Biomedical subjects

Jie Feng

Publications and source records attributed to Jie Feng.

62 records · Page 4Linked to original sources

PubMed2003

A novel antimicrobial protein isolated from potato (Solanum tuberosum) shares homology with an acid phosphatase.

The nucleotide and amino acids sequences for AP(1) will appear in the GenBank(R) and NCBI databases under accession number AY297449. A novel antimicrobial protein (AP(1)) was purified from leaves of the potato ( Solanum tuberosum, variety MS-42.3) with a procedure involving ammonium sulphate fractionation, molecular sieve chromatography with Sephacryl S-200 and hydrophobic chromatography with Butyl-Sepharose using a FPLC system. The inhibition spectrum investigation showed that AP(1) had good inhibition activity against five different strains of Ralstonia solanacearum from potato or other crops, and two fungal pathogens, Rhizoctonia solani and Alternaria solani from potato. The full-length cDNA encoding AP(1) has been successfully cloned by screening a cDNA expression library of potato with an anti-AP(1) antibody and RACE (rapid amplification of cDNA ends) PCR. Determination of the nucleotide sequences revealed the presence of an open reading frame encoding 343 amino acids. At the C-terminus of AP(1) there is an ATP-binding domain, and the N-terminus exhibits 58% identity with an/the acid phosphatase from Mesorhizobium loti. SDS/PAGE and Western blotting analysis suggested that the AP(1) gene can be successfully expressed in Escherichia coli and recognized by an antibody against AP(1). Also the expressed protein showed an inhibition activity the same as original AP(1) protein isolated from potato. We suggest that AP(1) most likely belongs to a new group of proteins with antimicrobial characteristics in vitro and functions in relation to phosphorylation and energy metabolism of plants.

Acid Phosphatase↗

PubMed2003

Inactivation of the nod box distal half-site allows tetrameric NodD to activate nodA transcription in an inducer-independent manner.

In Rhizobium leguminosarum, NodD can activate nodA transcription in response to inducer flavonoids. Here, we show that the inducible nodA promoter contains an intrinsic part through which NodD can activate nodA transcription in an inducer-independent manner. Evidence was provided that NodD binds to target DNA through anchoring the two half-sites of the nod box as a tetramer. An imperfect inverted repeat AT-N10-GAT was found in each half-site and is critical for NodD binding. Mutation of the inverted repeat of the nod box distal half-site allowed NodD to activate nodA transcription in an inducer-independent manner in vivo, and to modulate the DNA bending of the NodD-nod box complex in the absence of inducer in vitro.

Acyltransferases↗

PubMed2003

Preparation of humanized ovarian carcinoma anti-idiotypic minibody.

Murine anti-idiotypic monoclonal antibody (MAb) 6B11 mimicking the tumor-associated antigen OC166-9 is used as a vaccine for the induction of an anti-tumoral immunity in experiments of in vitro and in vivo animal model with ovarian carcinoma. In this article, we have humanized 6B11 anti-idiotypic minibody using overlap polymerase chain reaction (PCR) and DNA recombinant technique, prokaryotic expression vector was produced by genetic fusion of 6B11V(L)-V(H) to human IgG1 hinge and CH3 region. Transformed E. coli BL21(DE3) were propagated and induced by isopropyl-beta D-thiogalactopyranoside (IPTG). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that a protein band with molecular weight of 50kD appeared as the expected size after transformation. Molecular weight of 100 kDa may be examined by electrophoresis in nondenaturing systems. The fusion protein was analyzed with enzyme-linked immunosorbant assay (ELISA), inhibition ELISA tests and Western blot, respectively. The humanized anti-idiotype minibody showed capacity of bivalent binding to ovarian cancer MAb COC166-9 and goat anti-human immunoglobulin IgG1. It is useful reagents for clinical use.

Animals↗

PubMed2003

[Monitoring novel ovarian carcinoma associated genes using cDNA expression microarray].

OBJECTIVE: To explore the gene expression pattern of sample of human ovarian carcinoma. METHOD: The difference in gene expression between normal and neoplastic human ovarian tissues were investigated, we described the assembly and utilization of a 512 member cDNA microarray. RESULT: Thirty-seven genes expressed in ovarian cancer were screened out, 14 genes were up-regulated, 23 genes were down-regulated. CONCLUSION: cDNA microarray for analysis of gene expression pattern is an effective method to identify novel ovarian cancer associated genes.

Female↗

PubMed2003

[Screening for ovarian cancer-associated genes with cDNA microarrays].

BACKGROUND & OBJECTIVE: The molecular mechanism leading to the development and progression of ovarian carcinoma are not completely understood. It may be the result of a series of molecular changes in the cell caused by changes in the expression level of numerous genes of tumor. In this report the authors used cDNA microarray to identify differentially expressed genes between ovarian cancer tissue and normal ovary tissue to screen ovarian cancer-associated genes. METHODS: cDNA microarrays were prepared by spotting PCR products of 512 cDNA of human oncogene and tumor suppressor gene onto specially treated glass slides with robotics. The probes were prepared by labeling normal tissue mRNA and cancer tissue mRNA with Cy3-dUTP and Cy5-dUTP separately through reverse transcription PCR. The arrays were then hybridized against the cDNA probe mixture and the fluorescent signals were scanned. The obtained data were analyzed using ImaGene 3.0 software. RESULTS: Thirty-eight genes showed co-expression specificity in 3 or more than 3 cases. There were 15 upregulated genes and 23 downregulated genes in ovarian cancer tissues. CONCLUSION: Utilizing cDNA microarrays, 3 ovarian cancer-associated genes have been initially screened.

Female↗

PubMed2003

[Immuno-therapeutic study of anti-idiotype minibody (single chain Fv-CH3) on ovarian carcinoma bearing mice].

OBJECTIVE: An anti-idiotypic minibody with optimal antigenicity which mimicking ovarian cancer antigen was used for therapeutic research in mice model bearing ovarian cancer. METHODS: Using gene engineering technique, prokaryotic expression vector was constructed by genetic fusion of 6B11scFv to human IgG1 hinge and CH3 region. The fusion protein named minibody was induced with IPTG in E. coli and analyzed with Western blot and inhibition ELISA tests respectively. Twenty human-PBL-SCID mice bearing i.p. Skov3.ip1 cells were divided into two groups (10 per-group), 10 mice were immunized repeatedly by minibody every two weeks for three times. Indirect ELISA test was employed for analyzing the characterization of anti-anti-idiotypic scFv (Ab3). The latent period of ascites growth and the mean survival time were observed respectively. CD4+ and CD8+ T cells from the spleen of immunized mice were assayed by flow cytometry. RESULTS: SDS-PAGE gel electrophoresis showed that a protein band with molecular weight of 50,000 appeared as the expected size after transformation and induction the host bacteria BL21 (DE3). The expressed minibody could be reacted with COC166-9 (Ab1 of 6B11) and binding goat anti-human IgG1 antibody in Western blot. Inhibition ELISA showed minibody had the capacity of binding ovarian cancer monoclonal antibody COC166-9 instead of primal antigen. Ab3 could be detected in the sera of immunized mice with minibody by ELISA test. Ab3 reached the highest at the 14th day after last vaccination and lasted for 6 weeks. The ratio of CD4+/CD8+ was the highest at the 13th day after last vaccination. The latent period of ascites growth were (37.7 +/- 5.5) days and (48.6 +/- 14.3) days (P = 0.04) respectively; while the mean survival time were (42.5 +/- 1.8) days and (59.4 +/- 16.8) days (P = 0.011) in the control and minibody group respectively. CONCLUSIONS: These results demonstrate the successful construction and expression minibody with good immune activities of 6B11scFv and human IgG1 molecules function. Antigenicity is increased without adjuvants and partial humanization is realized. Minibody can induce humoral anti-idiotypic immunity responses against ovarian carcinoma in vivo. When ascites formation was delayed or prevented and the survival was prolonged in minibody group. We expect that minibody may be used as tumor vaccine to ovarian carcinoma in the future clinical trails.

Animals↗

PubMed2002

Expression and purification of Rhizobium leguminosarum NodD.

A Rhizobium expression system was constructed via introducing strong transcriptional elements to an IncP broad host range plasmid pKT230. Using this system, the nodD gene of Rhizobium leguminosarum biovar viciae was overexpressed in its own in vivo environment. Western blot showed that the NodD yield of the newly constructed expression vector pKNDT was much higher than that of a reported vector pIJ1518. Based on the result from quantitative gel retardation assay, the specific DNA-binding activity of NodD was estimated to increase by about 80-fold. Purification of NodD was performed by the sequential steps: polyethyleneimine treatment, ammonium sulfate fractional precipitation, and cellulose phosphate P11 chromatography. About 26 mg NodD with a purity about 90% was obtained from 14 g wet weight cell pellet within 2 days.

Bacterial Proteins↗

PubMed2002

[Establishment and biological characteristics of an immortalized human ovarian sarcomatoid carcinoma cell line BUPH:OVSC-2].

BACKGROUND & OBJECTIVE: Cell immortalization technique has been an important method to establish cell lines with expected phenotype. However, there was few report about the establishment of immortalized human ovarian carcinoma cell lines. The purpose of this study was to establish an immortalized human ovarian sarcomatoid carcinoma cell line, and to investigate its biological characteristics. METHODS: A specimen derived from metastatic tumor of a human ovarian sarcomatoid carcinoma in abdominal wall was obtained and cultured in vitro. The 10th passage was transfected with SV40 T gene, colonies were isolated by G418 selection and expanded to immortalized cell lines. The morphology of the cells was observed by microscope and electromicroscope, Growth curve, karyotype analysis, culturing in soft agar, nude mice transplantation, immunohistochemistry, etc. were used to investigate its biologic characteristics. The biologic characteristics of BUPH: OVSC-2 were compared with its original cells. RESULTS: After transfection and selection, an immortalized ovarian carcinoma cell line BUPH: OVSC-2 was established. The cell line has been maintained for over 90 passages. The H&E stain finding of transplanted tumor showed that the cells were morphologically sarcomatoid. However, the transmission electron microscopic observation exposed its epithelial origin. The cells grew exuberantly and had high malignant characteristics. Comparative studies revealed that the biological properties of BUPH: OVSC-2 and its original cells were identical. CONCLUSIONS: BUPH: OVSC-2 was demonstrated as an immortalized human ovarian sarcomatoid carcinoma cell line with high malignancy. It retains similar properties of their parental cells and could be a useful model for the study of human ovarian sarcomatoid carcinoma.

Cell Division↗