Kernel hierarchical gene clustering from microarray expression data

MOTIVATION: Unsupervised analysis of microarray gene expression data attempts to find biologically significant patterns within a given collection of expression measurements. For example, hierarchical clustering can be applied to expression profiles of genes across multiple experiments, identifying groups of genes that share similar expression profiles. Previous work using the support vector machine supervised learning algorithm with microarray data suggests that higher-order features, such as pairwise and tertiary correlations across multiple experiments, may provide significant benefit in learning to recognize classes of co-expressed genes. RESULTS: We describe a generalization of the hierarchical clustering algorithm that efficiently incorporates these higher-order features by using a kernel function to map the data into a high-dimensional feature space. We then evaluate the utility of the kernel hierarchical clustering algorithm using both internal and external validation. The experiments demonstrate that the kernel representation itself is insufficient to provide improved clustering performance. We conclude that mapping gene expression data into a high-dimensional feature space is only a good idea when combined with a learning algorithm, such as the support vector machine that does not suffer from the curse of dimensionality. AVAILABILITY: Supplementary data at www.cs.columbia.edu/compbio/hiclust. Software source code available by request.     

Rapid establishment of high-producing cell lines using dicistronic vectors with glutamine synthetase as the selection marker

Recombinant proteins are useful tools in biological research, drug development, and drug screening. Specially designed expression vectors have been developed to introduce cDNA for recombinant protein expression in mammalian cells. We have combined a discistronic mRNA design for expression of the recombinant protein, using glutamine synthetase (GS) for selection. A soluble form of human interleukin-4 receptor alpha chain was used as the model protein. The dicistronic vectors were compared to a standard expression vector in CHO-K1 cells in parallel experiments. Our data showed that a dicistronic vector containing an internal ribosome entry site (IRES) of the encephalomyocarditis virus (ECMV) was superior to a conventional expression vector in both levels of protein expression and amplification efficiency. The productivity of these clones was stable without selection pressure for an extended period of time. The GS selection system within a dicistronic vector design can achieve rapid and efficient gene amplification for protein production.     

High-frequency transformation of human repair-deficient cell lines by an Epstein-Barr virus-based cDNA expression vector.

We constructed a human cDNA expression vector by combining an episomal Epstein-Barr virus (EBV) vector with the expression cassette from the transient-expression vector, pCDM8. This new vector, designated pEBS7, exhibited high-level expression of reporter genes in normal and repair-deficient xeroderma pigmentosum cell lines. Reconstruction experiments indicated that marker genes diluted to a frequency of 10(-5) can be rescued on a single transfection dish. Moreover, derivative cell lines that constitutively express the gene encoding EBV nuclear antigen 1 exhibited a tenfold enhancement in the frequency of rescue of marker genes. The feasibility of preparing large-scale directional or nondirectional cDNA libraries in pEBS7 was demonstrated and reconstruction experiments indicated that marker genes could be rescued from either library with equal efficiency. These results establish a high-efficiency system for the isolation of genes by direct phenotypic selection in human mutant cell lines.     

Human constitutive androstane receptor mediates induction of CYP2B6 gene expression by phenytoin

Compared with its rodent orthologs, little is known about the chemical specificity of human constitutive androstane receptor (hCAR) and its regulation of hepatic enzyme expression. Phenytoin (PHY), a widely used antiepileptic drug, is a potent inducer of CYP2B6 in primary human hepatocytes, but does not activate human pregnane X receptor (PXR) significantly in cell-based transfection assays at the same concentrations associated with potent induction of CYP2B6. Based on this observation, we hypothesized that PHY may be a selective activator of hCAR. In primary human hepatocytes, expression of CYP2B6 reporter genes containing phenobarbital-responsive enhancer module (PBREM) or PBREM/xenobiotic-responsive enhancer module (XREM) response elements were activated up to 14- and 28-fold, respectively, by 50 microm PHY. By contrast, parallel experiments in HepG2 cell lines co-transfected with an hPXR expression vector did not show increased reporter activity. These results indicated that a PXR-independent pathway, which is retained in primary hepatocytes, is responsible for PHY induction of CYP2B6. Further experiments revealed that PHY effectively translocates hCAR from the cytoplasm into the nucleus in both primary human hepatocytes and CAR(-/-) mice. Compared with vehicle controls, PHY administration significantly increased CYP2B6 reporter gene expression, when this reporter construct was delivered together with hCAR expression vector into CAR(-/-) mice. However, PHY did not increase reporter gene expression in CAR(-/-) mice in the absence of hCAR vector, implying that CAR is essential for mediating PHY induction of CYP2B6 gene expression. Taken together, these observations demonstrate that, in contrast to most of the known CYP2B6 inducers, PHY is a selective activator of CAR in humans.     

Tryptophan promoter derivatives on multicopy plasmids: a comparative analysis of expression potentials in Escherichia coli

A collection of variant plasmids expressing either Escherichia coli galactokinase or human serum albumin under the control of several E. coli trp promoter derivatives were constructed and studied for both efficiency of expression and regulation by tryptophan. Several variables, including the length of the upstream region, tandem duplications of a core promoter, and the insertion of the trp repressor trpR gene onto the expression vector, were studied. It is shown that derivatives containing sequences upstream from the -35 region or multiple copies of the trp promoter produce twofold higher levels of protein than plasmids with a minimal trp promoter truncated at -40. We show that the expression of a heterologous protein such as albumin can be significantly improved (13% vs. 7% of total proteins) if both the upstream trp promoter region, which enhances promoter strength, and an intact trpR gene, are included on the plasmids.     

基于逆转录病毒载体的外源基因表达系统的评价和应用

逆转录病毒表达系统是基因治疗研究和RNA干扰技术广泛采用的外源基因表达系统。文中以增强型绿色荧光蛋白 (EGFP) 基因的表达水平和稳定性为指标,比较逆转录病毒表达载体pQCXIN和pcDNA3.1(+) 表达质粒介导的外源基因在HEK293细胞和CHO-K1细胞的表达效率。病毒感染HEK293细胞和CHO-K1细胞的相对荧光强度 (Relative fluorescence intensity,RFI) 均约为对应的质粒转染细胞的2倍。多轮反复感染逆转录病毒表达载体能有效提高HEK293细胞表达EGFP的效率。HEK293细胞经4轮病毒感染后的RFI值较1次病毒感染HEK293细胞的RFI值约提高2倍。此外,逆转录病毒表达载体介导的外源基因表达的稳定性优于质粒转染的外源基因表达。采用携带人重组活性蛋白C (Recombinant human activated protein C,rhAPC) 基因的pQCXIN和HEK293细胞进一步验证了逆转录病毒载体介导的外源基因表达效率,构建了rhAPC表达水平为10~15 mg/(106 cells·d) 的HEK293细胞系。研究结果表明,逆转录病毒表达系统是有应用价值的介导外源基因在哺乳动物细胞高效表达的技术途径。     

Manipulation of OCT4 levels in human embryonic stem cells results in induction of differential cell types

To fully understand self-renewal and pluripotency and their regulation in human embryonic stem cells (hESCs), it is necessary to generate genetically modified cells and analyze the consequences of elevated and reduced expression of genes. Genes expressed in hESCs using plasmid vectors, however, are subject to silencing. Moreover, hESCs have a low plating efficiency when dissociated to single cells, making creation of subcloned lines inefficient. In addition to overexpression experiments, it is important to perform loss-of-function studies, which can be achieved rapidly using RNA interference (RNAi). We report stable long-term expression of enhanced green fluorescent protein (eGFP) in hESCs using a lentiviral vector, and establishment of an eGFP-expressing subline (RG6) using manual dissection. To demonstrate the efficacy of RNAi in hESCs, an RNAi expression vector was used to achieve reduced expression of eGFP in hESCs. To evaluate the role of OCT4 in the regulation of hESC self-renewal and differentiation, a vector expressing a hairpin RNA targeting endogenous expression of OCT4 was constructed. In a novel experiment in hESCs, the OCT4 cDNA sequence was cloned into an expression vector to allow for the transient upregulation of OCT4 in hESCs. The ability to manipulate levels of OCT4 above and below enodogenous levels allows the determination of OCT4 function in hESCs. Specifically, reduced expression of OCT4 in hESCs promoted upregulation of markers indicative of mesoderm and endoderm differentiation, and elevated levels of OCT4 in hESCs promoted upregulation of markers indicative of endoderm derivatives. Thus, both upregulation and downregulation of Oct4 in hESCs results in differentiation, but with patterns distinct from parallel experiments in mice.     

Complete characterization of the gene coding for the Epstein-Barr virus major membrane antigen gp 220/340 and selective expression of a secreted form of gp 220

The gene which encodes the Epstein-Barr gp 220/340 was inserted into a eukaryotic expression vector. A cDNA clone corresponding to the mature mRNA coding for gp 220 was isolated from an Epstein-Barr virus cDNA library and inserted in the same expression vector, enabling us to identify the precise location of the intron within the gp 220/340 coding sequence. The recombinant plasmids direct the expression of membrane proteins detected by immunofluorescence experiments using an anti-gp 220/340 monoclonal antibody in transfected human cells. The region of the gp 220/340 gene encoding the domain for membrane anchorage was removed from the two recombinant plasmids and the sequence containing the intron produced secreted forms of both truncated gp 220 and gp 340 whereas only the former was obtained with the intronless sequence.     

Effects of pCIneo and pCEP4 expression vectors on transient and stable protein production in human and simian cell lines

To support and meet the demand for recombinant proteins early in the drug discovery process, much work has been directed toward improving the methods used for transient gene transfection and expression. A factor which could potentially affect the outcome of experiments is the choice of the expression vector. Conventional vectors such as pCIneo and pcDNA3 have been used frequently. Each of these places the gene of interest under the control of the CMV promoter. An interesting alternative is provided by episomal vectors. For example, the pCEP4 vector contains the gene coding for the Epstein Barr nuclear antigen as well as the EBNA ori P sequence. This combination allows for the episomal replication of the plasmid. In preliminary experiments, we compared transient secreted placental alkaline phosphatase production in 8 cell lines from 3 different species using the pCIneo vs. pCEP4 vectors and found the utility of the pCEP4 vector to be limited to the human 293 EBNA cell line. In this paper, we have compared the two vectors in six cell lines of simian and human origin, measuring the transient production of secreted placental alkaline phosphatase and human hepatocyte growth factor. In general, the pCEP4 vector produced higher amounts of both proteins in transient transfections. Results were particularly pronounced in the HEK 293 and 293 EBNA cell lines. Stable pools of cells (uncloned) expressing human hepatocyte growth factor were isolated using pCIneo and pCEP4 and protein production levels were compared to those seen in transient transfections. Stable expression with pCEP4 was found to produce the highest levels of human hepatocyte growth factor in 3 of 4 cell lines. Finally, electroporation and FuGENE^TM6(Roche, Indianapolis IN) as transfection methods were compared measuring transient production of secreted placental alkaline phosphatase, human hepatocyte growth factor, and green fluorescent protein. FuGENE produced higher protein concentrations in less time than electroporation for all 3 proteins. This revised version was published online in July 2006 with corrections to the Cover Date.     

Adeno-associated virus type 2-mediated transduction of murine hematopoietic cells with long-term repopulating ability and sustained expression of a human globin gene in vivo.

Adeno-associated virus type 2 (AAV), a nonpathogenic human parvovirus, is gaining attention as a vector for potential use in human gene therapy. We and others have described AAV-mediated beta-globin gene transfer and expression in established human and murine erythroleukemia cell lines in vitro. However, successful AAV-mediated globin gene transduction of hematopoietic stem cells and long-term expression in vivo in progeny cells have not been documented. We report here that infection of murine hematopoietic bone marrow cells ex vivo with a recombinant AAV vector containing the genomic copy of a normal human globin gene followed by transplantation of these cells into lethally irradiated congenic mice resulted in efficient gene transfer into hematopoietic cells with long-term repopulating ability as detected by the presence of the human globin gene sequences in bone marrow and spleen in primary recipient mice for at least 6 months. Long-term expression of the human globin gene was also detected in bone marrow, but not in spleen, in primary recipient mice. Furthermore, in secondary-transplant experiments, we were also able to document the presence as well as expression of the transduced human globin gene in mouse bone marrow for up to 3 months. These results provide further support for potential use of the AAV-based vector system in gene therapy of human hemoglobinopathies in general and sickle-cell anemia and beta-thalassemia in particular.     

High-throughput screening for soluble recombinant expressed kinases in Escherichia coli and insect cells

We have constructed a dual expression vector for the production of recombinant proteins in both Escherichia coli and insect cells. In this vector, the baculoviral polyhedrin promoter was positioned upstream of the bacteriophage T7 promoter and the lac operator. This vector, designated pBEV, was specifically designed to exploit the advantages that both hosts would provide. This vector also facilitates one-stop cloning, thereby simplifying the expression process for automation, and the development of a high-throughput method for protein expression. Utilizing the multi-system vector pBEV, a high-throughput process was developed with expression in deep-well blocks and purification in micro-titer plates enabling the identification of expression and solubility in both E. coli and insect cells. In this study, using pBEV, we have successfully expressed and purified multiple human kinases produced in E. coli and insect cells. Our results validate expression screening as a strategy to rapidly triage proteins identifying the optimum expression system and conditions for production.     

Functional expression and display of an antibody Fab fragment in Escherichia coli: study of vector designs and culture conditions

Several different vector designs are currently being used to display and express Fab molecules in Escherichia coli, but their relative efficiency in phage display and protein expression cannot be compared from the published data. We systematically investigated which vector design most effectively displays and expresses Fab molecules in E. coli using, as a model system, a human Fab against tetanus toxoid (tt). Three different vectors were used in this study: pFab1 where the VL-CL and VH-CH1 genes were driven by two promoters in two separate expression cassettes, and pFab2 and pFab3 that both contain one dicistronic expression cassette with two translation initiation sites and either VH-CH1 before VL-CL or VL-CL before VH-CH1, respectively. The display of tt-Fab on the surface of phage and the expression of tt-Fab protein in E. coli were compared for the aforementioned vectors. Our results showed that the pFab3 vector was most effective in Fab display. A 10-fold increase in the expression of secreted Fab was observed in pFab3 when compared with vectors pFab1 and pFab2. Further experiments were conducted using pFab3 to optimize expression levels using different strains of E. coli and various culture conditions. The highest expression of tt-Fab was obtained using the pFab3 vector in host strain JM105 with an induction temperature at 37 degrees C and IPTG concentration of 0.1 mM.     

Cyr61, a member of CCN family, is a tumor suppressor in non-small cell lung cancer

Cysteine-rich protein 61 (Cyr61) is a member of a family of growth factor-inducible immediate-early genes. It regulates cell adhesion, migration, proliferation, and differentiation and is involved in tumor growth. In our experiments, the role of Cyr61 in non-small cell lung cancer (NSCLC) was examined. Expression of Cyr61 mRNA was decreased markedly in four of five human lung tumor samples compared with their normal matched lung samples. NSCLC cell lines NCI-H520 and H460, which have no endogenous Cyr61, formed 60-90% fewer colonies after being transfected with a Cyr61 cDNA expression vector than cells transfected with the same amount of empty vector. After stable transfection of a Cyr61 cDNA expression vector, proliferation of both H520-Cyr61 and H460-Cyr61 sublines decreased remarkably compared with the cells stably transfected with empty vector. The addition of antibody against Cyr61 partially rescued the growth suppression of both H520-Cyr61 and H460-Cyr61 cells. Cell cycle analysis revealed that both H520-Cyr61 and H460-Cyr61 cells developed G(1) arrest, prominently up-regulated expression of p53 and p21(WAF1), and had decreased activity of cyclin-dependent kinase 2. The increase of pocket protein pRB2/p130 was also detected in these cells. Notably, both of the Cyr61-stably transfected lung cancer cell lines developed smaller tumors than those formed by the wild-type cells in nude mice. Taken together, we conclude that Cyr61 may play a role as a tumor suppressor in NSCLC.     

Efficient gene transfer by fiber-mutant adenoviral vectors containing RGD peptide

One of the hurdles to adenovirus (Ad)-mediated gene transfer is that Ad vectors mediate inefficient gene transfer into cells lacking in the primary receptors, Coxsackievirus and adenovirus receptor (CAR). We previously developed a fiber-mutant Ad vector containing the Arg-Gly-Asp (RGD)-containing peptide motif on the HI loop of the fiber knob, and showed that the mutant vector had enhanced gene transfer activity to human glioma cells, which showed little CAR expression, compared to the vector containing wild type fiber. In this study, the feasibility of the Ad vector containing RGD peptide on the fiber knob was examined in a wide variety of cell types: CAR-positive or -negative human tumor cells, mouse cells, and leukemia cells. The mutant vector infected the cells, which lacked CAR expression but showed αv integrin expression, about 10–1000 times more efficiently than the vector containing wild type fiber via an RGD-integrin (αvβ3 and αvβ5)-dependent, CAR-independent cell entry pathway. The results of this study indicate that Ad vector containing RGD peptide on the fiber knob could be of great utility for gene therapy and gene transfer experiments.     

Construction of a defective retrovirus containing the human hypoxanthine phosphoribosyltransferase cDNA and its expression in cultured cells and mouse bone marrow.

Defective ecotropic and amphotropic retroviral vectors containing the cDNA for human hypoxanthine phosphoribosyltransferase (HPRT) were developed for efficient gene transfer and high-level cellular expression of HPRT. Helper cell clones which produced a high viral titer were generated by a simplified method which minimizes cell culture. We used the pZIP-NeoSV(X) vector containing a human hprt cDNA. Viral titers (1 X 10(3) to 5 X 10(4)/ml) of defective SVX HPRT B, a vector containing both the hprt and neo genes, were increased 3- to 10-fold by cocultivation of the ecotropic psi 2 and amphotropic PA-12 helper cells. Higher viral titers (8 X 10(5) to 7.5 X 10(6] were obtained when nonproducer NIH 3T3 cells or psi 2 cells carrying a single copy of SVX HPRT B were either transfected or infected by Moloney leukemia virus. The SVX HPRT B defective virus partially corrected the HPRT deficiency (4 to 56% of normal) of cultured rodent and human Lesch-Nyhan cells. However, instability of HPRT expression was detected in several infected clones. In these unstable variants, both retention and loss of the SVX HPRT B sequences were observed. In the former category, cells which became HPRT- (6-thioguanine resistant [6TGr]) also became G418s, indicative of a cis-acting down regulation of expression. Both hypoxanthine-aminopterin-thymidine resistance (HATr) and G418r could be regained by counterselection in hypoxanthine-aminopterin-thymidine. In vitro mouse bone marrow experiments indicated low-level expression of the neo gene in in vitro CFU assays. Individual CFU were isolated and pooled, and the human hprt gene was shown to be expressed. These studies demonstrated the applicability of vectors like SVX HPRT B for high-titer production of defective retroviruses required for hematopoietic gene transfer and expression.     

A multisite gateway-based toolkit for targeted gene expression and hairpin RNA silencing in tomato fruits

A collection of fruit promoters, reporter genes and protein tags has been constructed in a triple-gateway format, a recombination-based cloning system that facilitates the tandem assembly of three DNA fragments into plant expression vectors. The new pENFRUIT collection includes, among others, the classical tomato-ripening promoters E8 and 2A11 and a set of six new tomato promoters. The new promoter activities were characterized in both transient assays and stable transgenic plants. The range of expression of the new promoters comprises strong (PNH, PLI), medium (PLE, PFF, PHD) and weak (PSN) promoters driving gene expression preferentially in the fruit, and covering a wide range of tissues and developmental stages. Together, a total of 78 possible combinations for the expression of a gene of interest in the fruit, plus a set of five reporters for new promoter analysis, was made available in the current collection. Moreover, the pENFRUIT promoter collection is adaptable to hairpin RNA strategies aimed at tissue/organ-specific gene silencing with only an additional cloning step. The pENFRUIT toolkit broadens the spectrum of promoter activities available for fruit biotechnology and fundamental research, and bypasses technical difficulties of current ligase-dependent cloning techniques in the construction of fruit expression cassettes. The pENFRUIT vector collection is available for the research community in a plasmid repository, facilitating its accessibility.     

Development and characterization of a binary gene expression system based on bacteriophage T7 components in adenovirus vectors

To explore the utility of the bacteriophage T7 binary system in adenovirus (Ad) vectors we constructed three Ad5-based vectors containing the T7 RNA polymerase (T7pol) gene in either early region 1 (E1) or E3. The recombinant Ad vectors were either deficient (AdT7pol1, AdT7pol2) or competent (AdT7pol3) for replication in human cells other than Ad5 transformed (293) cells. To test the ability of the T7 polymerase produced by these vectors to drive gene expression, a reporter vector was constructed with an E1 substitution comprising the bacterial β-galactosidase (βGal) (lacZ) gene under the control of the T7 gene 10 promoter (T7pro) and linked to the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) (AdBHG10T7βGal). Coinfections were performed with the various AdT7pol vectors and the reporter vector, and expression was analysed in three different human cell lines: 293, A549 and MRC-5. Depending on the AdT7pol vector used, different levels of expression were obtained from the reporter gene. In 293 cells, expression was detected following infection at very low multiplicities of infection (moi) with all of the T7pol vectors when coinfected with the reporter vector AdBHG10T7βGal. In A549 and MRC-5 cells very little expression was detected using AdT7pol1 or pol2 and efficient expression was only obtained when relatively high moi values of the replication-competent vector were used in the coinfections. We also constructed a single vector containing both elements of the T7 system (T7pol in E3 and T7 promoter driving expression of the chloramphenicol acetyl transferase (cat) gene in E1). This vector proved difficult to rescue but was stable once isolated. Finally, experiments performed to evaluate the `leakiness' of the Ad-T7 system detected very little expression from the T7pro in the absence of T7 polymerase suggesting this system may be useful for the cloning and expression of genes encoding cytotoxic proteins.     

A retroviral vector capable of targeted gene transfer into cells expressing HIV envelope glycoprotein

An expression vector encoding a chimeric envelope protein composed of CD4 and ecotropic retroviral envelope glycoprotein was constructed with the aim of accomplishing targeted gene transfer into HIV-1-infected cells. The chimeric protein was efficiently expressed and transported to the surfaces of various cell types and supported HIV-1 entry into human cells. A packaging cell line producing retroviral vectors carrying chimeric envelope proteins was then established. The vector particles produced were shown to be capable of specific gene transfer into human cells expressing HIV envelope glycoprotein. Blocking experiments confirmed that the vector particles entered the cells via an interaction between the chimeric and HIV envelope proteins. This targeting vector may thus be a useful tool with which to develop effective gene therapies against HIV infection.     

Expression of the fibronectin-binding components of Streptococcus pyogenes in Escherichia coli demonstrates that they are proteins

The fibronectin-binding components (fbcs) of two clinical isolates and a culture collection strain of Streptococcus pyogenes have been analysed. Western immunoblotting of bacterial lysates which had been fractionated on polyacrylamide gels revealed trypsin-sensitive fibronectin-binding species. The genes specifying the fbcs were cloned from all three strains and expressed in Escherichia coli using a lambda EMBL3 vector. An fbc gene from the culture collection strain was subcloned and expressed in the E. coli expression vector pJLA601, and subjected to deletion analysis. The fibronectin-binding domain was thereby localized within a 40 kDa truncated peptide encoded by the 1000 bp C-terminal region of the gene. Southern hybridization experiments demonstrated that the analysed gene was present in the parental S. pyogenes chromosome, but not in the DNA of fbc expressing lambda clones obtained from the two clinical isolates. Further evidence for the existence of at least two different types of fbcs in group A streptococci was provided by Western blot analysis of recombinant phage lysates which revealed a complex series of fibronectin-binding species ranging from 120 to 200 kDa in size and showing strain-dependent variation in their patterns. As was the case with parental streptococcal strains all of the recombinant fbcs were protease-sensitive, and treatment with trypsin or pronase resulted in a total loss of fibronectin-binding activity. Competitive inhibition experiments indicated that lipoteichoic acid was not a significant fbc in the tested streptococcal strains.(ABSTRACT TRUNCATED AT 250 WORDS)     

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。