Construction Of Eukaryotic Expression Vector Of Azurin Gene With HA Epitope Tag And The Experimental Study About Human Osteosarcoma Cell U2OS Transfected By Eukaryotic Expression Vector Of Azurin Gene

BackgroundThe osteosarcoma is the most common malignant tumor of bone. It occurs mostly in teenagers, with high maligancy, early metastasis,and poorly prognosis. At present for which the common treatment, such as surgical operation, chemotherapy, is suboptimal and the prognosis of patients remains dismal. Therefore, novel therapeutic strategies are constantly being pursued. Various candidates for osteosarcoma therapy include new chemotherapeutic drugs, differentiating agents, vaccines, monoclonal antibodies, and approaches using dendritic cells. Recently, interest was rekindled in the old idea of using live or attenuated pathogenic bacteria or their products in the treatment of cancer.The infection of tumor-bearing mice with live attenuated cells of Salmonella typhimurium has been reported to produce tumor regression. A significant regression of subcutaneous tumors in mice was observed by combining anaerobic bacteria withof subcutaneous tumors in mice was observed by combining anaerobic bacteria with various chemotherapeutic agents. The antitumor activity of these pathogens has been attributed to the activation of an immune response against tumor antigens, to the preferential growth of certain bacteria within the tumor, or to the inhibition of angiogenesis. However, live microorganisms with or without chemotherapeutic agents produce significant morbidity and mortality. Hence, studies are in progress to identify pure metabolites of microbial origin or any other components of the microbial cell that might have antitumor activity. Recently, azurin is one of representative bacterial products applied in the treatment of tumour.Azurin is a member of a group of copper-containing redox proteins called cupredoxins, which elaborated from the pathogenic bacteria Pseudomonas aeruginosa. Different cupredoxins are produced by different aerobic bacteria as agents of electron transfer. Recent evidence confirms azurin could selectively trigger cell death in some cancer cells, such as melanoma and human breast cancer. In vitro studies revealed a direct physical interaction between azurin and p53. After internalization within tumor cells, azurin forms a complex with the tumor suppressor protein p53, stabilizes it. and enhances its intracellular level, thereby inducing apoptosis via caspase-mediated mitochondrial pathways.In our research group, the pqe30/azurin vector which contains azurin cDNA has been constructed, and the engineering bacteria strain Ml5 which can express azurin has been obtained, and the active azurin protein has been purified. We recently reported that azurin can selectively induce the human osteosarcoma cell line U2OS apoptosis in vitro, but at present we were not able to obtain massive, active, purify azurin protein that used in the clinical long-term treatment of osteosarcoma. Therefore, in the present study, we first construct the eukaryotic expression vectorcontaining azurin gene with HA epitope tag with biological activities. The human osteosarcoma cell line U2OS were transfected by the eukaryotic expression vector of azurin gene with HA epitope tag, and the azurin protein could effectively expressed in the human osteosarcoma cell line U2OS, which provides the further studies for azurin gene therapy in osteosarcoma.Objective1. To construct the eukaryotic expression vector containing azurin gene with influenza virus haemagglutinin 9 peptides HA epitope tag with biological activities by directed cloning technology. The positive clones were screened and identified by endonuclease digestion and PCR amplification.2. The eukaryotic expression vector pcDNA3.1(+)/azurin was introduced into the human osteosarcoma cell line LJ2OS by liposome-mediated gene transfer method. We study the expressed azurin protein product whether has the biology activeness which induced osteosarcoma cell apoptosis. And we investigated the expression of pro- and anti-apoptotic genes including bcl-2, bax, bcl-xl, p53 and Survivin gene during the expressed azurin protein product induced osteosarcoma cell apoptosis.MethodsThis topic experiment is divided two partsThe first part of experiment: PCR produce of azurin was amplified using specific primers containing influenza virus haemagglutinin 9 peptides HA epitope tag (HA tag).The eukaryotic expression plasmid pcDNA3.1(+)/azurin was constructed by directional cloning the target gene into eukaryotic expression vectorpcDNA3.1(+). The recombinant plasmid was identified by PCR amplification, restriction endonuclease mapping and sequencing.The second part of experiment: Using liposome-mediated gene transfer method, the human osteosarcoma cell line U2OS was transfected by the recombinant plasmid pcDNA3.1(+)/azurin and control plasmid.was into the with. The azurin mRNA in the transfected cells was verified by RT-PCR, and the fusion azurin-HA protein expression in the transfected cells was verified by Western the blot. Cell apoptosis of the transfected cells was determined by flow cytometry (FCM). DNA ladder was observed through agarose gel electrophoresis. RT-PCR was performed to detect bcl-2, bax, bcl-x, p53 and Survivin mRNA expression.Results1. The recombinant plasmid pcDNA3.1(+)/azurin was confirmed with the correct sequence of azurin-HA by PCR amplification, restriction endonuclease mapping and sequencing.2. The recombinant plasmid pcDNA3.1(+)/azurin was transfected into the human osteosarcoma cell line U2OS with liposome LipofectAMINE 2.000-mediated gene transfer method. After 72 hours of transfection, the results include:1) RT-PCR: The 414bp goal gene was amplified by RT-PCR in the experimental group transfected with pcDNA3.1 (+)/azurin.2) Western blot: The fusion azurin-HA protein expression was verified in the experimental group transfected with pcDNA3.1 (+)/azurin. The transfected cells can expressed the fusion azurin-HA protein.3) flow cytometry (FCM): The percentage of Go/Gi phase. S phase and G2/M cells phase in the experimental group transfected with pcDNA3.1 (+)/azurinwere all significantly lower than that in the blank group and in the control group. The apoptosis index in the experimental group transfected with pcDNA3.1 (+)/azurin is 64.30%±13.12%. obviously is higher than that in the blank group( 10.16%±1.87% ) and in the control group (16.42%± 7.19%). The expressed azurin protein product from the transfected cells could effectively promote the human osteosarcoma cell line U2OS apoptosis.4) agarose gel electrophoresis: The typical DNA ladder bands were observed in the experimental group transfected with pcDNA3.1 (+)/azurin.5) RT-PCR: When the expressed azurin protein from the transfected cells could effectively promote the human osteosarcoma cell line U2OS apoptosis, the pro-apoptotic gene bax mRNA level in the experimental group transfected with pcDNA3.1 (+)/azurin is obviously higher than that in the blank group and in the control group, and the light density ratio of bax/beta-actin is respective 0.45. 0.22 and 0.13 in the experimental group, the blank group and the control group. The pro-apoptotic gene p53 mRNA level in the experimental group transfected with pcDNA3.1 (+)/azurin is also obviously higher than that in the blank group and in the control group, and the light density ratio of p53/beta-actin is respective 0.3, 0.2 and 0.12 in the experimental group, the blank group and the control group. But the anti-apoptotic gene bcl-2 mRNA level in the experimental group transfected with pcDNA3.1 (+)/azurin is obviously lower than that in the blank group and in the control group, and the light density ratio of bcl-2/beta-actin is respective 0.26. 0.41 and 1.02 in the experimental group, the blank group and the control group. There is no significant difference of bcl-x mRNA andSurvivin mRNA in the experimental group, the blank group and the control groupConclusion1. The recombinant plasmid pcDNA3.1(+)/azurin was confirmed with the correct sequence of azurin-HA by PCR amplification, restriction endonuclease mapping and sequencing.2. The recombinant plasmid pcDNA3.1(+)/azurin is successfully transfected into the human osteosarcoma cell line U2OS and expressed in mRNA and protein level.3. The expressed azurin protein from the transfected cells could effectively promote the human osteosarcoma cell line U2OS apoptosis. The induction of apoptosis by azurin may be closely associated with up-regulation the mRNA level of bax gene and p53 gene, down-regulation he mRNA level of bcl-2 gene.