylation sequencing)and transcriptome(RNA sequencing)of milk somatic cells from cows with naturally occurring S.uberis subclinical mastitis and healthy control cows(n=3/group)were studied.Results:Globally,the DNA methylation levels of CpG sites were low in the promoters and first exons but high in inner exons and introns.The DNA methylation levels at the promoter,first exon and first intron regions were nega‑tively correlated with the expression level of genes at a whole‑genome‑wide scale.In general,DNA methylation level was lower in S.uberis‑positive group(SUG)than in the control group(CTG).A total of 174,342 differentially methylated cytosines(DMCs)(FDR<0.05)were identified between SUG and CTG,including 132,237,7412 and 34,693 DMCs in the context of CpG,CHG and CHH(H=A or T or C),respectively.Besides,101,612 methylation haplotype blocks(MHBs)were identified,including 451 MHBs that were significantly different(dMHB)between the two groups.A total of 2130 differentially expressed(DE)genes(1378 with up‑regulated and 752 with down‑regulated expression)were found in SUG.Integration of methylome and transcriptome data with MethGET program revealed 1623 genes with signifi‑cant changes in their methylation levels and/or gene expression changes(MetGDE genes,MethGET P‑value<0.001).Functional enrichment of genes harboring≥15 DMCs,DE genes and MetGDE genes suggest significant involvement of DNA methylation changes in the regulation of the host immune response to S.uberis infection,especially cytokine activities.Furthermore,discriminant correlation analysis with DIABLO method identified 26 candidate biomarkers,including 6 DE genes,15 CpG‑DMCs and 5 dMHBs that discriminated between SUG and CTG.Conclusion:The integration of methylome and transcriptome of milk somatic cells suggests the possible involve‑ment of DNA methylation changes in the regulation of the host immune response to subclinical mastitis due to S.uberis.The presented genetic and epigenetic biomarkers could contribute to the design of management strategies of subclinical mastitis and breeding for mastitis resistance.

Analysis of heavy-ion-induced DNA strand breaks in plasmid pUC18

Plasmid DNA was irradiated or implanted by mixed particle field(CR) or lithium-ion-beam to detect strand breaks.The primary results showed that mixed particle field could induce single and double strand breaks with positive linear-dose-effects;most of sequence changes induced by CR were point mutant.Lithium-ion-beam could induce strand breaks also,but it was only at dose of 20Gy.

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.

Effects of medium composition on the production of plasmid DNA vector potentially for human gene therapy

Plasmid vector is increasingly applied to gene therapy or gene vaccine. The production of plasmid pCMV-AP3 for cancer gene therapy was conducted in a modified MBL medium using a recombinant E. coli BL21 system. The effects of different MMBL components on plasmid yield, cell mass and specific plasmid DNA productivity were evaluated on shake-flask scale. The results showed that glucose was the optimal carbon source. High plasmid yield (58.3 mg/L) was obtained when 5.0 g/L glucose was added to MMBL. Glycerol could be chosen as a complementary carbon source because of the highest specific plasmid pro- ductivity (37.9 mg DNA/g DCW). After tests of different levels of nitrogen source and inorganic phosphate, a modified MMBL medium was formulated for optimal plasmid production. Further study showed that the initial acetate addition (less than 4.0 g/L) in MMBL improved plasmid production significantly, although it inhibited cell growth. The results will be useful for large-scale plasmid production using recombinant E. coli system.

Optimization on cationic liposome-mediated cell transfection of plasmid DNA

Objective:The development of gene carriers for efficient gene delivery into cells has attracted growing attention in recent years.The aim of this study was to achieve a better outcome of AAV-293 cells transfection by plasmid DNA.Methods:We studied the optimal condition for higher efficiency of cationic lipid-mediated cell transfection.Four experimental groups were set.Plasmid DNA and liposome were mixed in each groups at different ratios(μg:μL),1:2.5,1:3.5,1:4.0 and 1:5.0,respectively.LacZ gene functioned as reporter gene,measuring the transfection efficiency of the four groups using the method of X-gal staining.Results:When the ratio was 1:3.5,the cell transfection rate was the highest.While the ratio of 1:2.5 recommended by product manual achieve the lowest transfection rate.Their difference had statistical significance.Conclusion:In order to obtain a higher transfection efficiency,optimization on conditions of the ratio of plasmid DNA to liposome is necessary in cell transfection.

Amplification of plasmid DNA bound on soil colloidal particles and clay minerals by the polymerase chain reaction

Polymerase chain reaction （PCR） was used to amplify a 600-base pair （bp） sequence of plasmid pGEX-2T DNA bound on soil colloidal particles from Brown soil （Alfisol） and Red soil （Ultisol）, and three different minerals （goethite, kaolinite, montmorillonite）. DNA bound on soil colloids, kaolinite, and montmorillonite was not amplified when the complexes were used directly but amplification occurred when the soil colloid or kaolinite-DNA complex was diluted, 10- and 20-fold. The montmorillonite-DNA complex required at least 100-fold dilution before amplification could be detected. DNA bound on goethite was amplified irrespective of whether the complex was used directly, or diluted 10- and 20-fold. The amplification of mineral-bound plasmid DNA by PCR is, therefore, markedly influenced by the type and concentration of minerals used. This information is of fundamental importance to soil molecular microbial ecology with particular reference to monitoring the fate of genetically engineered microorganisms and their recombinant DNA in soil environments.

Preparation of Ag/AgBr/TiO_2 as Catalyst Carriers and Its Damage to Plasmid DNA and Tetrahymena

The composites based on the Ti O2 are potentially used in wetland pollution control. In this work, the biological effect of the Ag/Ag Br/Ti O2/Active carbon(AC) composites was studied on the plasmid DNA and Tetrahymena membrane. The atomic force micrograph(AFM) images showed that, in the presence of the composites under illumination, most p UC18 DNA molecules showed quite different topography and were opened and relaxed circle shapes. After DNA was catalyzed for 40 min, all supercoiled and circular DNA were changed into the linear DNA molecules. The gel electrophoresis experiment confirmed the results and demonstrated the dynamic process of DNA degradation. ATR-FTIR spectra revealed that amide groups and PO2-of the phospho-lipid phospho-diester on Tetrahymena surface were oxidized in the presence of the composites under illumination. An increase in the fluorescence polarization of DPH was observed, reflecting a significant decrease in membrane fluidity of Tetrahymena.

Construction and in vitro Expression of Streptococcus Mutans Surface Protein Encoding DNA Vaccine

DNA vaccine plasmids were constructed that encoded two highly conservative regions of a surface protein, PAc, from the human major cariogenic bacterium, Streptococcus mutans . Antigen expression was evaluated in vitro by immunohistochemical analysis of human endothelial cells following cationic liposome mediated transient transfection with recombinant plasmid. The results of this study provided a basis for further testing of these recombinant plasmids in primates and for efficacy testing of dental caries DNA vaccines in human volunteers in future.

Isolation of T-DNA flanking plant DNA from T-DNAinsertional embryo-lethal mutants of Arabidopsis thaliana by plasmid rescue technique

Three T-DNA insertional embryonic lethal mutants from NASC (The Nottingham Arabidopsis Stock Center)were first checked with their segregation ratio of abortive and normal seeds and the copy number of T-DNA insertion. The N4081 mutant has a segregation ratio of 1:3.04in average and one T-DNA insertion site according to our assay It was therefore chosen for further analysis. To isolate the joint fragment of T-DNA and plan DNA, the plasmid rescue technique waJs used. pEL-7, one of plasmids from left border of T-DNA, which contained pBR322 was selected from ampicillin plate. The T-DNA fragment of pEL-7 was checked by restriction enzyme analysis and Southern Blot. Restriction analysis confirmed the presence of known sites of EcoRI, PstI and PvuII on it.For confirming the presence of flanking plant DNA in this plasmid, pEL-7 DNA was labeled and hybridized with wild type and mutant plant DNA. The Southern Blot indicated the hybridization band in both of them. Furthermore, the junction of T-DNA/plant DNA was subcloned into bluescript SK+ and sequenced by Applied Biosystem 373A Sequencer. The results showed the 822 bp fragment contained a 274 bp sequence, which is 99.6%homolog (273bp/274 bp) to Ti plasmid pTi 15955 DNA.Ten bp of left 25 bp border repeat were also found in the juction of T-DNA and Plant DNA.Taken together, pEL-7 should contain a joint fragment of T-DNA and flanking plant DNA. This plasmid DNA could be used for the isolation of plant gene, which will be helpful to elucidate the relationship between gene function and plant embryo development.

Delivery of Plasmid DNA into Tumors by Intravenous Injection of PEGylated Cationic Lipoplexes into Tumor-Bearing Mice

For systemic injection of cationic liposome/plasmid DNA (pDNA) complexes (cationic lipoplexes), polyethylene glycol (PEG)-modification (PEGylation) of lipoplexes can enhance their systemic stability. In this study, we examined whether intravenous injection of PEGylated cationic lipoplexes into tumor-bearing mice could deliver pDNA into tumor tissues and induce transgene expression. PEGylation of cationic liposomes could prevent their agglutination with erythrocytes. However, when PEGylated cationic lipoplexes were injected intravenously into tumor-bearing mice, they accumulated in tumor vascular vessels and did not exhibit transgene expression in tumors with both poor and well-developed vascularization. Furthermore, PEGylated cationic lipoplexes of CpG- free pDNA could not increase transgene expression in tumors after intravenous injection. These results suggested that PEGylation could not extravasate cationic lipoplexes from vascular vessels in tumors and abolished transgene expression although it enhanced the systemic stability of cationic lipoplexes by avoiding interactions with blood components such as erythrocytes. Successful delivery of pDNA to tumors by PEGylated cationic liposomes will require a rational strategy and the design of liposomal delivery systems to overcome the issue associated with the use of PEG.

Endocytosis and Vesicular Transport of Plasmid DNA in Cells During Electric Field-Mediated Gene Delivery

Pulsed electric field has been used widely as a nonviral approach to improving gene delivery in basic and translational research[1-2].The technique has been called electrotransfection(ET),electroporation,electrogene transfer,and gene electroinjection in the literature [1,3].It has a great potential to improve clinical treatment of diseases through delivery of vaccines and therapeutic genes,genome and epigenome editing,and generation of human induced pluripotent stem cells for tissue engineering[1-3].During ET,extracellular transport of plasmid DNA(pDNA)relies on electrophoresis,which is critical for applications in vivo.However,mechanisms of intracellular transport remain to be understood.The lack of understanding has hindered the translation of ET technology to the clinic.It is well known that pulsed electric field can generate transient hydrophilic pores in the plasma membrane(i.e.,electroporation)that permit membrane-impermeant molecules to enter cells.Although the pores have yet to be visualized directly under a microscope,the electric field-induced membrane permeabilization has been demonstrated through experimental measurements of electrical conductance of synthetic lipid membranes and plasma membranes,direct observation of fluorescent markers crossing the membranes facing both cathode and anode,and numerical simulations of the membrane permeabilization[1,3].Results from the simulations have predicted that the cutoff size of the pores is on the order of a few hundred nanometers,and the lifetime of the pores that are larger than 100 nm is on the order of 10 msec.Although these data provide a solid evidence of the membrane permeabilization,recent studies have demonstrated that the generation of the pores is insufficient for ET[1,4].The reasons are as follows.First,the lifetime of the pores is several orders of magnitude shorter than the time scale for pDNA uptake,which is on the order of 10 min.Second,complex formation between pDNA and plasma membrane is a necessary condition for successful gene transfer.Third,inhibition of clathrin mediated endocytosis or Rac-1 dependent micropinocytosis can reduce the amount of pDNA internalized by cells [1].Finally,we demonstrate that few pDNA molecules can be observed in the cytosol that are not associated with the intracellular vesicles[5],suggesting that pDNA uptake is mediated by endocytosis.In addition to the internalization,ET requires the pDNA in the cytoplasm to reach the nucleus.To understand mechanisms of intracellular trafficking of pDNA,we have examined time-dependent pDNA distributions in cells,quantitatively determined percentages of pDNA molecules associated with different endocytic compartments using transmission electron microscopy(TEM),and investigated different approaches to facilitate cytoplasmic transport and nuclear entry of pDNA.Our data have shown that electrotransfected pDNA is located in different vesicular ultrastructures at or near the plasma membrane at10 min post application of electric pulses[5].In the hard-to-transfect cells(e.g.,4T1),pDNA penetration from the cell surface is less active,and the total number of vesicular structures associated with pDNA is low,compared to those in the easyto-transfect cells(e.g.,COS7).Our data have also shown that macropinocytosis is the most common pathway shared by all types of cells.To investigate how improve pDNA transport in cells,we have photochemically treated cells to non-specifically induce pDNA escape from intracellular vesicles,or blocked endosome and autophagic vacuole maturation through treatment of cells with Bafilomycin Al,an inhibitor of vacuolar H+ATPase.Our data demonstrate that both treatments can lead to reduction of ET efficiency although the treatment for inducing endosomal escape can enhance poly-L-lysine mediated gene delivery.These data suggest that the vesicles play an important role in protecting the naked pDNA during intracellular trafficking.The nuclear envelope is another major barrier to ET.To facilitate the nuclear entry,we have examined three different approaches.One is to synchronize the nuclear envelope breakdown(NEBD)prior to ET;the second approach is to pre-treat cells with a nuclear pore dilating agent(i.e.,trans-1,2-cyclohexanediol);and the third one is to incorporate a nuclear targeting sequence(NTS)(i.e.,SV40)into the pDNA.Our data have shown that the synchronization of the NEBD can significantly improve the ET efficiency without compromising the cell viability.The nuclear pore dilation can improve the ET as well but the dilating agent is cytotoxic.The incorporation of NTS into pDNA can improve the gene delivery efficiency but the improvement is cell-type dependent,suggesting that the NTS has to be screened and optimized for the cells of interest.In summary,the transient pores in the plasma membrane induced by the electric pulses will enable cellular uptake of membrane-impermeant molecules up to the size of small proteins.Larger molecules(e.g.,pDNA)have to be internalized via endocytic processes triggered by the pulsed electric field.Within the cells,pDNA transport is mediated by vesicles and can be blocked by non-specific escape from vesicles or inhibition of vesicle maturation.The nuclear entry of pDNA can be enhanced,without compromising cell viability,through the use of the NTS or the synchronization of the NEBD.

Destruction of <i>Escherichia coli</i>and Broad-Host-Range Plasmid DNA in Treated Wastewater by Dissolved Ozone Disinfection under Laboratory and Field Conditions

Broad-host-range plasmids are frequently associated with antibiotic resistance genes and can quickly spread antibiotic resistant phenotypes among diverse bacterial populations. Wastewater treatment plants have been identified as reservoirs for broad-host-range plasmids carrying resistance genes. The threat of broad-host-range plasmids released into the environment from wastewater treatment plants has identified the need for disinfection protocols to target broad-host- range plasmid destruction. Here we evaluate the efficacy of dissolved ozone at 2 and 8 mg·L–1 as a primary means for the destruction of broad-host-range plasmid and chromosomal DNA in simulated effluent. Pilot-scale tests using an experimental unit were carried out in municipal wastewater treatment plant effluent and compared with ultraviolet (UV)-irradiation and chlorination methodologies. Genes specific to Escherichia coli (uidA) and IncP broad-host-range plasmids (trfA) were monitored using real-time quantitative polymerase chain reaction (qPCR), and total DNA was monitored using absorbance spectroscopy. In wastewater treatment plant experiments, E. coli qPCR results were compared to a recognized culture-based method (Colilert?) for E. coli. In laboratory experiments, dissolved ozone at 8 mg·L–1 significantly destroyed 93% total, 98% E. coli, and 99% of broad-host-range plasmid DNA. Ozonation, UV-irradiation, and chlorination significantly reduced DNA concentrations and culturable E. coli in wastewater treat- ment plant effluent. Chlorination and UV disinfection resulted in 3-log decreases in culture-based E. coli concentrations in wastewater treatment plant effluent while changes were not significant when measured with qPCR. Only ozonation significantly decreased the IncP broad-host-range plasmid trfA gene, although concentrations of 2.2 × 105 copies trfA·L–1 remained in effluent. Disinfection processes utilizing high dissolved ozone concentrations for the destruction of emerging contaminants such as broad-host-range plasmid and total DNA may have utility as methods to ensure downstream environmental health and safe water reuse become more important.

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.

Brain abscess caused by Streptococcus anginosus group:Three case reports

BACKGROUND This case series investigated the clinical manifestations,diagnoses,and treatment of cerebral abscesses caused by Streptococcus anginosus.We retrospectively analyzed the clinical characteristics and outcomes of three cases of cerebral abscesses caused by Streptococcus anginosus and conducted a comprehensive review of relevant literature.CASE SUMMARY Case 1 presented with a history of left otitis media and exhibited high fever,confusion,and vomiting as primary symptoms.Postoperative pus culture indicated a brain abscess caused by Streptococcus constellatus infection.Case 2 experienced dizziness for two days as the primary symptom.Postoperative pus culture suggested an intermediate streptococcal brain abscess.Case 3:Enhanced head magnetic resonance imaging(MRI)and diffusion-weighted imaging revealed occupancy of the left temporal lobe,initially suspected to be a metastatic tumor.However,a postoperative pus culture confirmed the presence of a brain abscess caused by Streptococcus anginosus infection.The three cases presented in this case series were all patients with community-acquired brain abscesses resulting from angina caused by Streptococcus group infection.All three patients demonstrated sensitivity to penicillin,ceftriaxone,vancomycin,linezolid,chloramphenicol,and levofloxacin.Successful treatment was achieved through stereotaxic puncture,drainage,and ceftriaxone administration with a six-week course of antibiotics.CONCLUSION Preoperative enhanced head MRI plays a critical role in distinguishing brain tumors from abscesses.Selecting the correct early diagnostic methods for brain abscesses and providing timely intervention are very important.This case series was in accordance with the CARE guidelines.

指数补料联合升温发酵法提高治疗性HPV DNA疫苗产量的研究进展

宫颈癌是全球女性第四大常见癌症。目前上市的HPV疫苗均为预防性疫苗,对已感染HPV人群无治疗效果。HPV感染率仍然很高。近年来,科学家日益关注治疗性HPV疫苗的研发,可诱导细胞免疫杀死已感染HPV的细胞。治疗性HPV DNA疫苗以其低成本、稳定性好的特点备受青睐。为降低治疗性HPV DNA疫苗的成本,目前多采用大肠杆菌高密度发酵生产。本研究使用具有热敏感特性的DNA质粒特性的治疗性HPV DNA疫苗,采用摇瓶培养载有DNA质粒的大肠杆菌,30℃培养一定时间后,分别升温至37℃或42℃,结果显示,升温至42℃组比升温至37℃组的质粒产量明显升高;采用发酵方式培养大肠杆菌,30℃培养一定时间后,升温至42℃进一步提高质粒的拷贝数;分别探究恒速补料和指数补料两种方式联合升温发酵策略对DNA质粒产量的影响,结果显示,相较于恒速补料,指数补料可显著提高升温发酵过程中DNA质粒的产量,从125.76 mg·L^(-1)提升至619.94 mg·L^(-1)。发酵得到的治疗性HPV DNA疫苗可在293T细胞中高效表达,并能在体内引起HPV抗原特异性细胞免疫反应。本研究认为指数补料联合升温发酵策略对其他DNA质粒的发酵也有一定的借鉴意义,也为CAR-T等细胞产品、RNA相关产品提供高质量DNA质粒提供参考。

In Vitro Antioxidant and Radio Protective Activities of Lycopene from Tomato Extract against Radiation—Induced DNA Aberration

Background: The accumulation of free radicals is linked to a number of diseases. Free radicals can be scavenged by antioxidants and reduce their harmful effects. It is therefore essential to look for naturally occurring antioxidants that come from plants, as synthetic antioxidants are toxic, carcinogenic and problematic for the environment. Lycopene is one of the carotenoids, a pigment that dissolves in fat and has antioxidant properties. Materials and Methods: The antioxidant and free radical scavenging activity were assessed using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. The impact of lycopene on bacteria (E. coli) susceptibility to γ-radiation was examined by radio sensitivity assay. The study also examined the induction of strand breaks in plasmid pUC19 DNA and how lycopene extract protected the DNA from γ-radiation in vitro. Results: At varying concentrations, lycopene demonstrated its ability to scavenge free radicals such as 2, 2-diphenyl-1-picrylhydrazyl (DPPH). IC50 for lycopene was determined at 112 μg/mL which was almost partial to IC50 of standard antioxidant L-ascorbic acid. The D10 value 180 Gy of E. coli was found to be >2-fold higher in the extract-containing lycopene sample than in the extract-free controls. The lycopene extracts inhibited the radiation-induced deterioration of the plasmid pUC19 DNA. At an IC50 concentration, lycopene provided the highest level of protection. Conclusion: Lycopene functions as an efficient free radical scavenger and possible natural antioxidant source. For cancer patients and others who frequently expose themselves to radiation, lycopene may be a useful plant-based pharmaceutical product for treating a variety of diseases caused by free radicals.

Construction of a recombinant plasmid harbouring the rhoptry protein 1 gene of Toxoplasma gondii and preliminary observations on DNA immunity

Objective To observe the immune responses elicited in BALB/c mice by a DNA vaccine. A gene encoding rhoptry protein 1 (ROP1) from Toxoplasma gondii (T. gondii) was cloned into vector pcDNA3. Methods Amplifyied gene fragments coding for ROP1 from the genomic DNA of T.gondii ZS2 were inserted into cloning vector, pUC18, and sub-cloned into pcDNA3. Mice were injected at a dosage of 100?μg recombinant plasmid DNA by intramuscular injection and boosted after 2 weeks. pcDNA3 and normal saline were used as control. 30, 50 and 70 days after the second immunization, NK cell activity, T lymphocyte proliferation and sub-clusters and serum IgG antibody were assayed.Results The specific gene fragment coding for ROP1 was amplified and a pcROP1 recombinant was constructed. At 30 days after immunization, the spleens of the mice were obviously enlarged evidently. NKC activity and the proliferation of spleen T lymphocytes seen on MTT assay were higher in pcROP1 group than in the controls. The number of CD4+ T cells exhibited no obvious increase compared with that of the control, but CD8+ T cells were obviously increased (P<0.05). At 90 days after vaccination, the titer of IgG antibody in the serum of vaccinated mice was positive (1∶100). Conclusion pcROP1 was constructed and it could elicit both cellular and humoral immune responses in immunized mice.

ANALYSIS OF N-METHYL-N-NITROSOUREA-INDUCED MUTATIONS IN A SHUTTLE VECTOR PLASMID PROPAGATED IN MOUSE O^6-METHYLGUANINE-DNA METHYLTRANSFERASE-DEFICIENT CELLS IN COMPARISON WITH PROFICIENT CELLS.

To investigate a contribution of O-methylguanineto mutagenesis in mouse cells,we constructed ashuttle vector plasmid,pYZ289,from a part ofpZ189 plasmid and polyoma virus DNA.The plasmidcontains a supF gene as a marker of mutation andcan replicate in both E.coli and mouse cells.ThepYZ289 treated with N-methyl-N-nitrosourea (MNU)

Construction of DNA Vaccine for FMDV P1 Gene and Immunization Experiment

[Objective] This study aimed to construct DNA vaccine of foot-and-mouth disease （FMD）.[Method] Plasmid carriers plESZP1 and pUTK3CP1 with PRV were constructed for FMDV P1 gene expression.Mice were immunized,and their antibody level was detected.The two eukaryotic expression plasmids constructed were transfected into Vero cells.PCR,IFA and Westem-blot were carried out to detect the transcription and expression of the objective gene.Balb/C mice were intramuscularly inoculated with the DNA plasmid which expressed the target gene correctly,and the antibody level in mice was detected by the means of ELISA and serum neutralization （SN）.[Result] DNA plasmid carrying P1 gene which encodes FMDV capsid protein caused specific body fluid immunoreaction in mice,and the antibody level of anti-FMDV had no difference in the mice induced by the two recombinant plasmids.[Conclusion] This study lays a foundation for evaluating the genetically modified vaccine by immunizing animals with recombinant PRV containing the FMDV P1 gene and recombinant virus.