Platelet membrane-camouflaged silver metal-organic framework drug system against infections caused by methicillin-resistant Staphylococcus aureus.

BACKGROUND: Due to the intelligent survival strategy and self-preservation of methicillin-resistant Staphylococcus aureus (MRSA), many antibiotics are ineffective in treating MRSA infections. Nano-drug delivery systems have emerged as a new method to overcome this barrier. The aim of this study was to construct a novel nano-drug delivery system for the treatment of MRSA infection, and to evaluate the therapeutic effect and biotoxicity of this system. We prepared a nano silver metal-organic framework using 2-methylimidazole as ligand and silver nitrate as ion provider. Vancomycin (Vanc) was loaded with Ag-MOF, and nano-sized platelet vesicles were prepared to encapsulate Ag-MOF-Vanc, thus forming the novel platelet membrane-camouflaged nanoparticles PLT@Ag-MOF-Vanc. RESULTS: The synthesized Ag-MOF particles had uniform size and shape of radiating corona. The mean nanoparticle size and zeta potential of PLT@Ag-MOF-Vanc were 148 nm and - 25.6 mV, respectively. The encapsulation efficiency (EE) and loading efficiency (LE) of vancomycin were 81.0 and 64.7 %, respectively. PLT@Ag-MOF-Vanc was shown to be a pH-responsive nano-drug delivery system with good biocompatibility. Ag-MOF had a good inhibitory effect on the growth of three common clinical strains (Escherichia coli, Pseudomonas aeruginosa, and S. aureus). PLT@Ag-MOF-Vanc showed better antibacterial activity against common clinical strains in vitro than free vancomycin. PLT@Ag-MOF-Vanc killed MRSA through multiple approaches, including interfering with the metabolism of bacteria, catalyzing reactive oxygen species production, destroying the integrity of cell membrane, and inhibiting biofilm formation. Due to the encapsulation of the platelet membrane, PLT@Ag-MOF-Vanc can bind to the surface of the MRSA bacteria and the sites of MRSA infection. PLT@Ag-MOF-Vanc had a good anti-infective effect in mouse MRSA pneumonia model, which was significantly superior to free vancomycin, and has no obvious toxicity. CONCLUSIONS: PLT@Ag-MOF-Vanc is a novel effective targeted drug delivery system, which is expected to be used safely in anti-infective therapy of MRSA.

Meropenem-induced immune thrombocytopenia and the diagnostic process of laboratory testing.

BACKGROUND: Drug-induced immune thrombocytopenia (DITP) is a serious, life-threatening clinical syndrome, the diagnosis of which is consistently difficult. In this report, we present a case of DITP caused by meropenem that was confirmed by laboratory tests. CASE REPORT: A 59-year-old male patient developed severe thrombocytopenia 8 days after the administration of meropenem and cefoperazone-sulbactam. After other causes were ruled out, DITP was suspected. Drug-induced platelet (PLT) antibodies were detected by enzyme immunoassay, flow cytometry, and monoclonal antibody immobilization of PLT antigens (MAIPA). All these tests were performed in the presence and absence of the associated drugs. RESULTS: PLT antibodies were detected in the patient's serum only in the presence of meropenem. MAIPA experiments demonstrated that glycoprotein IIb/IIIa was the binding site of the meropenem-induced PLT antibodies. CONCLUSIONS: Drug-induced immune thrombocytopenia should be considered in cases of acute thrombocytopenia in patients undergoing meropenem treatment. Clinicians should be cognizant of DITP, and a definitive diagnosis should be pursued, if feasible.

Electrochemical immunosensor of platelet-derived growth factor with aptamer-primed polymerase amplification.

A new method for the determination of platelet-derived growth factor BB (PDGF-BB) was developed using an electrochemical immunosensor with an aptamer-primed, long-strand circular detection probe. Rabbit anti-human PDGF-B polyclonal antibody was immobilized on the electrode to serve as the capture antibody. The detection probe was synthesized via polymerase extension along a single-stranded circular plasmid DNA template with a primer headed by the anti-PDGF-B aptamer. In the presence of the analyte, the aptamer-primed circular probe was captured on the electrode via the formation of an antibody/PDGF-BB/aptamer sandwiched complex. The electroactivity indicator methylene blue was adsorbed on the electrode surface via the analyte-sandwiched complex with long-strand circular DNA, thus yielding a strong electrochemical signal for the quantification of PDGF-BB. This strategy allowed electrochemical detection with enormous signal amplification arising from the long-strand localized circular probe. The oxidation peak current of methylene blue in square wave voltammetric measurements showed a linear dependence on the concentration of PDGF-BB in the range from 50 to 500 ng mL(-1), with a detection limit as low as 18 pg mL(-1).

Increased expression of heat shock protein 70 and heat shock factor 1 in chronic dermal ulcer tissues treated with laser-aided therapy.

BACKGROUND: Chronic dermal ulcers are also referred to as refractory ulcers. This study was conducted to elucidate the therapeutic effect of laser on chronic dermal ulcers and the induced expression of heat shock factor 1 (HSF1) and heat shock protein 70 (HSP70) in wound tissues. METHODS: Sixty patients with 84 chronic dermal ulcers were randomly divided into traditional therapy and laser therapy groups. Laser treatment was performed in addition to traditional therapy in the laser therapy group. The treatment efficacy was evaluated after three weeks. Five tissue sections of healing wounds were randomly collected along with five normal skin sections as controls. HSP70-positive cells from HSP70 immunohistochemical staining were counted and the gray scale of positive cells was measured for statistical analysis. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting were performed to determine the mRNA and protein expressions of HSF1 and HSP70. RESULTS: The cure rate of the wounds and the total efficacy in the laser therapy group were significantly higher than those in the traditional therapy group (P < 0.05, P < 0.01, respectively). Immunohistochemical staining revealed that the HSP70-positive cell count was significantly higher in laser therapy group than those in the traditional therapy group and controls (P < 0.01), and the gray scale of the cell signal was obviously lower than traditional therapy group and controls (P < 0.05). By contrast, the traditional therapy group and the control group were not significantly different. The RNA levels of HSF1 and HSP70 were higher in the laser therapy group by RT-PCR, but very low in normal skin and the traditional therapy group. The analysis on the gray scale of the Western blot bands indicated that the expression of HSF1 and HSP70 in the laser therapy group was significantly higher than in the traditional therapy group and the control group (P < 0.01), and the expression in the traditional therapy group was also higher than in the control group (P < 0.05). CONCLUSION: Laser-aided therapy of chronic dermal ulcers plays a facilitating role in healing due to the mechanism of laser-activated endogenous heat shock protection in cells in wound surfaces.

Oxidored-nitro domain-containing protein 1 expression is associated with the progression of hepatocellular carcinoma.

Hepatocarcinogenesis is a stepwise process during which multiple genes are altered. Understanding the molecular mechanisms that induce hepatocarcinogenesis may improve the screening, prevention and treatment of patients with hepatocellular carcinoma (HCC). In recent years, the oxidored-nitro domain-containing protein 1 (NOR1) gene has been identified to have an important role in the development of HCC in vitro experiments. The current study aimed to examine the expression of NOR1 mRNA and protein expression in specimens of normal liver, hepatitis, cirrhosis and HCC, together representing the process of HCC development. Furthermore, the association between NOR1 expression and clinicopathological parameters of HCC patients was analyzed. Tissue microarrays containing the specimens of human normal liver, hepatitis, cirrhosis and HCC were purchased, and in situ hybridization and immunohistochemistry were used to detect the expression of NOR1 mRNA and protein expression, respectively. It was revealed that the positive rate of NOR1 protein and mRNA expression in the specimens of hepatitis and cirrhosis were not significantly different from that in the normal liver samples. However, the specimens of HCC exhibited an increased positive rate of NOR1 protein and mRNA expression in comparison with the normal liver samples. In addition, a higher positive rate of NOR1 protein expression was observed in HCC patients with a poor pathological differentiation grade and high tumor node metastasis (TNM) stage. In conclusion, the present study provides evidence, for the first time, of the increased expression of NOR1 in human HCC tissues, and its correlation with the pathological stage and TNM status. These findings indicate that NOR1 may be involved in the progression of HCC and it could be employed as a predictive biomarker in HCC development.

MicroRNA-199a-5p inhibits VEGF-induced tumorigenesis through targeting oxidored-nitro domain-containing protein 1 in human HepG2 cells.

VEGF induces deterioration of hepatocellular carcinoma (HCC) by enhancing cell proliferation and migration. MicroRNAs regulate many cellular processes. In this study, we examined the regulation of tumorigenesis in HCC cells by microRNAs in relation to the effect of VEGF. Differences in microRNA expression between HepG2 and THLE-3 cells were characterized by microarray analysis. The results showed that miR-199a-5p expression was markedly downregulated in HepG2 cells and was inhibited in VEGF-overexpressing HepG2 cells in a dose- and time-dependent manner. This miRNA also inhibited cell proliferation and migration, as demonstrated by MTT and cell migration assays. Oxidored-nitro domain-containing protein 1 (NOR1), a nitroreductase, was identified as a downstream target gene of miR-199a-5p, and upregulation of NOR1 proved critical for the inhibition of VEGF-induced cell proliferation and migration in HepG2 cells by miR-199a-5p. These results indicate that miR-199a-5p is critical for regulating cell proliferation and migration by targeting and upregulating NOR1 in human HepG2 cells.

[Pathogenic characters of infected bacteria after liver transplantation].

OBJECTIVE: To analyze the main pathogens of infection after the liver transplantation and their antibiotic resistant patterns. METHODS: The main pathogens of infection after the liver transplantation were retrospectively analyzed. Using 3-dimensional tests, ESBLs (extended-spectrum beta-lactamase), and AmpC were detected among the Gram negative bacilli. beta-Lactamase and Van gene in Enterococcus were determined by the standard agar dilution susceptibility tests and Nitrocefin respectively. RESULTS: The main infected strains were Enterococcus faecalis (15.0%), Enterobacter cloacae (13.9%), fungus (13.3%), and Escherichia coli (10.7%) after the liver transplantation. Among them, 32.4% of Enterobacter cloacae and 36.8% of Escherichia coli produced ESBLs; 33.8% of Enterobacter cloacae and 10.5% of Escherichia coli. produced AmpC beta-lactamases. The detectable rate of VanA gene in Enterococcusfaecalis and Enterococcus faecium was 7.5% and 11.1%; VanB was 3.8% and 7.4%; VanC was 1.3% and 0, respectively. CONCLUSION: The infection mainly occurs in the intestinal tract after the liver transplantation. The production of ESBLs and AmpC beta-lactamases is the main mechanism of antibiotic resistance. The increased detectable rate of vancomycin-resistant Enterococcus should be paid attention to.

Therapeutic effects of acetylspiramycin and garlicin on cryptosporidiosis among drug users.

Cryptosporidiosis affects humans of all ages, particularly malnourished children and those with compromised immune systems such as HIV/AIDS. This study investigated the therapeutic effects of acetylspiramycin and garlicin on Cryptosporidium infection in institutionalized male drug users receiving rehabilitative treatment. Examination of stool specimens from 903 drug users via modified acid-fast bacilli staining resulted in 172 positive cases. Among them 151 subjects consented to participate in a randomized trial of acetylspiramycin and garlicin in four groups: acetylspiramycin plus garlicin, acetylspiramycin only, garlicin only, and placebo control. The cryptosporidiosis rate was higher in younger subjects with longer drug use history than subjects who are older with shorter history of drug use. After two segments of treatments, 76.2% of the cases achieved negative test results, with the four groups achieving the rates of 92.1%, 76.7%, 72.2%, and 61.8%, respectively (χ(2) = 9.517, P = 0.023). These results indicate clinical potential of garlicin in conjunction with acetylspiramycin in treating cryptosporidiosis.

[In vitro gene transfection by magnetic iron oxide nanoparticles and magnetic field increases transfection efficiency].

OBJECTIVE: To evaluate the feasibility of using iron oxide nanoparticles as gene vector and the effect of magnetic field on efficiency of transfection. METHODS: Iron oxide nanoparticles were prepared by alkaline precipitation of divalent and trivalent iron chloride. The surface of iron oxide nanoparticles was modified by self-assembled poly-L-lysine to form particle complexes (IONP-PLL). Transfection was determined by delivering reporter gene, PGL2-control encoding luciferase, to different cell lines using IONP-PLL as vector. The effect of magnetic field on efficiency of transfection was determined using Nd-Fe-B permanent magnet. RESULTS: Foreign gene could be delivered to various cell lines by IONP-PLL and expressed with high efficiency, but the transfection efficiency and time course varied in the different cell lines studied. Magnetic field could enhance the efficiency of transfection by 5 - 10 fold. CONCLUSION: IONP-PLL can be used as a novel non-viral gene vector in vitro, which offers a basis for gene delivery in vivo.

[Polymorphism of two novel SNPs, which locate on chromosome 9p21-22, in Han Chinese of Hunan].

OBJECTIVE: To search novel SNPs in exons and regulatory regions of CDKN2A and two novel putative tumor suppressor genes NGX6 and UBAP1, which all reside on chromosome 9p21-22. METHODS: The exons and regulatory regions of those genes were amplified and sequenced in 96 subjects. RESULTS: Two novel SNPs were found, one resides on the sixth exon of UBAP1 gene and the other on the fourth exon of CDKN2A gene. Two novel SNPs were submitted to the dbSNP database, and their access ID are rs3135929 and rs3088440. The polymorphic information contents of them are 0.102 and 0.213 respectively. There is linkage equilibrium between them, and the polymorphic information content of their haplotype is 0.302, higher than any of them individually. CONCLUSION: The polymorphic information content can be improved by using haplotype analysis of several SNPs.

Plasmonic ELISA for the ultrasensitive detection of Treponema pallidum.

In this report, we have developed a plasmonic ELISA strategy for the detection of syphilis. Plasmonic ELISA is an enzyme-linked immunoassay combined with enzyme-mediated surface plasmon resonance (SPR) of gold nanoparticles (AuNPs). Immune response of the Treponema pallidum (T. pallidum) antibodies triggers the acetylcholinesterase-catalyzed hydrolysis of acetylthiocholine to produce abundant thiocholine. The positive charged thiol, in turn, alters the surface charge distribution the AuNPs and leads to the agglomeration of the AuNPs. The induced strong localized SPR effect of the agglomerate AuNPs can, thus, allow the quantitative assay of T. pallidum antibodies due to the remarkable color and absorption spectral response changes of the reaction system. The plasmonic ELISA exhibited a quasilinear response to the logarithmic T. pallidum antibody concentrations in the range of 1pg/mL-10ng/mL with a detection limit of 0.98pg/mL. Such a low detection limit was 1000-fold improvements in sensitivity over a conventional ELISA. The results of plasmonic ELISA in syphilis assays of serum specimens from 60 patients agreed with those obtained using a conventional ELISA method. The plasmonic ELISA has characteristics (analyte specific, cost-effective, ease of automatic, low limit of detection) that provide potential for diagnosis and therapeutic monitoring of syphilis.

[Analysis on the effectiveness in 727 times of platelet transfusion].

This study was purposed to analyze the efficiency of platelet transfusion and to explore factors influencing platelet transfusion efficiency. 727 times of platelets transfusion in 254 patients in The Third Xiangya Hospital from September 2010 to May 2011 were analyzed retrospectively. Moreover, according to symptoms, times of platelet transfusion, blood types and splenomegaly, the corrected count of increment (CCI) and percentage of platelet recovery (PPR) were calculated for evaluation of platelet transfusion efficiency. The results showed that there were 456 effective transfusions out of 727 transfusions (62.72). Among them, the therapeutic effect of platelet transfusion for patients with acute blood loss anemia and chronic systemic diseases was relatively obvious, specially for chronic renal disease, the effective efficiency of them was 94.12. The patients with splenomegaly showed a significant impact on platelet transfusion efficiency (41.07). Analysis found that the frequency of platelet transfusion negatively correlated with transfusion efficiency. It is concluded that the transfusion frequency and splenomegaly are factors influencing the transfusion efficiency.

The study of the combination detection of HPV-DNA and p16INK4a in cervical lesions.

The objective of this study is to detect the infection of human papillomavirus (HPV) and the expression of p16(INK4a) in cervical lesions and to investigate the interaction between hrHPV and p16(INK4a) for cervical lesions and its diagnostic efficiency. hrHPV-DNA was detected by the hybrid capture II (HC-II) system. Immunochemical method was used to detect the expression of p16(INK4a), and histopathologic test was performed to identify cervical lesions. χ(2) test and Spearman's rank correlation were used for statistical analysis. Additive effects model was used to analyze the interaction. The diagnostic sensitivity, specificity, positive predictive values, negative predictive values, accuracy, and the area under the receiver operating characteristic curve were calculated with SPSS 13.0. hrHPV and p16(INK4a) positive rate increased (P < 0.05) with histopathologic diagnosis increasing. The positive rates of hrHPV and p16(INK4a) in negative or chronic inflammation were statistically lower than that in cervical intraepithelial neoplasia (CIN)1, CIN2, CIN3, and squamous-cell carcinoma (SCC) (P < 0.05), respectively. There was a positive interaction between hrHPV and p16(INK4a), relative excess risk of interaction (RERI) was 52.49, attributable proportions of interaction (API) were 72.34%, and the synergy index (S) was 3.75. The specificity and AUC of combining hrHPV with p16(INK4a) were statistically higher than hrHPV or p16(INK4a) alone (P < 0.05). hrHPV and p16(INK4a) are useful markers for the early diagnosis of cervical lesions. A positive interaction between hrHPV and p16(INK4a) is seen. The combination of hrHPV and p16(INK4a) has a higher diagnostic accuracy than hrHPV or p16(INK4a) alone in diagnosis of cervical lesions.

An analysis on the combination expression of HPV L1 capsid protein and p16INK4a in cervical lesions.

Human papillomavirus (HPV) infection in cervix is the most important reason for cervical cancer, but only 2% cervical HPV infection will develop into cervical cancer. So how to identify patients at risk of progressive cervical lesions from those infected with HPV to avoid over treatment is a big issue in clinic. The aims of this study were to detect the expression of HPV L1 capsid protein and p16(INK4a) in cervical lesions and to investigate the combination expression of HPV L1 capsid protein and p16(INK4a) in cervical lesions and its diagnostic efficiency in clinic. Immunochemical method was used to detect the expression of HPV L1 capsid protein and p16(INK4a) in 169 cases of abnormal cytology. Histopathologic test was performed to identify cervical lesions of all the cases. chi(2) test and spearman's rank correlation were used for statistical analysis. The diagnostic sensitivity, specificity, positive predictive values (PPV), negative predictive values (NPV), accuracy, and the area under the receive operating characteristic (ROC) curve (denoted by A(Z)) were calculated with SPSS 13.0. All the statistical tests were two sided at the 5% level of significance. L1 expression decreased (P < 0.001), but p16(INK4a) expression increased (P < 0.001) with histopathologic diagnosis increasing. The expression rates of HPV L1 capsid protein, p16(INK4a), and L1(-)/p16(+) in cervical intraepithelial neoplasia (CIN)2, CIN3, and squamous-cell carcinoma were statistically different from those in CIN1 (P < 0.001). The expressions of HPV L1 capsid protein, L1(+)/p16(+), L1(+)/p16(-), and L1(-)/p16(-) were negatively correlated with the severity of cervical lesions (P < 0.001), whereas the expressions of p16(INK4a) and L1(-)/p16(+) were positively correlated with the severity of cervical lesions (P < 0.001). The specificity and A(Z) of combining L1 with p16 (INK4a) were statistically higher than L1 or p16 (INK4a) alone (P < 0.05). L1 and p16(INK4a) are useful biomarkers for the early diagnosis of cervical lesions. The combination of L1 and p16(INK4a) has a higher diagnostic accuracy than L1 or p16(INK4a) alone in diagnosis of cervical lesions.

Analysis of the dose-response relationship between high-risk human papillomavirus viral load and cervical lesions.

The aims of this study were to explore the dose-response relationship between high-risk human papillomavirus (hrHPV) load and cervical lesions; the relationship between hrHPV viral load and the severity of cervical lesions; and the clinical application of the hybrid capture II (HC-II) system in the secondary prevention of cervical cancer. HrHPV viral load was detected by the HC-II system and cervical lesions were diagnosed from biopsied tissue. Curve estimation and Mantel trend analysis were used to explore the dose-response relationship between hrHPV viral load and cervical lesions. Spearman's rank correlation analysis and ordinal regression model were used for the analysis of hrHPV viral load and the severity of cervical lesions. Curve estimation showed good correlation between cervical lesion rates and hrHPV viral load (r=0.775, P=0.008); the rate of cervical lesions increased with hrHPV viral load (chi(trend)=8.000, P<0.001). Medium intensity rank correlation was found between hrHPV viral load grades and the severity of cervical lesions (r(s)=0.321, P<0.001); a correlation appeared between hrHPV viral load and the severity of cervical lesions (P<0.001). These results suggest a dose-response relationship between hrHPV viral load and the severity of cervical lesions. This dependence has important clinical applications and shows the potential value of the HC-II system in cervical cancer prevention.

Tyrosine phosphorylation of Grb2: role in prolactin/epidermal growth factor cross talk in mammary epithelial cell growth and differentiation.

Characterizing mechanisms regulating mammary cell growth and differentiation is vital, as they may contribute to breast carcinogenesis. Here, we examine a cross talk mechanism(s) downstream of prolactin (PRL), a primary differentiation hormone, and epidermal growth factor (EGF), an important proliferative factor, in mammary epithelial cell growth and differentiation. Our data indicate that EGF exerts inhibitory effects on PRL-induced cellular differentiation by interfering with Stat5a-mediated gene expression independent of the PRL-proximal signaling cascade. Additionally, our data show that PRL is a potent inhibitor of EGF-induced cell proliferation. We identify tyrosine phosphorylation of the growth factor receptor-bound protein 2 (Grb2) as a critical mechanism by which PRL antagonizes EGF-induced cell proliferation by attenuating the activation of the Ras/mitogen-activated protein kinase (MAPK) pathway. Together, our results define a novel negative cross-regulation between PRL and EGF involving the Jak2/Stat5a and Ras/MAPK pathways through tyrosine phosphorylation of Grb2.

[Effects of a novel gene NOR(1) on the protein expression of HepG2 cell line].

AIM: To investigate the mechanism of NOR(1) gene, a novel gene associated with liver cancer. METHODS: NOR(1) was introduced into HepG2 cells by liposome transfection. After staining and image analysis, 6 differential expression spots which were up-regulated in NOR(1) transfected cells were isolated and identified by two-dimensional polyacrylamide gel eletrophoresis (2D PAGE) and MALDI-TOF. RESULTS: The proteins which included zinc finger protein, tumor necrosis factor receptor, and protein tyrosine phosphatase receptor were involved in gene transcription and signal transduction associated with cancer. CONCLUSION: NOR(1) gene may have some effects on liver cancer by up-regulating the expression of these proteins.

[Expression of BRD7-interacting proteins,BRD2 and BRD3, in nasopharyngeal carcinoma tissues].

BACKGROUND & OBJECTIVE: BRD7 is a novel gene tightly associated with nasopharyngeal carcinoma(NPC) cloned by cDNA representational difference analysis (cDNA RDA). Two proteins,BRD2 and BRD3, including bromodomain and interacting with BRD7 protein had been screened from human fetal brain cDNA library by yeast two-hybrid system. This study was designed to further identify the interactions of BRD2 and BRD3 with BRD7 respectively and to investigate the expression and action pattern of BRD2 and BRD3 in NPC. METHODS: BRD2 and BRD3 genes were respectively co-transformed to yeast Y187 with BRD7 gene, then the yeast cotransformers were blotted to nylon membrane. And then the expression of report gene Lac Z by beta-Gal was determined and the interactions of BRD2 and BRD3 proteins with BRD7 protein were identified. Besides,reverse transcription-polymerase chain reaction (RT- PCR) was used to examine the expression of BRD2 and BRD3 genes in normal nasopharyngeal epithelium and NPC biopsies, and to detect the effect of re-expression of BRD7 gene on the expression of BRD2 and BRD3 genes in HNE1 stably transfected BRD7 gene. RESULTS: The yeast transformers showed all blue by yeast two-hybrid system, which further identified that BRD2 and BRD3 proteins could respectively interact with BRD7 protein. Down-expression or loss of BRD2 and BRD3 genes were detectable in NPC biopsies. The expression levels of BRD2 and BRD3 were increasing with the re-expression of BRD7 gene in HNE1 stably transfected with BRD7. CONCLUSION: BRD7 protein could respectively interact with proteins, BRD2 and BRD3, and BRD7 could up-regulate the expression levels of BRD2 and BRD3 genes in mRNA level to some extent. Each of these three homolog proteins might be capable of forming heteromers with the others, which play important roles in the suppression of growth of NPC cells.

[Expression of a new cloned nitroreductase gene NOR1 and purification of expressed product].

BACKGROUND & OBJECTIVE: NOR(1)is a good candidate of tumor suppressor/susceptibility gene associated with nasopharyngeal carcinoma. This study was designed to construct the prokaryotic expression vector and to investigate the expression of nitroreductase gene NOR(1)in Escherichia coli and to purify expressed product. METHODS: Total RNA was subtracted from normal nasopharyngeal carcinoma tissue. The full length of NOR(1)gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and digested with BamHIand XhoI restriction endonucleases. The plasmid pGEX-4T-2 was also digested with BamHI and XhoI,then the NOR(1)gene was inserted into vector pGEX-4T-2. The recombinant expression vector pGEX-4T-2/NOR(1)was identified by sequencing and digested with restriction enzymes. E.coli Jm105 transformed with the recombinant plasmid was induced by IPTG to express GST fusion protein. The result was confirmed by Western blot analysis and the purified targeted protein was obtained by affinity chromatography. RESULTS: The 1.25kb NOR(1)gene was successfully isolated. After induction, a new anticipated protein of 74 kDa appeared on sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). It existed not only in supernatant but also in precipitation of broken bacteria. The result was confirmed by Western blot analysis,and the purified targeted protein was obtained by affinity chromatography. CONCLUSION: The successes in construction of expression vector of NOR(1), expression and purification of GST/NOR(1)fusion protein make it possible to prepare for the polyantibodies for NOR(1).