DNA-templated silver nanoclusters for fluorescence turn-on assay of acetylcholinesterase activity.

We have developed a fluorescence turn-on assay using DNA-templated silver nanoclusters (Ag NCs) (i.e., 12 polycytosine-templated silver nanoclusters, dC12-Ag NCs), which is amenable to rapid, ultrasensitive assay of acetylcholinesterase (AChE). The detection mechanism is based on the concept, that is, AChE hydrolyzes the acetylthiocholine (ATCh) chloride to produce thiocholine (TCh). Subsequently, TCh sensitively and rapidly reacts with dC12-Ag NCs via Ag-S bond forming and enhances the fluorescence of dC12-Ag NCs. Using dC12-Ag NCs, detection of TCh has a linear concentration range of 2.0 nM to 16.0 nM and a detection limit of 0.3 nM. Due to the sensitive and rapid fluorescence turn-on response of dC12-Ag NCs to TCh, AChE with activity as low as 0.5 × 10(-4) U/mL (signal/noise = 3) can be analyzed with a dynamic range of 0.1 to 1.25 × 10(-3) U/mL. The promising application of the proposed method in AChE inhibitor screening was demonstrated. AChE concentrations were determined in human blood red cell (RBC) membranes from clinical specimens using dC12-Ag NCs, and the quantitative results were validated with Ellman's method. Aside from the ease of manufacture, reduction of matrix effect, and low background noise, the continuous detection format and detection sensitivity can open up to wider applications to AChE activity assay in neurobiology, toxicology, and pharmacology, among other fields.

Association between maternal short-term exposure to ambient air pollution and the risk of fetal distress: A matched case-control study.

BACKGROUND: Ambient air pollution has been linked to gestational complications. However, the evidence on the relationship between air pollution and fetal distress is limited. OBJECTIVES: To investigate the relationship between maternal short-term air pollution exposure and fetal distress, and to identify a potential susceptible population. METHODS: This matched case-control study, involving 313 pregnancy women with fetal distress was conducted in Xi'an, the largest city in Northwest China from 2013 to 2016. Each woman with fetal distress was randomly matched with four women without fetal distress of the same age, same gestational week, and registration in the same period (n = 1252). Inverse distance-weighted (IDW) interpolation was applied to estimate maternal air pollution exposure based on the residential addresses. We employed conditional logistic regression model to evaluate the relationship between air pollutants and fetal distress. Distributed lag nonlinear model (DLNM) was performed to examine the exposure-response relationship between air pollutants and fetal distress. RESULTS: Maternal short-term exposure to PM10, PM2.5-10 (PMc), SO2, NO2, and CO was associated with increased risk of fetal distress. Each 10 µg/m3 increment in PM10, PMc, SO2 at lag 014, and NO2 at lag 010, the odds ratio (ORs) of fetal distress were 1.027 (95 % confidence interval (CI): 1.004, 1.050), 1.058 (95 % CI: 1.014, 1.105), 1.140 (95 % CI: 1.029, 1.264), and 1.158 (95 % CI: 1.046, 1.283), respectively. Similarly, with a 0.1 mg/m3 increment in CO at lag 014, the OR of fetal distress was 1.029 (95 % CI: 1.002, 1.058). Stratified analyses showed that the estimate associations of PM10, PM2.5 and CO appeared to be stronger, although not statistically significantly, among women with gestational complications. CONCLUSION: Maternal short-term exposure to ambient air pollution may increase the risk of fetal distress. Understanding the detrimental role of air pollution in fetal distress can help us better develop preventative methods in reducing its' impact on maternal and fetal health.

[Inhibitory effect of icariin on acetylcholinesterase].

Acetylcholinesterase (AChE) inhibitors are mainly used in the treatment of Alzheimer's disease (AD). The inhibitory effect of icariin on the activity of AChE was investigated by inhibition kinetics. The binding interaction and binding sites between icariin and AChE were also studied by using fluorimetry and molecular docking, respectively. The results showed that icariin could potently inhibit the activity of AChE, the IC50 value was determined to be 3.50 x 10(-8) mol x L(-1), and the determined IC50 value to tacrine was 0.75 x 10(-8) mol x L(-1). Kinetic analyses showed that icariin is a reversible and mixed type AChE inhibitor. The inhibition constants K1 and K(IS) were determined to be 2.67 x 10(-8) and 4.43 x 10(-8) mol x L(-1), respectively. Icariin binds selectively to the AChE peripheral anionic site via hydrogen bonds and Van der Waals forces.

Effect of simultaneous silencing of HPV-18 E6 and E7 on inducing apoptosis in HeLa cells.

The objective of this investigation was to determine if simultaneous silencing of the human papillomavirus type 18 (HPV-18) E6 and E7 oncogenes using RNA interference (RNAi) would be a potential therapeutic approach against the carcinogenic activity of this virus. Two synthetic double-stranded oligonucleotides, encoding short hairpin transcripts corresponding to HPV-18 E6 and E7 genes, were cloned into pGenesilence (pGS) 1.0 vectors to produce pGS-E6, pGS-E7, and pGS-(E6+E7), respectively. Our results showed that the expression of HPV-18 E6 class 1 and HPV-18 E7 in HeLa cells was markedly decreased after being transfected with pGS-E6, pGS-E7, and pGS-(E6+E7) vectors. Of the three vectors, pGS-(E6+E7) had a greater ability to decrease the growth rate of HeLa cells, inhibit colony formation in soft agar, and significantly reduce tumor growth in nude mice. We also found that depletion of HPV-18 E6 and E7 in this manner promoted apoptosis of HeLa cells. Our data showed that simultaneously decreasing HPV-18 E6 and E7 gene expression in HeLa cells by RNAi could significantly inhibit tumor growth under in vitro conditions and in nude mice. These data suggest that gene therapy may be a possible therapeutic approach for HPV-positive cervical cancers.

Lead and cadmium synergistically enhance the expression of divalent metal transporter 1 protein in central nervous system of developing rats.

Divalent metal transporter 1 (DMT1) can transport a large range of ions, including toxic lead (Pb) and cadmium (Cd), across membranes. In this study, a total of 24 rats were divided into four groups for intragastrical perfusion treatment: control, Pb alone, Cd alone, and Pb + Cd. Pb and Cd contents in blood were detected, and the mRNA and protein levels of DMT1 were analyzed in the cerebellum, cortex, and hippocampus. Both Pb and Cd levels were elevated in all groups perfused with Pb and/or Cd, except for Pb level in the Cd-alone group (P < 0.05). The mRNA level of DMT1 did not differ among the four groups (P > 0.05). However, the DMT1 protein expression was significantly increased by 0.9-, 1.0-, and 1.1-fold in cerebellum, cortex, and hippocampus of the Pb + Cd group than in controls, respectively. Pb and Cd exposure can synergistically induce DMT1 protein synthesis and has implications for transportation of toxic ions in the developing rat's brain.

Blood lead and cadmium levels and relevant factors among children from an e-waste recycling town in China.

BACKGROUND: Primitive electronic waste (e-waste) recycling is ongoing in Guiyu, and thus toxic heavy metals may keep on threatening to the health of local children. Some related factors may contribute to the elevation of blood lead levels (BLLs) or blood cadmium levels (BCLs). OBJECTIVE: To investigate the children's BLLs and BCLs in Guiyu and Chendian as compare to discuss the effects of primitive e-waste recycling activities on children's health. METHODS: Two hundred and seventy-eight children less than 8 years who lived in Guiyu and Chendian were observed, and their BLLs and BCLs were determined by graphite atomizer absorption spectrophotometer. Questionnaire survey for risk factors was also performed and data were analyzed using spearman correlation analyses and logistic regression analyses. RESULTS: Children living in Guiyu had significantly higher BLLs and BCLs as compared with those living in Chendian (p<0.01). In Guiyu, 70.8% of children (109/154) had BLLs>10 microg/dL, and 20.1% of children (31/154) had BCLs>2 microg/L, compared with 38.7% of children (48/124) had BLLs>10 microg/dL and 7.3% of children (9/124) had BCLs>2 microg/L in Chendian (p<0.01, respectively). We also observed a significant increasing trend in BLLs with increasing age in Guiyu (p<0.01). Mean height of children in Guiyu was significantly lower than that in Chendian (p<0.01). The risk factors related to children's BLLs and BCLs mainly included father's engagement in the work related to e-waste, children's residence in Guiyu and the amount of time that children played outside near the road everyday. CONCLUSIONS: There are close relationships between the BLLs, BCLs in children and the primitive e-waste recycling activities in Guiyu. Environmental pollution, especially lead pollution, has threatened the health of children living around e-waste recycling site.

Identification and characterization of epitopes from influenza A virus hemagglutinin that induce broadly cross-reactive antibodies.

Influenza is the most common infectious disease and is caused by influenza A virus (IAV) infection. Hemagglutinin (HA) is an important viral protein of influenza A and is a major component of current IAV vaccines. The side effects associated with IAV vaccination are well studied; however, the HA­induced immunopathological changes have remained largely elusive. The primary objective of the present study was to determine the tissue cross­reactive epitopes of HA proteins. Monoclonal antibodies (McAbs) were generated according to traditional methods using purified HA proteins from influenza vaccine lysates. The specificity of these McAbs was analyzed using western blot analysis and ELISA. Human tissue microarrays were employed for immunohistochemical staining to screen these McAbs. Rat brain tissues were subjected to immunohistochemical staining and electron microscopy to demonstrate the subcellular localization of antibodies targeting specific antigens. A total of 67 hybridoma cell lines positive for McAb against HA antigen were obtained. Three cross­reactive McAbs (H1­13, H1­15 and A1­10) were discovered through tissue screening. Based on the 3 cross­reactive McAbs and the amino acid sequence of HA, the presence of two broadly cross­reactive HA epitopes, 194­WGIHH­198 and 365­WYGYHH­370, was assumed. McAbs against these synthetic epitope peptides were obtained. They reacted with porphyrin ring­containing molecules, including hemoglobin (Hb) and protoporphyrin, and with numerous types of normal tissue. In conclusion, the present study identified two broadly cross­reactive epitopes on HA (194­WGIHH­198 and 365­WYGYHH­370). Antibodies against these epitopes react with Hb and numerous types of important normal tissues/organs. These newly identified cross­reactive epitopes from IAV HA may provide crucial information for influenza research.

Antibodies against H1N1 influenza virus cross-react with α-cells of pancreatic islets.

Epidemiological studies have documented that the incidence of human type 1 diabetes was significantly increased after H1N1 epidemic. However, a direct link between human type 1 diabetes and virus infection remains elusive. We generated 84 clones of murine monoclonal antibodies against the H1N1, and carried out immunohistochemistry in normal human tissue microarray. The results showed that two clones specifically cross-reacted with human α-cells of pancreatic islets. Reverse transcription polymerase chain reaction and deoxyribonucleic acid sequencing showed that the amino acid sequences of light and heavy chains of these clones were different. Importantly, the expression profiles of two monoclonal antibodies were individual different. For the first time, we provide direct evidence that monoclonal antibodies against H1N1 can cross-react with human pancreas α-cells, another source of ß-cells, suggesting α-cells might be a novel target to be investigated in diabetes research.

Elevated blood lead levels of children in Guiyu, an electronic waste recycling town in China.

BACKGROUND: Electronic waste (e-waste) recycling has remained primitive in Guiyu, China, and thus may contribute to the elevation of blood lead levels (BLLs) in children living in the local environment. OBJECTIVES: We compared the BLLs in children living in the e-waste recycling town of Guiyu with those living in the neighboring town of Chendian. METHODS: We observed the processing of e-waste recycling in Guiyu and studied BLLs in a cluster sample of 226 children < 6 years of age who lived in Guiyu and Chendian. BLLs were determined with atomic absorption spectrophotometry. Hemoglobin (Hgb) and physical indexes (height and weight, head and chest circumferences) were also measured. RESULTS: BLLs in 165 children of Guiyu ranged from 4.40 to 32.67 microg/dL with a mean of 15.3 microg/dL, whereas BLLs in 61 children of Chendian were from 4.09 to 23.10 microg/dL with a mean of 9.94 microg/dL. Statistical analyses showed that children living in Guiyu had significantly higher BLLs compared with those living in Chendian (p < 0.01). Of children in Guiyu, 81.8% (135 of 165) had BLLs > 10 microg/dL, compared with 37.7% of children (23 of 61) in Chendian (p < 0.01). In addition, we observed a significant increasing trend in BLLs with increasing age in Guiyu (p < 0.01). It appeared that there was correlation between the BLLs in children and numbers of e-waste workshops. However, no significant difference in Hgb level or physical indexes was found between the two towns. CONCLUSIONS: The primitive e-waste recycling activities may contribute to the elevated BLLs in children living in Guiyu.

[Preparation of monoclonal antibodies against His-tag and epitope analysis of cross antigens].

OBJECTIVE: To explore the influence of His-tag on recombinant proteins in vaccination, immunization and pathogenesis. METHODS: Multiple mouse monoclonal antibodies (mAb) against His-tag were prepared. The biological and immunoreactive characteristics of these mAbs and their cross-reactivity with the normal human tissues were investigated by ELISA, Western blotting and immunohistochemistry (IHC), respectively. RESULTS: The binding activity of these anti-His mAbs was associated with the steric configuration of the his-tagged antigen. In addition, most of these mAbs reacted with human hemoglobin and some normal human tissues. CONCLUSION: Anti-His antibodies could be elicited by His-tagged recombinant proteins in vivo experiments. Moreover, the functional studies of the His-tagged recombinant proteins might be affected by the reactions of anti-His6 antibodies with human hemoglobin and normal human tissues.

Conditionally replicating oncolytic adenoviral vector expressing arresten and tumor necrosis factor-related apoptosis-inducing ligand experimentally suppresses lung carcinoma progression.

Current methods of treatment for lung carcinoma are ineffective for the majority of patients. Conditionally replicating adenoviruses (CRAds) represent a potential novel treatment for a number of neoplastic diseases, including lung carcinoma. The present study aimed to investigate the synergistic mechanisms underlying the anti-angiogenesis gene, arresten, and the apoptosis-inducing gene, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), in order to evaluate their therapeutic potential in lung cancer. The two genes were expressed by CRAd, which was confirmed using reverse transcription-polymerase chain reaction and western blotting. In vitro analyses demonstrated that CRAd adenoviruses are capable of selectively inhibiting A549 lung cancer cell growth and replication but not in that of healthy cells. In vivo analyses demonstrated that the infection of A549 cell lines using CRAd armed with the two genes (CRAd-arresten-TRAIL) enhanced the tumor inhibition, compared with cells infected with CRAd-arresten, CRAd-TRAIL or CRAd, and with the control group. CRAd-arresten-TRAIL may therefore be useful in the treatment of lung cancer.

In situ induced metal-enhanced fluorescence: a new strategy for biosensing the total acetylcholinesterase activity in sub-microliter human whole blood.

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities (i.e., total AChE) in human blood are biomarkers for theranostic monitoring of organophosphate neurotoxin-poisoned patients. We developed an ultra-sensitive method to detect the total AChE activity in sub-microliter human whole blood based on in situ induced metal-enhanced fluorescence (MEF). Both AChE and BChE can catalyze the hydrolysis of the acetylthiocholine (ATCh) substrate and produce positively-charged thiocholine (TCh). TCh can reverse the negatively-charged surface of core-shell Ag@SiO2 nanoparticles (NPs). The negatively-charged fluorescent dye (8-hydroxypyrene-1,3,6-trisulfonic acid, HPTS) is then confined to the surface of Ag@SiO2 NPs and generates an enhanced fluorescence signal in situ. Changes in the surface charge of Ag@SiO2 NPs are monitored by Zeta potential, and the MEF effect is confirmed by the measurements of fluorescence time decay. AChE activity has a dynamic range of 0 U/mL to 0.005 U/mL and a detection limit of 0.05 mU/mL. The total AChE activity in the sub-microliter human whole blood could be determined; the results were further validated. Therefore, combining the AChE catalytic reaction with MEF provides a simple, ultra-sensitive, and cost-effective "in situ MEF" approach to determine the total AChE activity in human whole blood sample down to sub-microliters without matrix interferences. The strategy also allows potential usage in other tissues and other fields.

Development and characterization of a panel of cross-reactive monoclonal antibodies generated using H1N1 influenza virus.

To characterize the antigenic epitopes of the hemagglutinin (HA) protein of H1N1 influenza virus, a panel consisting of 84 clones of murine monoclonal antibodies (mAbs) were generated using the HA proteins from the 2009 pandemic H1N1 vaccine lysate and the seasonal influenza H1N1(A1) vaccines. Thirty-three (39%) of the 84 mAbs were found to be strain-specific, and 6 (7%) of the 84 mAbs were subtype-specific. Twenty (24%) of the 84 mAbs recognized the common HA epitopes shared by 2009 pandemic H1N1, seasonal A1 (H1N1), and A3 (H3N2) influenza viruses. Twenty-five of the 84 clones recognized the common HA epitopes shared by the 2009 pandemic H1N1, seasonal A1 (H1N1) and A3 (H3N2) human influenza viruses, and H5N1 and H9N2 avian influenza viruses. We found that of the 16 (19%) clones of the 84 mAbs panel that were cross-reactive with human respiratory pathogens, 15 were made using the HA of the seasonal A1 (H1N1) virus and 1 was made using the HA of the 2009 pandemic H1N1 influenza virus. Immunohistochemical analysis of the tissue microarray (TMA) showed that 4 of the 84 mAb clones cross-reacted with human tissue (brain and pancreas). Our results indicated that the influenza virus HA antigenic epitopes not only induce type-, subtype-, and strain-specific monoclonal antibodies against influenza A virus but also cross-reactive monoclonal antibodies against human tissues. Further investigations of these cross-reactive (heterophilic) epitopes may significantly improve our understanding of viral antigenic variation, epidemics, pathophysiologic mechanisms, and adverse effects of influenza vaccines.

[Expression and significance of B-cell-specific activator protein of H/RS cell in classical Hodgkin's Lymphoma].

OBJECTIVE: To explore the expression of B-cell-specific activator protein (BSAP) of H/RS cell in classical Hodgkin's lymphoma (cHL). METHODS: Immunohistochemical method was used to detect the expression of BSAP in 33 samples of formalin-fixed, paraffin-embedded tissues of cHL. Nine samples of lymph node of reactive hyperplasia, 10 samples of B-cell lymphoma, and 10 samples of T-cell lymphoma were also detected as BSAP controls. Mouse-anti-human monoclonal antibodies CD20, CD30 and CD15 were detected among the cHL cases as routine comparison. RESULTS: 30 of 33 (90.91%) cases of cHL were BSAP expression positive in H/RS cells, while all of the 33 (100%) cases of background B-lymphocytes were BSAP positive. Almost all B cells of lymph node reactive hyperplasia were BSAP positive. All malignant cells in B-cell lymphoma were BSAP positive, while all malignant cells in T-cell lymphoma were BSAP negative. Among the 33 cases of cHL there was a significant difference between the expression of BSAP and the expression of CD20 (30.30%) in H/RS cells (P = 0.000), and no significant difference between the expression of CD30 (93.94%) and CD15 (75.75%, P = 0.082). CONCLUSION: The frequent expression of BSAP in H/RS cells of classical Hodgkin's disease provides further evidence for its B-cell origin and helps to identify H/RS cells. The expression of BSAP in H/RS cells can be used to distinguish HL from anaplastic large cell lymphoma (ALCL).

Touch-down PCR and single-strand conformation polymorphism for detecting clonal T cell receptor gamma gene rearrangement in lymphoid leukemia.

OBJECTIVE: To explore the value of detecting clonal T cell receptor gamma (TCR-gamma) gene rearrangement with touch-down PCR and single-strand conformational polymorphism analysis (SSCP) in the diagnosis of lymphoid leukemia. METHODS: The DNA of peripheral blood leucocytes from lymphoid leukemia patients were extracted for amplification of the TCR-gamma gene rearrangement with the consensus primers and touch-down PCR. The PCR products were analyzed by agarose gel electrophoresis, direct DNA sequencing and SSCP analysis. The positive control cell line DNA was mixed in different proportions with the DNA extracted from reactive lymphoid tissue to test the sensitivity of the touch-down PCR. RESULTS: Fifteen of 18 T lymphoid leukemia and 2 of the 4 B lymphoid leukemia patients were identified to be positive by agarose electrophoresis. The positive PCR products were further analyzed by SSCP analysis, which showed discrete bands. Direct DNA sequencing confirmed the clones to be TCR-gamma gene rearrangement, and the sensitivity of touch-down PCR was 1%. CONCLUSION: Consensus primers for studying TCR-gamma gene rearrangement in combination with touch-down PCR can effectively amplify the clonal TCR-gamma gene rearrangement in T lymphoid leukemia.

Expressions of latent membrane protein 1, p53 and bcl-2 proteins and their significance in Hodgkin's lymphoma.

OBJECTIVE: To study the expressions of latent membrane protein 1 (LMP1), p53 and bcl-2 proteins and investigate their significance in the pathogenesis of Hodgkin's lymphoma. METHODS: Immunohistochemical staining was used to examine the expressions of LMP1, bcl-2, and p53 proteins in 31 paraffin-embedded tissue samples from Hodgkin's lymphoma. RESULT: The positivity rates of LMP1, p53 and bcl-2 proteins were 58.1% (18/31), 61.3% (19/31) and 35.5% (11/31), respectively, showing a statistically significant difference between the expressions of the former 2 proteins. CONCLUSION: p53 may be involved as an important agent in the pathogenesis of Hodgkin's lymphoma induced by LMP1, whereas bcl-2 appears unrelated to the development of this disease.

Detection of Epstein-Barr virus in human colorectal cancer by in situ hybridazition.

OBJECTIVE: To investigate the relationship between Epstein-Barr virus (EBV) and the pathogenesis of human colorectal cancer. METHODS: The small-molecule RNA fragments of EBV was examined by in situ hybridization in colorectal cancer specimens obtained from 130 patients. RESULTS: EBV was found in the 6 of 130 colorectal cancer cases. EBV positivity was more prevalent in male patients (4/6), and the tumor tissues of the 4 EBV-positive cases were featured by obvious stromal infiltration by the lymphocytes (4/6). CONCLUSION: EBV infection might be related to the pathogenesis of some colorectal adenocarcinomas in China, and intensive lymphocyte infiltration in the stroma of the tumor tissues can be an important pathological indication of EBV infection in colorectal tumors.

Comparison of three methods for the detection of Epstein-Barr virus in Hodgkin's lymphoma in paraffin-embedded tissues.

The percentage rate of Epstein-Barr virus (EBV)-positive cases of Hodgkin's lymphoma (HL) ranges between 20 and 70% in various studies worldwide. To further explore the definite rate in China, three methods, including immunohistochemistry for EBV latent membrane protein 1 (LMP1), in situ hybridization (ISH) for EBV-encoded RNA (EBER)-1 and polymerase chain reaction (PCR) for EBV BamHI­W fragment, were employed to detect EBV in 59 cases of HL in China using paraffin-embedded tissue samples. Our results revealed that the PCR method presented the highest (44/59, 74.6%) detection rate among the three methods. The other two methods identified 66.1% (39/59, LMP1) and 67.8% (40/59, EBER1 ISH) EBV-positive results, respectively. Three samples were positive for LMP1 but negative when using EBER1 ISH, while another four samples were EBER1-positive but LMP1-negative. Of the four major histopathological subtypes of HL, the lymphocyte predominant (LR) subtype is the one most frequently associated with EBV, followed by the mixed cellularity (MC), nodular sclerosis (NS) and lymphocyte depletion (LD) subtypes. Our results also indicated the seldomly reported fact that EBV-positive cases in children were more numerous than those of adults with HL.

The DG75 B-cell lymphoma line exhibits biclonal immunoglobulin gene rearrangement.

Immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangement (GR) studies have been successfully employed to investigate the clonality and cell lineage of various lymphoid malignancies. Several lymphoma cell lines, such as BJAB, RAJI, DG75 and Jurkat cell lines, were often used as the positive controls in GR detection assays. Of those, the DG75 B-cell lymphoma line was found to exhibit biclonality [two or more homoduplex and heteroduplex bands in a polymerase chain reaction (PCR) product of clonality assay] in the PCR of GR detection assays. To further explore these characteristics of the biclonal phenomenon, the PCR products were purified and cloned into a pEGM-T clone vector. The sequences were analyzed using DNA analysis software. The results demonstrated that the two bands originated from two forms of GR of DG75 cell lines, i.e., DG75 is a biclonal cell line in Ig GRs, which has not been reported before.

[Construction and expression of recombinant adenovirus vector carrying human endostatin, K5 and endostatin-K5 gene].

AIM: To construct the recombinant adenoviral vectors expressing human endostatin, K5 and endostatin-K5 gene respectively, and study their bioactivity in vitro. METHODS: Human endostatin, K5 and endostatin-K5 gene were amplified by PCR, which were then subcloned into shuttle vector pAd5-CMV-H1H2-MCS-6His by enzyme and ligation respectively. The positive recombinant plasmids linearized by Pac I were cotransfected into HEK 293 cells with the Pac I linearized adenoviral backbone plasmid using calcium phosphate precipitation method. The recombinant viruses were purified by CsCl density gradient centrifugation. The protein expression at different time points (24 h, 48 h and 72 h) was determined by Western blot. The inhibitory effect of the protein on ECV-304 growth was detected by MTT. RESULTS: The recombinant adenoviral vectors expressing human endostatin, K5 and endostatin-K5 gene were successfully constructed in HEK293 cells. Protein expression was detected by Western blot. The level of protein expression was increased with the prolonged incubation of the infected HeLa cells. Three kinds of the protein expressed by the recombinant adenoviral vectors showed obvious inhibitory effect on ECV-304 cell growth. CONCLUSION: The protein expressed by adenoviral vectors carrying endostatin, K5 and endostatin-K5 gene has an obvious inhibitory effect on ECV-304 cell growth.