Separation and identification of differentially expressed nuclear matrix proteins in breast carcinoma forming.

BACKGROUND: Breast carcinoma is one of most prevalent malignant tumors occurring in women. Short of prevention, detection of breast carcinoma at an early, still curable stage would offer the best route to decrease its mortality rates. This highlights the urgent need for suitable biomarkers for early diagnosis and a better understanding of the disease pathogenesis. MATERIAL AND METHODS: NMPs were extracted from normal human breast tissue (Group I), from hyperplastic mammary tissue specimens (Group II), from atypical epithelial hyperplasia specimens (Group III), and from breast carcinoma (Group IV) tissue. Differential proteome profiles were established and analyzed by means of immobilized pH gradient-based two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The different NMPs were analyzed in the National Center for Biotechnology Information (NCBI) database with Mascot software. RESULTS: Well-resolved, reproducible 2-DE profiles of human breast tissues were obtained. Average protein spots were 904 +/- 58, 912 +/- 51, 931 +/- 63, 944 +/- 70 in Group I, Group II, Group III, and Group IV, respectively. Several different proteins were analyzed using mass spectrometry and bioinformation. Of these, 12 were well characterized. Compared to Group I, three proteins were up-regulated in Groups II, III, and IV, including Hsp27, prohibitin, and laminA/C. Upregulation was confirmed using Western blotting and immunohistochemical analysis. The correlation of prohibitin expression with clinicopathological features was also investigated. DISCUSSION: The proteins identified in this study may potentially prove to be useful markers for breast carcinoma diagnosis.

Expressions of nucleoside diphosphate kinase (nm23) in tumor tissues are related with metastasis and length of survival of patients with hepatocellular carcinoma.

OBJECTIVE: To evaluate the relationship of expressions of nucleoside diphosphate kinase (nm23) and proliferating cell nuclear antigen (PCNA), as well as apoptosis, with the prognosis of HCC patients by analyzing their pathological and clinical data. METHODS: The expressions of nm23 and PCNA were analyzed by immunohistochemistry and the apoptotic phenomena were detected by TUNEL technique in the liver samples from 43 HCC tissues, 39 para-neoplastic tissues, and 10 normal tissues. The mean apoptosis index (AI) and proliferative index (PI) in individual sample were calculated. RESULTS: As shown by the detection, 32.6% of carcinomas had negative nm23 signal in tumor tissues, whereas all para-neoplastic and normal tissues had positive nm23. The AI in nm23 positive HCC was significantly higher than that in nm23 negative one, with statistical difference (P<0.05). Furthermore, the expressions of nm23, and the values of AI and PI were contrastively analyzed with some main pathological and clinical data of HCC. It revealed that HCC with extrahepatic metastasis showed remarkable correlation with the negative nm23 (P=0.013) and higher PI values of HCC (P=0.015). The disease-free survival in HCC patients with negative nm23 expression was significantly poorer than that in patients with positive nm23 expression. CONCLUSIONS: These data suggest that expressions of nm23 protein in tumor tissues are correlated with occurrences of metastasis and length of survival of the HCC patients, which may be an indicator for their prognosis.

[Expression of keratin 8 in carbon tetrachloride-induced liver injury of mice].

OBJECTIVE: To investigate the expression of keratin 8 (K8) in carbon tetrachloride (CCl(4))-induced liver injury of mice. METHODS: Forty ICR mice were divided into four groups. CCl(4) 300 microl/kg body weight in olive oil was injected intraperitoneally for 0, 2, 4, 6 weeks in group A, B, C and D, respectively. Mice were sacrificed 3 d after the last injection and then the vital organs were collected and weighed. RT-PCR and immunohistochemistry methods were used to analyze the expression of keratin 8 in the liver. RESULTS: The ratios of liver and body weight were increased significantly after administration of CCl(4), which were 5.60 %, 6.87%, 7.83 % and 7.76% at 0, 2, 4 and 6 weeks after injection, respectively. The expression of K8 was increased at the 2 w, 4 w and 6 w after CL(4) administration. CONCLUSION: The expression of K8 is positively correlated with the liver injury induced by CCl(4). The accumulation of K8 may be involved in the mechanism of liver injury.

Anticancer activity of PDSS2, prenyl diphosphate synthase, subunit 2, in gastric cancer tissue and the SGC7901 cell line.

The aim of this study was to assess whether PDSS2 (prenyl diphosphate synthase, subunit 2), a candidate tumor suppressor protein, has a potential anticancer role in human gastric cancer tissue and the SGC7901 gastric cell line. A PDSS2 eukaryotic expression vector was constructed and introduced into SGC7901 cells. The relationship between PDSS2 expression and cell proliferation, cell cycle distribution, and apoptosis in tumor cells was analyzed by RT-PCR, western blotting, the MTT colorimetric assay, flow cytometry, and immunohistochemistry. Increased exogenous PDSS2 expression in vitro is associated with decreased cellular proliferation of the gastric cancer cell line SGC7901. PDSS2 also induced apoptosis in SGC7901 cells by causing cell cycle arrest in the G0/G1 phase. Moreover, a significantly low expression level of PDSS2 protein was found in gastric cancer. Decreased or absent expression of PDSS2 was showed in the gastric tumor biopsy samples analyzed, correlating with cancer differentiation. PDSS2 has potent anticancer activity in gastric cancer tissues and the SGC7901 cell line and is possibly involved in apoptosis in SGC7901 cells.

[Meta-analysis on peginterferon plus ribavirin in treatment of hepatitis C virus genotype 1 or 4 infection in HIV patients].

OBJECTIVE: To perform a Meta-analysis on peginterferon with interferon in treatment of HIV patients coinfected with refractory genotype HCV. METHODS: A literature search of Medline was conducted to identify eligible randomized controlled trials. Meta analysis was conducted to evaluate peginterferon and interferon in treatment of coinfected HCV genotype 1 or 4 in HIV patients. RESULT: Six trials of 88 matched the selection criteria. Total 1,131 patients with coinfection of HCV genotype 1 or 4 and HIV were included. Sustain viral response was higher in patients treated with peginterferon plus ribavirin compared with that of interferon plus ribavirin (26 % compared with 8 %) or peginterferon alone (26 % compared with 13 %). Severe adverse effects and withdrawal rates were similar for patients treated with peginterferon and patients treated with interferon. CONCLUSION: Peginterferon plus ribavirin in treatment of patients with coinfection of genotype 1 or 4 HCV and HIV can achieve higher sustain viral response and the likelihoods of serious adverse effects and withdrawal rates are similar to other therapies.

Proteomic analysis of HuH-7 cells harboring in vitro-transcribed full-length hepatitis C virus 1b RNA.

AIM: The present study examined the differential expression of proteins in HuH-7 cells and HuH-7 cells harboring in vitro-transcribed full-length hepatitis C virus 1b RNA (HuH-7-HCV), and elucidated the cellular responses to HCV replication. METHODS: The protein profiles of matched pairs of HuH-7-HCV cells and HuH-7 mock cells were analyzed by 2-D electrophoresis (2DE). Solubilized proteins were separated in the first dimension by isoelectric focusing, and by 12.5% SDS-PAGE in the second dimension. The differential protein expression was analyzed by use of image analysis software to identify candidates for HCV infection-associated proteins. RESULTS: In total, 29 protein spots showed increases and 25 protein spots showed decreases in signal in HuH-7-HCV cell 2DE profiles as compared with HuH-7 mock cells. In the next step, the 10 spots showing the greatest increase and the 10 spots showing the greatest decrease were excised from gels and the proteins present were identified by Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometer (MALDI-TOF MS) or MALDI-TOF/TOF MS. In total, 13 proteins were identified successfully. The potential significance of the differential expression due to HCV replication was discussed. CONCLUSION: Our study identifies changes in the proteome of HuH-7 cells in the presence of HCV replication and yields information of the mechanism of HCV pathogenesis. These results will be useful for the identification of HCV infection-associated proteins that could be molecular targets for treatment.

Over-expression of nm23-H1 in HeLa cells provides cells with higher resistance to oxidative stress possibly due to raising intracellular p53 and GPX1.

AIM: To determine whether the antitumor factor nm23 is related with antioxidation. METHODS: Full-length human nm23-H1 was cloned into a mammalianexpressing vector and transiently introduced into HeLa cells. RESULTS: A remarkably low level of reactive oxygen species (ROS) was detected in the cells overexpressing nm23-H1. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and trypan blue assays found that the cells transfected with a nm23- H1-expressing plasmid had higher viability and stronger resistance to oxidative stress. Immunoprecipitation tests revealed that endogenous nm23-H1 formed a protein complex with p53. Furthermore, the intracellular levels of p53 and p53- regulated gene GPX1 were obviously increased in the cells overexpressing nm23- H1. The downregulation of p53 in the cells overexpressing nm23-H1 resulted in a higher cellular ROS level and lower cell viability. CONCLUSION: The findings suggest that nm23-H1 may act as a cellular protector against oxidative stress, possibly triggering the p53-related antioxidative pathway.

T-2 toxin-induced apoptosis involving Fas, p53, Bcl-xL, Bcl-2, Bax and caspase-3 signaling pathways in human chondrocytes.

OBJECTIVE: To investigate the effects of T-2 toxin on expressions of Fas, p53, Bcl-xL, Bcl-2, Bax and caspase-3 on human chondrocytes. METHODS: Human chondrocytes were treated with T-2 toxin (1-20 ng/ml) for 5 d. Fas, p53 and other apoptosis-related proteins such as Bax, Bcl-2, Bcl-xL, caspase-3 were determined by Western blot analysis and their mRNA expressions were determined by reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: Increases in Fas, p53 and the pro-apoptotic factor Bax protein and mRNA expressions and a decrease of the anti-apoptotic factor Bcl-xL were observed in a dose-dependent manner after exposures to 1-20 ng/ml T-2 toxin, while the expression of the anti-apoptotic factor Bcl-2 was unchanged. Meanwhile, T-2 toxin could also up-regulate the expressions of both pro-caspase-3 and caspase-3 in a dose-dependent manner. CONCLUSION: These data suggest a possible underlying molecular mechanism for T-2 toxin to induce the apoptosis signaling pathway in human chondrocytes by regulation of apoptosis-related proteins.

[Differential expression of genes related to transcription in cultured hepatoma cells with intact genome of hepatitis C virus].

OBJECTIVE: To investigate the effects of hepatitis C virus (HCV) on transcription regulation genes of host cells by gene chip assays in cultured cells with intact HCV genome. METHODS: Huh-7 hepatoma cells were cultured and infected with in vitro constructed HCV. The total RNAs, proteins and cell culture supernatants of HCV infected cells and control cells were isolated. Proteins and cell culture supernatants were used to detect the HCV replication and protein expression in cell culture system. The HCV protein expression was detected with Western blotting. Released HCV from infected cells was analyzed by real-time fluorescence quantitative PCR. Total RNA was qualified using 10 g/L agarose gel electrophoresis. cRNA was synthesized, fluorescence labeled and purified, then hybridized with Agilent oligo microarray (20173 probes). Differential expression of genes related to transcription in cell culture system was analyzed. RESULT: HCV was positive in cell culture supernatants and HCV protein expression was also positive according to Western blotting results. Eleven up-regulated and 11 down-regulated genes related to transcription were found after Agilent gene chip screening. CONCLUSION: Intact hepatitis C virus cell culture system provides an useful tool for study on the affects of HCV infection on transcription regulation genes in host cells.

Detection of human papillomavirus type 16 DNA in formalin-fixed, paraffin-embedded tissue specimens of gastric carcinoma.

OBJECTIVES: Human papillomavirus type 16 (HPV16) is regarded as one of the important tumor-related viruses, which are known to have a role in cervical carcinoma; however, there are few reports on HPV16 in gastric carcinoma (GC). Our study aimed to investigate the relationship between HPV16 and the occurrence of GC. METHODS: Liquid PCR (LPCR) and in-situ PCR (ISPCR) methods were carried out to detect the HPV16 oncogene E6 cell-type-specific enhancer in the long control region of HPV16 in 40 GCs and corresponding gastric adjacent normal mucosa (GANM). The patients were from Shaanxi Province in China; Helicobacter pylori (Hp) was detected by immunohistochemistry and by hematoxylin and eosin staining in their gastric tissues. RESULTS: The HPV16 E6 gene was detected in 37.5% (15/40) of the GCs and 5% (2/40) of the GANMs with LPCR, as was the cell-type-specific enhancer; however, the positive rate of E6 was 27.5% (11/40) in GCs and 0% (0/40) in GANMs, respectively, with ISPCR. HPV16 DNA was mainly located in the nucleus of gastric glandular epithelium cells. The infection rate of HPV16 DNA in GCs was higher than that in GANMs (P=0.0004), and the HPV16 had no statistical correlations with sex, age, invasion, grading or lymph node metastasis (P>0.05). The infection rate of HPV16 in cardiac GCs was significantly higher than that in noncardiac ones (P=0.0136), and HPV16 had no correlation with Hp in GCs (P=0.0829). Receiver operator characteristic curve analysis indicated that there was no statistical difference between the LPCR and ISPCR methods in our study through optimizing parameters in ISPCR procedures (P=0.768). CONCLUSIONS: These findings suggested that HPV16 can infect gastric glandular epithelium cells and that viral infection might play a role in the occurrence of GCs independent of or without the cooperation of an Hp infection.

[Current status and perspective of hepatitis C virus models].

Lack of proper study models has brought difficulties in the study of the mechanism of viral infection, life cycle and pathogenic mechanism of hepatitis C virus (HCV) and also become the major obstacles in development of efficient vaccine and new drugs for hepatitis C. In recent years, the establishment of robust HCV cell culture infection system and HCV transgenic animal provide powerful tools for the analysis of host virus interactions, which facilitate the discovery of antiviral drugs and vaccines for this important human pathogen.

[Effect of NO and Fas pathway on T-2 induced apoptosis in chondrocytes].

OBJECTIVE: To investigate the relationship of T-2 toxin-induced chondrocytes apoptosis with nitric oxide(NO) and Fas apoptosis pathway. METHODS: Human chondrocytes cultured in vitro were treated with different concentrations of T-2 toxin at different time (1-5 d). Cell viability of the treated cells was measured by MTT assay. Apoptotic ultrostructural changes of the treated cells were observed with electron microscopy. Biological changes of apoptosis were detected by annexin V/PI Flow cytometer (FCM). The levels of NO in culture media were detected by colorimetric method of Griess assay. Nitric oxide synthase (iNOS) and Fas protein were measured by Western blot. RESULTS: In this study the results shown the dose-dependent and time-dependent effects of T-2 toxin, within a range of concentration (1-2000 ng/mL) and a period of time (1-5 d), on the T-2 toxin-treated chondrocytes. Apoptotic body was found in T-2 toxin-treated chondrocytes by electron microscopy. Early-stage apoptosis rate and late-stage apoptosis rate were both increased in T-2 toxin-treated cells when compared with non-treated cells in a dose-dependent manner. The levels of NO in T-2 toxin-treated culture media were higher than that of normal control. Over-expressions of iNOS and Fas protein were detected in T-2 toxin-treated cells. T-2 toxin-induced apoptosis was noted to be significtnly correlated with the level of NO production and the levels of iNOS and Fas protein expression. CONCLUSION: T-2 toxin can enhance NO production and upregulate the expression of iNOS and Fas protein. Both NO and Fas signaling pathway are involved in T-2 toxin-induced apoptosis.

[Inheritance instability of chromosome 6q in patients with gastric cancer].

OBJECTIVE: To investigate the possible correlation between the microsatellite DNA instability (MSI), loss of heterozygosity (LOH ) on the long arm of chromosome 6 (6q) and the clinicopathologic features in Chinese patients with gastric cancer. METHODS: PCR-SSLP-Silver Stain method was used to detect four loci MSI and LOH in gastric tumor and paired normal control tissues of twenty and seven cases. RESULTS: In our study, the positive of MSI at one or more loci were observed in 16 out of 27 informative individuals (59.2%). 7 cases of 11 informative individuals without MSI were detected to happen the LOH at one or more loci (63.6%). And both MSI and LOH were frequently resulting at loci D6S434 (6q16.3-q21) and D6S404 (6q16.3-q23.2). MSI and LOH occurred in both well-differentiated and poor- differentiated gastric cancers, with a slight tendency of high grade MSI (MSI-H) existing chiefly in well-differentiated gastric cancer. CONCLUSION: The existence of MSI and/or LOH on 6q in 80% gastric carcinoma suggests that the genetic instability of 6q plays an important role in Chinese gastric cancer. The frequency of MSI and LOH has no significant relationship to clinicopathologic features such as age, gender, histological grade of differentiation and stages of tumor. The critical region of allelic deletion on 6q in Chinese gastric cancer is similar to those in other countries, and further confirms the presence of a putative tumor suppressor gene in this region.

Correlation of serum leptin levels with anthropometric and metabolic parameters and biochemical liver function in Chinese patients with chronic hepatitis C virus infection.

AIM: To determine serum leptin levels and investigate their correlations with anthropometric and metabolic parameters and biochemical liver function in patients with chronic hepatitis C virus (HCV) infection and their potential clinical implications. METHODS: Forty-two chronic HCV-infected patients without anti-viral treatment were enrolled in this study, 30 patients had chronic hepatitis C, 10 had cirrhosis, and 2 had hepatocellular carcinoma (HCC). Thirty age- and sex-matched healthy individuals served as controls. Serum leptin levels were determined by ELISA. The biochemical liver function and serum lipids were determined at the same time. The height and body weight of patients and controls were measured, and body mass index (BMI) and body fat were calculated simultaneously. The correlations of serum leptin levels with anthropometric and metabolic parameters and biochemical liver function were assessed statistically. RESULTS: The mean of serum leptin levels in patients with chronic hepatitis C, HCV-associated cirrhosis, HCV-associated HCC and control groups was (6.13+/-3.94), (5.25+/-4.21), (4.17+/-0.28), and (3.59+/-3.44) ng/mL, respectively. The serum leptin level in patients with chronic hepatitis C was significantly higher than that in controls. The serum leptin levels between cirrhotic patients and controls and between male and female cirrhotic patients had no significant difference. Serum leptin levels were positively-correlated with body fat, BMI, and apolipoprotein B (Apo B) in patients with chronic HCV infection. The serum alanine aminotransferase (ALT) levels were closely-correlated with BMI in patients with chronic hepatitis C. CONCLUSION: HCV infection interferes with fat and lipid metabolism in patients with chronic HCV infection and leptin may play a role in hepatosteatosis.

Transcriptional activation function of hepatitis B virus Pre S1 protein in yeast.

OBJECTIVE: To explore the feasibility of cloning of the hepatocyte receptor interacting with the Pre S1 protein of HBV by two-hybrid system. METHODS: Yeast expression plasmids encoding fusion proteins of full length or portions of Pre S1 of HBV and DNA binding domain of yeast protein GAL4 were constructed and used to transform yeast reporter strain SFY526. Reporter gene product beta-galactosidase activity was assayed as a measure of transcriptional activation in yeast. Mammalian expression plasmid encoding fusion proteins of full length Pre S1 and DNA binding domain of GAL4 was constructed and used to cotransfect hepatoma cell line Huh-7 together with CAT reporter plasmid. Cell extracts were assayed for CAT activity by thin-layer chromatography. RESULTS: The fusion proteins of full length Pre S1 protein and GAL4 DNA binding domain presented transcriptional activation function in yeast. The transcription activating sequence was localized to the 21 to 47 amino acids of Pre S1 protein. Fusion proteins of full length Pre S1 and GAL4 DNA binding domain did not show transcriptional activation function in mammalian cells. CONCLUSIONS: The transcription activating sequence of HBV Pre S1 protein in yeast overlaps the hepatocyte receptor binding site. The transcriptional activation function of HBV Pre S1 protein in yeast may prevent researchers from using yeast two-hybrid system to clone HBV receptor interacting with Pre S1 protein. However, the Pre S1 protein does not show transcriptional activation function in mammalian cells. Mammalian two-hybrid system may be a practical method to clone the HBV hepatocyte receptor interacting with Pre S1 protein.

[Bioinformatics analysis on CK8 full length coding sequence and eukaryotic expression in SMMC7721 cells].

AIM: To construct an eukaryotic expression vector containing the coding region of human full length cytokeratin 8 gene and to detect its expression in SMMC7721 cells. METHODS: CK8 cDNA was amplified by RT-PCR and cloned to pMD18-T simple vector. After confirming the sequence, the cDNA was inserted into pEGFP-C1 and the positive clone pEGFP-CK8 was obtained. The recombinant plasmid was transfected into SMMC7721 cells with Lipofectamine(TM);2000 and the expression was detected by fluorescence microscope, real time PCR and Western blot. The physical-chemical properties, signal peptide and functional motifs were predicted by the bioinformatics software. RESULTS: PCR, restriction enzyme digestion and DNA sequencing showed that the recombinant plasmid contained the coding region of full length CK8 gene. Observation under fluorescence microscope and the results of real time PCR and western blot indicated CK8 was over-expressed in SMMC7721 cells. CONCLUSION: The eukaryotic expression vector containing the CK8 gene was successfully constructed and expressed, which provides a basis for the study for biological function of CK8.

Human prion protein mutants with deleted and inserted octarepeats undergo different pathways to trigger cell apoptosis.

Octarepeats region sequence is one of the most important characteristics of PrP topology. To explore the mechanism of deleted and inserted octarepeats mutants PrP-caused apoptosis, wild-type PrP (PrP-PG5), and PrP with deleted octarepeats (PrP-PG0) and with four (PrP-PG9) and seven (PrP-PG12) extra octarepeats were transiently induced into SH-SY5Y cell. The results indicated PrP-PG9 and PrP-PG12 mainly retained in fraction of cytoplasm, while PrP-PG5 and PrP-PG0 presented both in cell membrane and cytoplasm. Cells expressing PrP-PG9 and PrP-PG12 were sensitive to endoplasmic reticulum (ER) stimuli, tunicamycin, and brefeldin A. ER-stress-related proteins, Grp94, XBP1, TRAF2, and CHOP, were significantly increased in cells expressing PrP-PG9 and PrP-PG12, while Grp78 increased markedly 12 h and pro-caspase-12 decreased sharply 20 h post-transfection. It indicates that expressions of PrP mutants with inserted octarepeats cause ER stress and lead to cell apoptosis lately. Meanwhile, cellular Cytochrome C increased and Bcl-2 decreased obviously in cells expressing PrP-PG0, indicating triggering a mitochondrial-related apoptosis. These data highlight that PrP mutants in region of octarepeats may undergo different pathways to trigger cell apoptosis, in which PrPs with inserted octarepeats via ER stress and PrP mutant without octarepeats via mitochondrial-related pathway.

[Cloning and expression of human keratin 8 gene cDNA in E.coli].

AIM: To clone and express human keratin 8 gene cDNA in E.coli. METHODS: Human cytokeratin 8 gene cDNA was amplified by RT-PCR from genomic RNA of human cell line 7721. The amplified cytokeratin 8 gene cDNA was cloned into pMD18-T vector. Then, the CK8 cDNA was amplified by PCR from recombinant plasmid pMD18-CK8, and was subcloned into pET-28a(+) expression vector. The recombinant plasmid pET-28a-CK8 DNA was transformed into E.coli DH5alpha strain. RESULTS: Human cytokeratin 8 gene cDNA was cloned, and the recombinant plasmid pMD18-CK8 was transformed into E.coli. The CK8 cDNA was subcloned into E.coli DH5alpha strain, and successfully expressed in E.coli. CONCLUSION: Human cytokeratin 8 gene cDNA is cloned, and successfully expressed in E.coli, which lay the foundation of further study on the CK8 biological properties and functions.

The correlations between HPV16 infection and expressions of c-erbB-2 and bcl-2 in breast carcinoma.

Breast carcinoma (BC) is a prevalent malignant tumour occurring in women. Many studies have indicated the role of human papilloma virus type 16 (HPV16) in the pathogenesis of BC; however, the correlations of HPV16 infection with the clinicopathologic features of BC and the expressions of c-erbB-2 and bcl-2 have not yet been elucidated. In this study, HPV16 was detected by amplifying the HPV16 E6 gene by the polymerase chain reaction method, and the expressions of c-erbB-2 and bcl-2 in 40 BCs and 20 normal breast tissue samples, obtained from Shaanxi Province, were examined using the streptavidin-peroxidase method with monoclonal antibodies specific to c-erbB-2 and bcl-2. The infection rate of HPV16 E6 and the positive expression rate of c-erbB-2 were significantly higher in the BCs than in the normal tissues (HPV16 E6: 60% vs. 5%; c-erbB-2: 42.5% vs. 5%, P < 0.05). However, the positive expression rate of bcl-2 was significantly lower in the BCs than in the normal tissues (67.5% vs. 95%, P < 0.05). The infection rate of HPV16 did not correlate with any of the pathological features observed (P > 0.05). HPV16 infection correlated with bcl-2 expression (P = 0.015) but not with c-erbB-2 expression (P = 0.747) in the BCs. Interestingly, HPV16 infection correlated with bcl-2 expression in grade I BCs (P = 0.018) but not in grade II-III BCs (P = 0.633). Our data suggest that HPV16 infection is correlated with bcl-2 expression in BCs.

[Prokaryotic expression and immunogenicity of hWAPL, a norel oncogene related to cervical cancer].

AIM: To construct the prokaryotic expression plasmid of hWAPL, induce the expression of the protein and prepare polyclonal antibody. METHODS: The hWAPL cDNA was amplified by RT-PCR from HeLa cells derived RNA and then cloned to pMD18-T according to A-T. The DNA fragment was isolated and linked to prokaryotic expression vector pET28a. The recombinant protein was induced by IPTG and identified by SDS-PAGE. Then it was injected into mice to prepare polyclonal antibody. The immunogenicity and specificity of the recombinant protein were identified by ELISA and Western blot. RESULTS: The sequencing, PCR and endonucleases digestion results showed that the hWAPL fragment was correctly inserted into pMD18-T and pET28a vectors. SDS-PAGE showed 25,000 fusion hWAPL protein was expressed in BL21 cells. The titer of polyclonal antibody was 1:3 200 by indirect ELISA. The 25,000 fusion hWAPL protein was detected by SDS-PAGE and Western blot. CONCLUSION: The hWAPL protein can be expressed by prokaryotic vector pET28a. Furthermore, the expression protein can be used as immunogen to generate antibodies with specificity.