Biomedical subjects

Ling-Xia Zhang

Publications and source records attributed to Ling-Xia Zhang.

8 recordsLinked to original sources

PubMed2005

Cloning and characterization of a novel hepatitis B virus core binding protein C12.

AIM: To elucidate the biological function of HBV core antigen (HBcAg) on pathogenesis of hepatitis B, a novel gene C12 coding for protein with unknown function interacting with HBcAg in hepatocytes was identified and characterized. METHODS: HBcAg bait plasmid pGBKT7-HBcAg was constructed and transformed into yeast AH109, then the transformed yeast was mated with yeast Y187 containing liver complementary DNA (cDNA) library plasmid in 2XYPDA medium. Diploid yeast was plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) and synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing X-alpha-gal for screening twice. After extracting and sequencing of plasmid from blue colonies, we isolated a cDNA clone encoding a novel protein designated as C12 that directly interacted with HBcAg. The interaction between HBcAg and C12 was verified again by re-mating. pEGFP-N1-C12 fluorescent protein fusion gene was transfected in 293 and L02 cell, and observed by fluorescent microscope. MTT reduction assay was used to study the action of C12 protein effect on metabolism of mammal cell. Yeast two-hybrid and cDNA microarray were performed to search binding protein and differential expression genes regulated by C12 protein. RESULTS: C12 gene was screened and identified by yeast two-hybrid system 3. The interaction between HBcAg and the novel protein coded by the new gene C12 was further confirmed by re-mating. After 48 h, fluorescence of fusion protein could be observed steadily in the 293 and L02 cell plasma. Under MTT assay, we found that the expression of C12 did not influence the growth of liver cells. Seventeen differential expression genes in HepG2 cells transfected with C12 protein expression plasmid by cDNA microarray, of which 16 genes were upregulated and 1 gene was downregulated by C12 protein. Twenty-one colonies containing 16 different genes coding for C12 protein binding proteins were isolated by yeast two-hybrid, there were 2 new genes with unknown function. CONCLUSION: The novel protein C12 is located in cell plasma, and its overexpression has no significant effect on the metabolism of liver cell. It interacts with many proteins in hepatocytes and may be involved in many processes of gene expression.

Amino Acid Sequence↗

PubMed2005

A microarray-based gastric carcinoma prewarning system.

AIM: To develop a microarray-based prewarning system consisting of gastric cancer chip, prewarning data and analysis software for early detection of gastric cancer and pre-cancerous lesions. METHODS: Two high-density chips with 8,464 human cDNA sites were used to primarily identify potential genes specific for normal gastric mucosa, pre-cancerous lesion and gastric cancer. The low-density chips, composed of selected genes associated with normal gastric mucosa, precancerous lesion and gastric cancer, were fabricated and used to screen 150 specimens including 60 specimens of gastric cancer, 60 of pre-cancerous tissues and 30 of normal gastric mucosa. CAD software was used to screen out the relevant genes and their critical threshold values of expression levels distinguishing normal mucosa from pre-cancerous lesion and cancer. All data were stored in a computer database to establish a prewarning data library for gastric cancer. Two potential markers brcaa1 and ndr1 were identified by Western blot and immunohistochemistry. RESULTS: A total of 412 genes associated with three stages of gastric cancer development were identified. There were 216 genes displaying higher expression in gastric cancer, 85 genes displaying higher expression in pre-cancerous lesion and 88 genes displaying higher expression in normal gastric mucosa. Also 15 genes associated with metastasis of gastric cancer and 8 genes associated with risk factors were screened out for target genes of diagnosis chip of early gastric cancer. The threshold values of 412 selected genes to distinguish gastric cancer, pre-cancerous lesion from normal gastric mucosa were defined as 6.01+/-2.40, 4.86+/-1.94 and 5.42+/-2.17, respectively. These selected 412 genes and critical threshold values were compiled into an analysis software, which can automatically provide reports by analyzing the results of 412 genes obtained by examining gastric tissues. All data were compiled into a prewarning database for gastric cancer by CGO software. Northern blot and immunohistochemistry analysis confirmed that gene and protein of brcaa1 displayed lower expression in normal gastric mucosa and higher expression in gastric cancer tissues, conversely, ndr1 displayed lower expression in gastric cancer and higher expression in normal gastric mucosa. CONCLUSION: The microarray-based prewarning system for gastric cancer was developed. This system consisted of gastric cancer-associated gene chip, prewarning data and analysis software, which has a high potential for applications in the early detection of gastric cancer. The two potential markers brcaa1 and ndr1 identified may be used to distinguish cancer status fand non-cancer status.

Antigens, Neoplasm↗

PubMed2004

[Mutual interaction between hepatitis B virus antigen and metallothionein].

OBJECTIVE: To screen and identify the protein interacting with HBV antigen in hepatocytes. Then investigate the biological functions of hepatitis B virus antigen in the pathogenesis of hepatitis B and seek effective methods to prevent and treat it. METHODS: The yeast two-hybrid system-3 technique was used to construct HBV PreS2, HBeAg, HBcAg, HBxAg bait plasmids. The bait plasmids transformed the yeast AH109 and expressed themselves in it. After being identified by SDS-PAGE and Western blot, the AH109 yeast was mated with yeast Y187 containing liver cDNA library plasmid in 2 x YPDA medium to form diploid yeast and was then plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) and synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing x-alpha-gal for screening. Plasmids of blue colonies were extracted and transformed into Escherichia coli, then analyzed by DNA sequencing and bioinformatics. To further prove the interaction between HBV antigen and metallothionein, translation was performed by using reticulocyte lysate and coimmunoprecipitation was displayed in vitro. RESULTS: Genes coding for HBV antigen binding protein were successfully cloned and metallothionein was found in that protein. The interaction between HBeAg, HBcAg and HBxAg and metallothionein were further proved by coimmunoprecipitation in vitro. CONCLUSION: The interaction between HBV antigen and metallothionein indicates that metallothionein may participate in the pathogenesis of hepatitis B

Hepatitis B↗

PubMed2004

[Genetic variation analysis of SARS coronavirus].

Severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV) has been verified as the causative agent of a worldwide outbreak of SARS. Since the observed per-base replication error-rate of RNA polymerase is about 3 x 10(-5), RNA virus populations typically contain genetic variants that form a heterogeneous virus pool. This feature confers great adaptability on viruses and is partly responsible for current difficulties of viral disease prevention and control, such as of HIV and HCV infections. SARS-CoV, a newly-identified single-strand RNA virus, has been identified with common variable property of RNA viruses. In this study, genetic variation of SARS-CoV is analyzed based on available documented data and our study. It suggests that SARS-CoV is of similar mutation frequency and dynamic mutant distributions like other RNA viruses, though relatively less identified variation number than those causing chronic infections. In contrast, the viral spike (S) gene exhibits high variation, while nucleocapsid (N) gene shows relative sequence conservation. No obvious correlation is observed between viral variation and clinical disease manifestation from present analyzed data.

Genetic Variation↗

PubMed2003

Screening of augmenter of liver regeneration-binding proteins by yeast-two hybrid technique.

OBJECTIVE: To investigate the biological function of augmenter of liver regeneration (ALR), we used yeast-two hybrid technique to detect proteins in hepatocytes interacting with ALR. METHODS: ALR bait plasmid was constructed by using yeast-two hybrid system 3, then transformed into yeast AH109. The transformed yeast was mated with yeast Y187 containing liver cDNA library plasmid in a 2XYPDA medium. Diploid yeast was plated on a synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing x-alpha-gal for selection and screening. After extracting and sequencing of the plasmid from blue colonies. Analysis was performed by bioinformatics. RESULTS: Of 36 colonies sequenced, 14 are metallothionein, 12 albumin, and 3 selenoprotein P. One colony is a new gene with unknown function. CONCLUSION: The successful cloning of gene of ALR interacting protein has paved the way for studying the physiological function of ALR and associated proteins.

Animals↗

PubMed2002

Preparation of human single chain Fv antibody against hepatitis C virus E2 protein and its identification in immunohistochemistry.

AIM: To screen human single chain Fv antibody (scFv) against hepatitis C virus E2 antigen and identify its application in immunohistochemistry. METHODS: The phage antibody library was panned by HCV E2 antigen, which was coated in microtiter plate. After five rounds of biopanning,56 phage clones were identified specific to HCV E2 antigen. The selected scFv clones were digested by SfiI/NotI and DNA was sequenced. Then it was subcloned into the vector pCANTAB5E for expression as E-tagged soluble scFv. The liver tissue sections from normal person and patients with chronic hepatitis B and chronic hepatitis C were immunostained with HCV E2 scFv antibody. RESULTS: The data of scFv-E2 DNA digestion and DNA sequencing showed that the scFv gene is composed of 750 bp. ELISA and immunohistochemistry demonstrated that the human single chain Fv antibody against hepatitis C E2 antigen has a specific binding character with hepatitis virus E2 antigen and paraffin-embedded tissue, but did not react with liver tissues from healthy persons or patients with chronic hepatitis B. CONCLUSION: We have successfully screened and identified HCV E2 scFv and the scFv could be used in the immunostaining of liver tissue sections from patients with chronic hepatitis C.

Amino Acid Sequence↗

PubMed2002

Cloning and expression of the gene of augmenter of liver regeneration in yeast cells.

OBJECTIVE: To study the function of augmenter of liver regeneration (ALR) as a regulatory factor that specifically stimulates hepatic cell regeneration, we constructed yeast expressive vector of ALR and expressed it in yeast cells. METHODS: Total RNA was extracted from HepG2 cells, and reverse transcription polymerase chain reaction (RT-PCR) was performed to amplify the coding region of ALR. The products were cloned into PGEM-T vector and sequenced, then cloned into PGBK T7 vector. The recombinant plasmid PGBK T7-ALR was transformed into yeast AH109. The yeast protein was extracted and analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting hybridization technique. RESULTS: DNA sequencing results confirmed that the coding region of ALR was correctly inserted into the yeast expression vector, and Western blotting assay showed that recombinant ALR was successfully expressed in yeast. Its molecular weight was identical to the theoretical value of 15 000 Da; the protein was found inside the yeast cells. CONCLUSION: The successful expression of ALR in yeast cells makes it possible to study further on its biological function.

Blotting, Western↗

PubMed2002

Cloning and expression of the preS1 gene of hepatitis B virus in yeast cells.

OBJECTIVE: To investigate the complex functions of HBV preS1 protein, we constructed HBV preS1 gene expression vector and expressed it in yeast cells. METHODS: Polymerase chain reaction (PCR) was performed to amplify the gene of HBV preS1 from the plasmid pCP10 containing the whole DNA fragment of HBV ayw subtype as template and the PCR product was cloned into the pGEM-T vector for sequencing. After being identified, the HBV preS1 gene was cut from the pGEM-T vector by EcoR I and Pst I restriction enzymes, and cloned into yeast expressive plasmid pGBKT7 to construct pGBKT7-preS1 recombinant expressive plasmid. This plasmid was transformed into yeast cell AH109 and expressed in it. The yeast protein was isolated and analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) and Western blotting. RESULTS: The HBV preS1 gene was amplified successfully and identified by DNA sequencing. The PCR products were coincided completely with the reported sequence. The digested fragments were cloned into the pGBKT7 vector and transformed into yeast cell AH109. The results of SDS-PAGE and Western blotting assay showed: (1) The HBV preS1 protein was expressed and existed in yeast cells; (2) The molecular weight of the expression product was about 30 000 D. CONCLUSION: The HBV preS1 gene was successfully cloned and expressed in yeast cells.

Blotting, Western↗