Retroviral shuttle vectors as a tool for the study of mutational specificity (base substitution/deletion/mutational hotspot)

This paper summarizes the use of the retroviral shuttle vector pZipGptNeo for studies of mutational specificity in mammalian cells. This vector was constructed by the introduction of a DNA fragment containing the E. coli gpt gene into the retroviral shuttle vector pZipNeoSV(X)1. The pZipGptNeo vector was then introduced into mouse L cells to construct the A9I2 cell line. Studies utilizing the A9I2 cell line to determine the specificity of spontaneous and chemically-induced mutations are summarized. The construction of a new retroviral shuttle vector and its introduction into the CHO-K1 cell line is described. Preliminary experiments suggest that spontaneous gpt gene mutations arising in CHO cells are similar to those seen in the mouse L cells.     

乙型肝炎病毒S基因与截短C基因融合的穿梭表达载体的构建

研究目的是构建HBVS基因和截短C基因融合的胞壁型和分泌型大肠杆菌和分枝杆菌(E．coli-BCG)穿梭质粒。采用PCR方法从结核分枝杆菌MTB毒株H37Rv的基因中扩增出相对分子质量为19000的抗原胞壁区及其上游调控元件基因,克隆入穿梭载体pOLYG中。以含HBV基因组的质粒pCP10序列为模板,PCR扩增获得5基因片段Sw和C基因编码氨基端的部分基因片段Ct,克隆入胞壁型穿梭表达载体pCW和分泌型穿梭表达载体pDE22。经酶切和序列测定证实,胞壁型和分泌型载体pCW-Sw-Ct和pDE22-Sw-Ct构建成功。为进一步研究含S基因和截短C基因融合的重组BCG活疫苗奠定了基础。     

Construction of a new high-copy number shuttle vector of Bacillus thuringiensis

AIMS: Construction and characterization of a new cloning shuttle vector for gene transfer and expression in Bacillus thuringiensis. METHODS AND RESULTS: A novel short and high-copy number shuttle vector called pHBLBIV, was constructed for gene transfer and expression in Bacillus thuringiensis. A 1.6-kbp replicon of a relatively high-copy number endogenous plasmid of a selected B. thuringiensis strain was ligated to Escherichia coli pUC18 replicon containing the ampicillin and the erythromycin resistance genes used for the selection of respectively E. coli and B. thuringiensis transformants. The constructed vector was shown to have a high copy number compared with the conventional B. thuringiensis vectors, and used successfully for the transfer of vegetative insecticidal protein-encoding gene (vip) in between B. thuringiensis strains. CONCLUSIONS: A new shuttle vector of B. thuringiensis-E. coli named pHBLBIV was constructed. It was characterized by its high copy number, small size and segregational stability. This vector was successfully used for vip gene cloning and transfer in B. thuringiensis. SIGNIFICANCE AND IMPACT OF THE STUDY: A novel shuttle vector has been constructed, which has demonstrated potential for the cloning and expression of genes in B. thuringiensis.     

利用枯草芽孢杆菌ytkA和ywoF基因启动子与信号肽重组分泌表达mpd基因

用大肠杆菌-枯草芽孢杆菌穿梭载体pNW33N和去除了信号肽编码序列的成熟mpd基因构建了穿梭启动子探针pNW33N-mpd。用该探针从质粒pMPDP3和pMPDP29上克隆来自于枯草芽孢杆菌ytkA和ywoF基因上游的启动子功能片段,构建了穿梭表达载体pNYTM和pNYWM。将表达载体pNYTM和pNYWM转入枯草芽孢杆菌1A751获得表达菌株1A751(pNYTM)和1A751(pNYTM),mpd基因在ytkA和ywoF基因的启动子和信号肽的带动下实现了分泌表达且具有天然活性,结果表明ytkA基因的启动子强度强于ywoF基因的启动子。利用ytkA基因的强启动子和nprB基因的分泌型信号肽编码序列构建了新的穿梭分泌表达载体pYNMK,并使mpd基因在枯草芽孢杆菌WB800中得到了更高水平的分泌表达,表达菌株WB800(pYNMK)在培养到第84h时甲基对硫磷水解酶酶活达到最高值为10.40u/mL,是出发菌株邻单胞菌M6表达量的10.8倍,重组表达产物有91.4%分泌在培养基中。     

大肠杆菌-双歧杆菌穿梭表达载体的构建及内皮抑素基因的表达

目的:构建大肠杆菌-长双歧杆菌穿梭表达载体,并通过此载体使人内皮抑素基因在大肠杆菌和长双歧杆菌中得到表达。方法:以质粒pDG7、pBCSK( )、pET-9C为基础,构建大肠杆菌-长双歧杆菌穿梭表达载体pET-1128,并将人内皮抑素基因插入到新构建的表达载体中,分别转化大肠杆菌BL21(DE3)和长双歧杆菌NQ-1501。诱导表达,表达产物经SDS-PAGE和WesternBlot鉴定。结果:成功构建了大肠杆菌-双歧杆菌穿梭载体,人内皮抑素基因在大肠杆菌和长双歧杆菌中均可表达。结论:构建的穿梭载体为今后用双歧杆菌作为生理菌载体进行肿瘤的基因治疗奠定了基础。     

昆虫杆状病毒系统表达口蹄疫病毒3ABC基因

以O型口蹄疫病毒为研究对象,经过RTPCP扩增得到非结构蛋白3ABC基因,克隆到转移载体pFastbacHT,将其转入含穿梭载体Bacmid的DH10Bac,与Bacmid发生位点特异性转座作用,得到3ABC的重组穿梭载体Bacmid3ABC,再将其转染昆虫细胞HiFive。PCR鉴定证实3ABC基因正确地插入到病毒基因组的多角体蛋白基因启动子下游,经过SDSPAGE和Westernblot检测,3ABC基因在昆虫细胞中表达了大小约为50kDa的蛋白条带,3ABC基因在BactoBac系统中的成功表达为建立以基因工程产品为抗原、鉴别诊断自然感染和免疫动物的方法提供了技术条件。     

携带PTEN基因的重组腺病毒表达载体构建的研究

构建携带抑癌基因PTEN(Phosphatase and temin homolog deleted on chromosome ten)的重组腺病毒表达裁体,为研究PTEN的功能和作用机制奠定基础.采用RT-PCR法从大鼠海马神经元扩增目的基因PTEN,克隆人含绿色荧光蛋白(Green fluorescence protein),GFP基因的pAdTrack-CMV穿梭质粒,在含有腺病毒骨架质粒pAdEasy-1的BJ5183大肠杆菌内进行同源重组;获得重组腺病毒质粒,经Pacl线性化后,转染AD293细胞.结果表明,感染腺病毒载体的AD293细胞表达GFP基因,随着时间逐渐增强,并且出现明显的细胞病变效应(Cytopathic effect,CPE),经PCR对传代的Ad-PTEN分析证实得到目的基因.成功构建了携带PTEN基因的腺病毒表达载体,为研究PTEN的功能和作用机制奠定了基础.     

抗辐射菌属-大肠杆菌间的穿梭载体的构建及荧光基因的表达

摘要：【目的】构建抗辐射菌属一大肠杆菌间的穿梭载体,通过此载体使荧光素酶基因在大肠杆菌中得到表达。【方法】以质粒pUE30、pGBM5及pKatCAT为基础,构建抗辐射菌属一大肠杆菌间的穿梭载体,将groEL启动子和荧光素酶基因lux+插入到构建的穿梭载体中得到穿梭表达载体,并将该载体转化大肠杆菌诱导荧光素酶基因的表达。【结果】成功构建了大小约为5.8 kb的抗辐射菌属一大肠杆菌间的穿梭载体pZT17,该载体在没有抗生素的非选择性培养基中能稳定存在。在穿梭载体pZT17的EcoRV部位插入含有groEL启动子和荧光素酶基因lux+的DNA片段,构建得到了穿梭表达载体pZTGL2;利用该表达载体在大肠杆菌中可诱导表达荧光素酶基因。【结论】构建的穿梭表达载体为以后用大肠杆菌高效表达来源于抗辐射菌的基因、特别是DNA损伤修复蛋白基因,提供了可能。     

GFP为报告基因的酵母哺乳动物细胞穿梭载体的构建及其在人血管内皮细胞中的表达

为研究酵母作为载体在口服基因治疗及免疫中的作用 ,需要一种能够在酵母中复制而在哺乳动物细胞中表达的穿梭载体 .利用通用载体质粒融合系统 (UPS)构建了一种以GFP为报告基因的新载体 ,以常规的氯化锂法对酿酒酵母进行转化 ,证明该载体能够在酵母中复制 ;以脂质体介导向人血管内皮细胞进行了转染 ,有绿色荧光 ,证明该载体能够在哺乳动物细胞中表达 .所获得的新型的穿梭载体为口服酵母在基因治疗中的应用提供了物质准备     

Ethylnitrosourea-induced mutation and molecular analysis of transgenic mice containing the gpt shuttle vector

Novel transgenic mice were developed in order to study the in vivo mutagenesis. The transgenic mice carried pCGK shuttle vector, which contained the Escherichia coli gpt gene as a mutational target, the kanamycin-resistant gene (Kanr) and cos region derived from bacteriophage lambda. The shuttle vector can be recovered from the transgenic mouse genome into the gpt-deficient E. coli by an in vitro packaging method and is selectable as a Kanr phenotype. Mutations induced at the gpt gene can be easily detected with a selective agent, 6-thioguanine (6-TG). In the previous study, the pCGK shuttle vector was incorporated into Chinese hamster CHL/IU cells and the resultant transgenic cell line was shown to be a useful system to study in vitro mutagenesis at the gpt gene. Therefore, an advantage of the shuttle vector is that in vivo mutational data obtained from the transgenic mouse can be compared with those of transgenic cell line in vitro. A transgenic CD-1 mouse line, designated as #128, that carried approximately 50 copies of pCGK shuttle vectors, was selected among 4 transgenic mouse lines. To investigate the sensitivity of the #128 line, the transgenic mice were treated with a single intraperitoneal injection of 250 mg/kg of N-ethyl-N-nitrosourea (ENU) or with 50 mg kg-1 day-1 of ENU for 5 consecutive days, and bone marrow, spleen and liver were dissected to investigate their mutational responses. The background mutant frequency was between 18x10(-6) and 75x10(-6) among all tissues tested. ENU induced significant increases in the mutant frequency above the background level in all three tissues at 14 days after single or 5-day treatment with the chemical. The increases in the mutant frequencies in bone marrow, spleen and liver were 6.4- to 6.8-fold, 3.0- to 5.6-fold and 3.0- to 3.3-fold, respectively. The shuttle vector DNA was recovered from the bone marrow of both spontaneous and ENU-treated mice and the gpt gene was amplified by polymerase chain reaction. The amplified DNA was subject to DNA sequence analysis. Out of 79 spontaneous and 52 ENU-induced mutants, the gpt gene could be amplified from 28 spontaneous and 46 ENU-induced mutants. DNA sequence analysis showed that predominant mutations were identified as A:T to T:A transversions (22 out of 46 sequenced mutants) and G:C to A:T transitions (9/46) in ENU-induced mutants, whereas G:C to T:A transversions (7 out of 28 sequenced mutants) were predominant in spontaneous mutants. These results demonstrate that this transgenic mouse, in combination with the transgenic CHL/IU cell line, is a useful system to study in vivo and in vitro mutational events at the same target gene.     

Shuttle vector plasmid propagation in human peripheral blood lymphocytes facilitated by liposome-mediated transfection

Transfection of human peripheral blood lymphocytes facilitated by a positively charged liposome preparation (Lipofectin, BRL) is 100-fold more efficient than the DEAE dextran technique for the uptake and replication of shuttle vector plasmid DNA. The yield of progeny plasmids obtained from 10 ml of blood was high enough for mutational analysis. A marked increase in the mutation frequency of the shuttle vector marker gene was noted in response to the induction of psoralen adducts in the vector. By using normal human lymphocytes, this method will permit shuttle vector analysis of DNA repair and mutagenesis in a large number of individuals. This method could also prove useful for studies of human lymphotropic viruses.     

Construction of a Helicobacter pylori-Escherichia coli shuttle vector for gene transfer in Helicobacter pylori.

In this study, a Helicobacter pylori-Escherichia coli shuttle vector was constructed for transferring DNA into H. pylori. The smallest cryptic plasmid (1.2 kb), pHP489, among those harbored by 77 H. pylori isolates was selected as a base replicon for constructing vectors. HindIII-digested pHP489 was ligated with a kanamycin resistance gene [aph(3')-III], which originated from Campylobacter jejuni, to produce the recombinant plasmid pHP489K. pHP489K was efficiently transformed into and stably maintained in H. pylori strains. The shuttle vector pBHP489K (3.6 kb) was constructed by the recombination of pHP489, ColE1, and aph(3')-III sequences. pBHP489K was reciprocally transformed into and maintained in both H. pylori and E. coli. Introduction of the shuttle vector clone DNA (pBHP489K/AB; 6.7 kb), containing the ureA and ureB genes of H. pylori, into urease-negative mutants of H. pylori led to the restoration of their urease activity. The transformants were confirmed to contain the incoming plasmid DNA. pBHP489K satisfied the requirements for an H. pylori-E. coli shuttle vector, implying that it might be a useful vector for investigating pathogenicity and restriction-modification systems of H. pylori.     

谷氨酸棒杆菌/大肠杆菌穿梭型启动子探测载体构建

从质粒pXZ10145和pUC19出发,构建了一个谷氨酸棒杆菌/大肠杆菌穿梭载体pAK6。pAK6的大小为5684bp,带有卡那霉素和氨苄青霉素抗性选择标记,以及多克隆位点。在pAK6基础上,构建了以氯霉素乙酰转移酶为报告基因的启动子探测载体pAKC6,pAKC6的大小为6474bp。采用鸟枪法,将经Sau3AI消化的谷氨酸棒杆菌基因组片段连入pAKC6;根据谷氨酸棒杆菌对氯霉素的抗性,从中分离出两个具有启动子功能的插入片段。通过测定报告基因氯霉素乙酰转移酶的活性,对两个启动子片段在谷氨酸棒杆菌中的强度进行了初步的判断;测序后,用启动子预测软件对其结构进行了预测,证实了启动子序列的存在。     

Construction of an EBV-derived shuttle vector for studying the influence of transcription on mutagenesis

We have constructed an EBV-derived shuttle vector, pF1-EBV, which replicates in human cells as an extrachromosomal element. The structural sequences of the gene encoding the bacterial xanthine-guanine-phosphoribosyltransferase (gpt) were fused to the promoter and presumptive control region of the mouse metallothionein I (MT-I) gene. Human 293 cells transformed with the recombinant plasmid synthesized gpt mRNA and the expression of the gene was inducible by zinc. The gpt gene offers a convenient system of selection for mutant plasmids by transformation into the appropriate gpt- E. coli strain. A clonal cell line created by establishment of the pF1-EBV shuttle vector showed a spontaneous gpt- frequency of 2.10(-5). An increase in mutation frequency above background was induced by mutagenizing this cell line with the alkylating agent N-methyl-N-nitrosourea (MNU). The recombinant molecule that we have constructed should provide a tool for studying the role of gene expression in DNA repair and mutagenesis.     

Construction and evaluation of shuttle vector, pGYC4α, based on pYC2 from Lactobacillus sakei

The shuttle vector pGYC4α (6,157 bp) was constructed based on the sigma-replicon plasmid pYC2 from Lactobacillus sakei BM5 isolated from kimchi. The vector contained inserts of the ColE1 replicon, α-amylase gene from Bacillus licheniformis containing its own signal peptide, and lactococcal promoter P32. Transformation and expression of a selection marker gene (α-amylase) with pGYC4α were demonstrated in Escherichia coli and several lactic acid bacteria (LAB). The highest α-amylase activity in LAB transformants was obtained in M17/0.25% glucose media with 0.5% CaCO(3). The segregational stability of the shuttle vector in LAB was 100% for more than 100 generations in the absence of antibiotic pressure. The developed vector might be useful as a genetic tool for food industries.     

Construction of an inducible expression shuttle vector for Laribacter hongkongensis, a novel bacterium associated with gastroenteritis

An Escherichia coli-Laribacter hongkongensis shuttle vector (pPW380) was constructed by ligating the 4701-bp EcoRI digested fragment of pHLHK8 to EcoRI digested pBK-CMV. An E. coli-L. hongkongensis inducible expression shuttle vector was further constructed by ligating a 2105-bp fragment that contains the tetracycline repressor and tetracycline-inducible promoter region of pALC2084 to the 8897-bp fragment of pPW380, deletion of the green fluorescent protein gene, and insertion of a multiple cloning site. This inducible expression system was able to express two commonly used reporter genes, the green fluorescent protein gene and the glutathione S-transferase gene, efficiently in E. coli and L. hongkongensis.     

Site-specific gene modification by PNAs conjugated to psoralen

DNA-binding molecules, including triplex-forming oligonucleotides (TFOs) and peptide nucleic acids (PNAs), can be utilized to introduce site-specific mutations or to promote recombination at selected genomic sites. To further evaluate the utility of PNAs for site-specific gene modification, we tested dimeric bis-PNAs conjugated to psoralen. These PNAs are designed to form a triplex-invasion complex within the supF reporter gene in an episomal shuttle vector and to direct site-specific photoadduct formation by the conjugated psoralen. The psoralen-bis-PNA conjugate was found to direct photoadduct formation to the intended 5'-TpA base step next to the PNA-binding site, and the photoadduct formation efficiency displayed both concentration and UVA irradiation dependence. The effect of PNA-targeted photoadducts in a mammalian system was tested by SV40-based shuttle vector assay. After in vitro binding, we found that photoadducts directed by PNAs conjugated to psoralen-induced mutations at frequencies in the range of 0.46%, 6.5-fold above the background. In a protocol for intracellular gene targeting in the episomal shuttle vector, the psoralen-PNA-induced mutation frequency was 0.13%, 3.5-fold higher than the background. Most of the induced mutations were deletions and single-base-pair substitutions at or adjacent to the targeted PNA-binding and photoadduct-formation sites. When the results are taken together, they demonstrate the ability of bis-PNAs conjugated with psoralen to mediate site-specific gene modification, and they further support the development of PNAs as tools for gene-targeting applications.     

Recombination between irradiated shuttle vector DNA and chromosomal DNA in African green monkey kidney cells.

An autonomously replicating shuttle vector was used to investigate enhancement of plasmid-chromosome recombination in mammalian host cells by gamma irradiation and UV light. Sequences homologous to the shuttle vector were stably inserted into the genome of African green monkey kidney cells to act as the target substrate for these recombination events. The shuttle vector molecules were irradiated at various doses before transfection into the mammalian host cells that contained the stable insertions. The homologous transfer of the bacterial ampicillin resistance gene from the inserted sequences to replace a mutant ampicillin sensitivity gene on the shuttle vector was identified by the recovery of ampicillin-resistant plasmids after Hirt extraction and transformation into Escherichia coli host cells. Gamma irradiation increased homologous shuttle vector-chromosome recombination, whereas UV light did not increase the frequency of recombinant plasmids detected. Introducing specific double-strand breaks in the plasmid or prolonging the time of plasmid residence in the mammalian host cells also enhanced plasmid-chromosome recombination. In contrast, plasmid mutagenesis was increased by UV irradiation of the plasmid but did not change with time. The ampicillin-resistant recombinant plasmid molecules analyzed appeared to rise mostly from nonconservative exchanges that involved both homologous and possibly nonhomologous interactions with the host chromosome. The observation that similar recombinant structures were obtained from all the plasmid treatments and host cells used suggests a common mechanism for plasmid-chromosome recombination in these mammalian cells.     

整合型碱性蛋白酶基因工程菌中抗性基因的敲除*

要：利用大肠杆菌载体pET—韶a和穿梭载体PHY3肋队构建了敲除载体p10c,通过DP4A变性技术和同源重组技术成功地敲除了整合型碱性蛋白酶基因工程茵BP旧1中的卡那霉素抗性基因(M),得到11株敲除卡那霉素抗性基因的阳性克隆,并使产酶水平保持稳定。该方法的建立为基因敲除技术在工业微生物研究中应用提供了经验,并为微生物来源的转基因产品安全性的研究提供了模型。