Level of CYP4G19 Expression Is Associated with Pyrethroid Resistance in Blattella germanica.

German cockroaches have become a large problem in the Shenzhen area because of their pesticide resistance, especially to pyrethroid. A pyrethroid called "Jia Chong Qing" to prevent pests for a long time were found to be resistant to "Jia Chong Qing" with resistance index of 3.88 measured using RT-PCR and immunohistochemistry analysis showed that both CYP4G19 mRNA and CYP4G19 protein expression levels in the wild strain were substantially higher than that of a sensitive strain. dsRNA segments derived from the target gene CYP4G19 were prepared using in vitro transcription and were microinjected into abdomens of the wild strain. Two to eight days after injection, the result showed that CYP4G19 mRNA expressions were significantly reduced in the groups injected with dsRNAs.

A promising alternative anti-HBV agent: the targeted ribonuclease.

HBV-targeted ribonuclease (TR) is a fusion of HBV core protein (HBVc) and human eosinophil-derived neurotoxin (hEDN). Introduction of TR by transfection or transduction into HepG2.2.15 cells (a cell model of HBV infection) revealed that it significantly reduces serological markers of HBV replication (including HBsAg, HBeAg and HBV DNA) in cell supernatants, suggesting that the targeted ribonuclease inhibits HBV replication. To further our understanding of the molecular mechanism of the anti-HBV effect of TR, we expressed TR in E. coli and found that purified TR possesses RNase activity and targeting activity. Furthermore, the antiviral effect of TR depends both on an enzymatically active hEDN and on the core domain. In or out of HepG2.2.15 cells, TR coassembles with the wild-type capsid protein into particles with internal hEDN domains. Our data suggest an intracellular ribonuclease activation mechanism that, owing to the characteristics of HBV morphogenesis, is highly virus specific. HBV may therefore be particularly vulnerable to the capsid-targeted viral inactivation approach.

[Prediction and modification analysis of low-affinity HLA-A*0201 restricted CTL epitopes derived from HIV-1 pol antigen].

AIM: To screen the possible HLA-A*0201 restricted low-affinity CTL epitopes derived from HIV-1 pol antigen and to predict and identify the possible change of the affinity between epitope and the HLA-A*0201 molecule when the epitope is modified. METHODS: HLA-A*0201 restricted low-affinity CTL epitopes were predicted by CTL epitope prediction software based on super motif, proteasome cleavage probability, HLA affinity and so on. The candidates were modified acid substitution and analyzed by computer. T2 cells were used to determine the peptide by amino binding affinity and HLA-A*0201-peptide complex stability. RESULTS: Among the three predicted peptides by softer ware, YVSLSFPQI (pol52-60Y1), YVSQIIEQL pol(673-681Y1), YIQKETWEA(pol548-556Y1)could bind to HLA-A*0201 with high affinity, and the dissociation time of 50% HLA-A*0201 peptide complex was over to 8 h. CONCLUSION: Our results suggest that the three predicted peptides, as modification, might be HLA-A*0201 restricted CTL epitopes.

Adenoviral-vector mediated transfer of HBV-targeted ribonuclease can inhibit HBV replication in vivo.

Hepatitis B virus (HBV)-targeted ribonuclease (HBV-TR) is a fused protein of HBV core protein and a ribonuclease, human eosinophil-derived neurotoxin (hEDN). Our previous results showed that HBV-TR could effectively inhibit HBV replication in vitro. To test whether HBV-TR can inhibit HBV replication in vivo, we constructed a recombinant adenoviral vector expressing HBV-TR (Ad-TR) and used it to treat HBV-transgenic mice. Immunohistochemical staining showed that TR was expressed at varied levels in different tissues of Ad-TR-treated mice. Serum HBsAg concentration was decreased by 64.8% for the Ad-TR-treated mice compared with empty adenoviral vector-treated control mice. The amount of HBV-DNA in the livers of the Ad-TR-treated mice was 0.74 x 10(7) copies/mug of genomic DNA while the amount of HBV-DNA in the livers of the empty adenoviral vector-treated control mice was 2.86 x 10(7) copies/mug of genomic DNA. Serum HBV-DNA of Ad-TR-treated mice was also decreased by 71.4% compared with empty adenoviral vector-treated control mice. In addition, for some Ad-TR-treated mice, the expression of HBsAg in the liver cells turned negative. No discernible adverse effects were observed for Ad-TR-treated mice. Taken together, our results indicated that adenovirus mediated transfer of HBV-TR can inhibit HBV replication in vivo.

Characterization of the genome sequence of an oncolytic Newcastle disease virus strain Italien.

We determined the complete genome sequence of strain Italien, a virulent and oncolytic strain of Newcastle disease virus (NDV) by direct nucleotide sequencing of RT-PCR products, a size of 15,186 nucleotides (nt). Comparison of six coding genes and non-coding regions of Italien with those of the other 25 sequenced strains revealed NDV Herts/33 was the most similar strain with Italien. The gene encoding the RNA dependent RNA polymerase was the most highly conserved, while the gene encoding phosphoprotein was the most highly variable. The HN and F proteins of Italien have been modeled on the crystal structure in order to study the structural characteristics. Interaction between the HN protein and the heptad repeat B (HRB) region of F protein was analyzed in silico by molecular docking predicted five critical residues I133, V142, D143, R480, and K567 on HN protein. Identification of amino acid residues that could be crucial for this interaction provides working hypotheses for subsequent studies.

[Cloning and expression of PbTIP analogous gene from Plasmodium berghei ANKA and preparation of its polyclonal antibody].

OBJECTIVE: To clone and express a novel protein analogous to TIP (T cell immunomodulatory protein) of Plasmodium berghei ANKA and prepare its polyclonal antibody. METHODS: The PbTIP encoding nucleotide sequence was searched from the Plasmodium berghei genomic database and amplified by PCR. The gene was sub-cloned into prokaryotic expression vector pGEX4T-1 and expressed in E.coli BL21 (DE3). After induction by IPTG, the expression of PbTIP-GST fusion protein was characterized by SDS-PAGE and Western blotting. The inclusion bodies of GST-PbTIP fusion protein were injected into BALB/c mouse. Anti-sera were identified by indirect fluorescent antibody test and western blotting. RESULTS: The PbTIP-GST fusion protein was successfully expressed in the form of inclusion bodies, by controlling the temperature and concentration of IPTG. Anti-PbTIP-GST sera were acquired with high titer. The sera specifically recognized the PbTIP with a band of 60 000 in P.berghei infected erythrocyte protein. CONCLUSION: PbTIP/GST fusion protein and polyclonal antibody have been obtained.

Targeted inhibition of HBV gene expression by single-chain antibody mediated small interfering RNA delivery.

UNLABELLED: RNA interference is highly effective at inhibiting HBV gene expression and replication. However, before small interfering RNA (siRNA) can be used in the clinic, it is essential to develop a system to target their delivery. Antibody-mediated delivery is a novel approach for targeting siRNA to appropriate cells. In this report, we asked whether this siRNA delivery strategy would be effective against HBV. Of 5 candidates, a specific siRNA that effectively inhibited HBV gene expression and replication was determined. Two fusion proteins, s-tP and sCkappa-tP, were constructed to contain a single chain of the human variable fragment, scFv, against hepatitis B surface antigen (HBsAg), a truncated protamine (tP), and in the case of sCkappa-tP, a constant region of the kappa chain (Ckappa). S-tP and sCkappa-tP were developed to provide targeted delivery of the siRNA, siRNA expressing cassettes (SEC), and siRNA-producing plasmids. Fluorescein isothiocyanate-siRNA, fluorescein isothiocyanate-SEC, and plasmid DNA were specifically delivered into HBsAg-positive cells using the sCkappa-tP fusion protein, and effectively inhibited HBV gene expression and replication. HBV gene expression was also inhibited by siRNA or siRNA-producing plasmids in HBV transgenic mice. CONCLUSION: Our results describe a potential method for the targeted delivery of siRNA or siRNA-producing plasmids against HBV, using anti-HBsAg fusion proteins.

Nickel compounds render anti-apoptotic effect to human bronchial epithelial Beas-2B cells by induction of cyclooxygenase-2 through an IKKbeta/p65-dependent and IKKalpha- and p50-independent pathway.

The carcinogenicity of nickel compounds has been well documented both in vitro and in vivo; however, the molecular mechanisms by which nickel compounds cause cancers are far from understood. Because suppression of apoptosis is thought to contribute to carcinogenesis, we investigated the mechanisms implicated in nickel-induced anti-apoptotic effect in human bronchial epithelial (Beas-2B) cells. We found that exposure of Beas-2B cells to nickel compounds resulted in increased cyclooxygenase-2 (COX-2) expression and that small interfering RNA (siCOX-2) knockdown of COX-2 expression resulted in increased cell sensitivity to nickel-triggered cell apoptosis, demonstrating that COX-2 induction has an anti-apoptotic effect on Beas-2B cells. Overexpression of IKKbeta-KM, a kinase inactive mutant of IKKbeta, blocked NF-kappaB activation and COX-2 induction by nickel compounds, indicating that activated NF-kappaB may be a mediator for COX-2 induction. To further explore the contribution of the NF-kappaB pathway in COX-2 induction and in protection from nickel exposure, mouse embryonic fibroblasts deficient in IKKbeta, IKKalpha, p65, and p50 were analyzed. Loss of IKKbeta impaired COX-2 induction by nickel exposure, whereas knockout of IKKalpha had a marginal effect. Moreover, the NF-kappaB p65, and not the p50 subunit, was critical for nickel-induced COX-2 expression. In addition, a deficiency of IKKbeta or p65 rendered cells more sensitive to nickel-induced apoptosis as compared with those in wild type cells. Finally, it was shown that reactive oxygen species H(2)O(2) were involved in both NF-kappaB activation and COX-2 expression. Collectively, our results demonstrate that COX-2 induction by nickel compounds occurs via an IKKbeta/p65 NF-kappaB-dependent but IKKalpha- and p50-independent pathway and plays a crucial role in antagonizing nickel-induced cell apoptosis in Beas-2B cells.

Cyclooxygenase-2 induction by arsenite is through a nuclear factor of activated T-cell-dependent pathway and plays an antiapoptotic role in Beas-2B cells.

Arsenite is a well known metalloid human carcinogen, and epidemiological evidence has demonstrated its association with the increased incidence of lung cancer. However, the mechanism involved in its lung carcinogenic effect remains obscure. The current study demonstrated that exposure of human bronchial epithelial cells (Beas-2B) to arsenite resulted in a marked induction of cyclooxygenase (COX)-2, an important mediator for inflammation and tumor promotion. Exposure of the Beas-2B cells to arsenite also led to significant transactivation of nuclear factor of activated T-cells (NFAT), but not activator protein-1 (AP-1) and NFkappaB, suggesting that NFAT, rather than AP-1 or NFkappaB, is implicated in the responses of Beas-2B cells to arsenite exposure. Furthermore, we found that inhibition of the NFAT pathway by either chemical inhibitors, dominant negative mutants of NFAT, or NFAT3 small interference RNA resulted in the impairment of COX-2 induction and caused cell apoptosis in Beas-2B cells exposed to arsenite. Site-directed mutation of two putative NFAT binding sites between-111 to +65 in the COX-2 promoter region eliminated the COX-2 transcriptional activity induced by arsenite, confirming that those two NFAT binding sites in the COX-2 promoter region are critical for COX-2 induction by arsenite. Moreover, knockdown of COX-2 expression by COX-2-specific small interference RNA also led to an increased cell apoptosis in Beas-2B cells upon arsenite exposure. Together, our results demonstrate that COX-2 induction by arsenite is through NFAT3-dependent and AP-1- or NFkappaB-independent pathways and plays a crucial role in antagonizing arsenite-induced cell apoptosis in human bronchial epithelial Beas-2B cells.

Immune responses in mice induced by prime-boost schemes of the Plasmodium falciparum apical membrane antigen 1 (PfAMA1)-based DNA, protein and recombinant modified vaccinia Ankara vaccines.

The apical membrane antigen 1 (AMA1) of malaria parasites is a leading vaccine candidate. Its expression in merozoites and sporozoites and its importance for erythrocyte and hepatocyte invasion underline the significance of both humoral and cellular immunities against this antigen in malaria protection. We have generated a DNA construct and a recombinant poxvirus (rMVA) for expressing the Plasmodium falciparum AMA1 ectodomain, produced recombinant AMA1 protein (rAMA1) and evaluated their antigenicity in mice using single and combinatory vaccine schemes. Our results showed that although vaccinations of mice by either DNA or rMVA alone did not yield high antibody responses, they had primed significant numbers of rAMA1-responsive splenocytes. Under heterologous prime-boost schemes, priming with DNA followed by boosting with rMVA or rAMA1 protein resulted in a significant increase in antibody titers. In addition, the antibody titers to AMA1 appeared to be correlated with the levels of inhibition of merozoite invasion of erythrocytes in vitro. Furthermore, different prime-boost schemes resulted in different AMA1-specific antibody isotype (IgG1/IgG2a) ratios, providing us with an indication about Th1 or Th2 responses the vaccination regimens have induced. This study has yielded useful information for further in vivo evaluation of the suitability and effectiveness of the heterologous prime-boost strategy in AMA1 vaccination.

Involvement of nuclear factor of activated T cells 3 (NFAT3) in cyclin D1 induction by B[a]PDE or B[a]PDE and ionizing radiation in mouse epidermal Cl 41 cells.

The results from animal studies have shown that mouse skin is highly susceptible to both ionizing radiation and benzo[a]pyrene-7,8-diol-9,10-epoxide (B[a]PDE). Previous studies have also indicated that cyclin D1 plays a crucial role in controlling cell proliferation and tumorigenesis. We, therefore, investigated here the effect of ionizing radiation and B[a]PDE on cyclin D1 transcription and potential involvement of NFAT3 in regulation of cyclin D1 transcription in mouse epidermal Cl 41 cells. We found that B[a]PDE exposure induced a high level of NFAT activation and cyclin D1 transcription in mouse epidermal Cl 41 cells. Ionizing radiation exhibited an enhancement for NFAT activation and cyclin D1 induction by B[a]PDE, even though ionizing radiation by itself had only a marginal effect. By stably knockdown of NFAT3 protein expression using specific NFAT3 small interfering RNA (siRNA), we found that cyclin D1 induction by B[a]PDE or B[a]PDE plus ionizing radiation was dramatically impaired. These results indicate that ionizing radiation is able to enhance cyclin D1 transcription induced by B[a]PDE, and NFAT3 is involved in the regulation of cyclin D1 transcription by B[a]PDE or B[a]PDE plus ionizing radiation.

[Adenovirus-mediated delivery of targeted ribonuclease against HBV replication in vitro].

OBJECTIVE: To observe the inhibitory effect of targeted ribonuclease delivered via adenovirus against HBV replication in vitro. METHODS: The shuttle plasmids pDC316 were constructed on the basis of the previous plasmids pcDNA3.1(-)/TRL, pcDNA3.1(-)/TR, pcDNA3.1(-)/TRmut, pcDNA3.1(-)/HBVc, and pcDNA3.1(-)/hEDN by subcloning the target gene sequences of TRL, TR, HBVc, and hEDN, respectively. HEK 293 cells were cotransfected with the pDC316 plasmids respectively in the presence of the rescue plasmid pBHGlox(delta)E1,3Cre to yield the recombinant adenoviral vectors which comprised the above genes. After transfection of HepG2.2.15 cells with the vectors, RAd/TRL expression was detected by indirect immunofluorescence staining and RT-PCR. Radioimmunoassay was used to analyse anti-HBV activity of RAd/TRL. RESULTS: Recombinant RAd vectors were prepared successfully. Effective expression of RAd/TRL in HepG2.2.15 cells resulted in a significant decrease of HBsAg and HBeAg concentration in comparison with the controls. CONCLUSION: Adenoviral vector-mediated targeted ribonuclease can effectively inhibit HBV replication.

Effects of polycyclic aromatic hydrocarbons (PAHs) on vascular endothelial growth factor induction through phosphatidylinositol 3-kinase/AP-1-dependent, HIF-1alpha-independent pathway.

Previous studies have demonstrated that exposure to polycyclic aromatic hydrocarbons (PAHs) and its derivatives is associated with an increased risk of skin cancers, and the carcinogenic effect of PAHs is thought to involve both tumor initiation and promotion. Whereas PAH tumor initiation is well characterized, the mechanisms involved in the tumor promotion of PAHs remain elusive. In the present study, we investigated the effects of PAHs on vascular endothelial growth factor (VEGF) expression by comparison of its induction between the active metabolite and its parent compound (B[a]PDE versus B[a]P) or between active compound and its relatively inactive analog (5-MCDE versus CDE). We found that exposure of cells to (+/-)-anti-benzo-[a]pyrene-7,8-diol-9,10-epoxide (B[a]PDE) or (+/-)-anti-5-methylchrysene-1,2-diol-3,4-epoxide (5-MCDE) led to marked induction of VEGF in Cl41 cells, whereas benzo[a]pyrene (B[a]P) or chrysene-1,2-diol-3,4-epoxide (CDE) did not exhibit significant inductive effects. Exposure of cells to B[a]PDE and 5-MCDE did not induce HIF-1alpha activation, whereas AP-1 was significantly activated. Moreover, overexpression of TAM67 (a dominant-negative mutant c-Jun) dramatically blocked that VEGF induction. Electrophoretic mobility shift assay showed that AP-1 was only able to specifically recognize and bind to its AP-1 potential binding site within -1136 and -1115 of the VEGF promoter region. Site-directed mutation of this AP-1 binding site eliminated the VEGF transcriptional activity induced by B[a]PDE, suggesting that the AP-1 binding site between -1136 and -1115 in the VEGF promoter region is critical for VEGF induction by B[a]PDE. In addition, overexpression of Deltap85 (a dominant-negative mutant PI-3K) impaired B[a]PDE- and 5-MCDE-induced VEGF induction. Considering our previous findings that PI-3K is an upstream mediator for c-Jun/AP-1 activation, we conclude that the VEGF induction by B[a]PDE and 5-MCDE is through PI-3K/AP-1-dependent and HIF-1alpha-independent pathways. These findings may help us to understand the mechanisms involved in PAH carcinogenic effects.

[Expression, purification and transduction of Tat-p53 fusion protein].

AIM: To express and purify Tat-p53 fusion protein and investigate its transduction efficiency. METHODS: The gene encoding wide-type p53 was isolated using RT-PCR from A549 cell line and cloned into pTAT-HA and pET32a prokaryotic expression vectors. Recombinant plasmids were transformed into E.coli BL21(DE3)LysS, then the transformed cells were induced with IPTG. The expression and purification of the Tat-p53 and p53 were analyzed by SDS-PAGE. BALB/c mice were immunized with purified p53 protein. The serum was isolated and the antibody specific to p53 was measured by ELISA. The transduction efficiency of Tat-p53 was detected using indirect immunofluorescence assay. RESULTS: Prokaryotic expression vectors of Tat-p53 and p53 were constructed correctly. Tat-p53 fusion protein and p53 protein were successfully expressed and purified. p53 specific mouse antiserum was obtained. IFA result indicated that Tat-p53 fusion protein transduced into HepG2 cells efficiently. CONCLUSION: The obtained Tat-p53 fusion protein may be valuable for the basic research on therapy for liver carcinoma.

[DNA/MVA combined immunization: antibody response to Plasmodium falciparum merozoite surface protein 1 in mice].

OBJECTIVE: To explore the effect of DNA/MVA combined immunization in enhancing antibody response to MSP1. METHODS: DNA vaccine and recombined MVA were constructed based on synthesized MSP1 gene (3D7). BALB/c mice were primed with DNA solely or together with GM-CSF expressing plasmid and then boosted with rMVA/ 190. Serum IgG and subtype IgG1 and IgG2a were assayed by ELISA. All mice were challenged with allelic replaced Plasmodium berghei. RESULTS: Antibodies to MSP1-190 were detected after DNA immunization with an end-point dilution titer of 1:2500. When GM-CSF plasmid was added, the antibody end-point dilution titer reached 1:11150, with an increase of 53 and 10 times respectively after MVA boosting. Among them anti-19000 antibodies were prominent, 1/4-1/3 of total IgG in serum. However, when the mice were challenged with Pb-PfM19 no prolonged survival was observed (P>0.05). CONCLUSION: High titer antibodies can be elicited in mice by using codon optimized MSPI gene and DNA/MVA combined immunization. The specificity and protection of these antibodies is being further investigated.

Effect of vector-expressed shRNAs on hTERT expression.

AIM: To study the effect of short hairpin RNAs (shRNAs) expressed from DNA vector on hTERT expression. METHODS: Oligonucleotides coding for four shRNAs against hTERT were cloned into a mammalian shRNA expression vector pUC18U6 to form pUC18U6ht1-4, which were then introduced into HepG2 cells by using liposome-mediated transfection. HepG2 cells transfected by pUC18U6 and pUC18U6GFPsir, which expressed shRNA against green fluorescent protein (GFP), were used as controls. hTERT mRNA in the transfected cells were quantified by using real-time fluorescent RT-PCR. RESULTS: Among the four shRNAs against hTERT, two decreased the hTERT mRNA level. Compared with the controls, pUC18U6ht which expressed the two shRNAs reduced hTERT mRNA by 39% and 49% (P<0.05). CONCLUSION: hTERT expression is inhibited by the shRNAs expressed from the DNA vector.

Anti-HBV activity of TRL mediated by recombinant adenovirus.

AIM: To investigate the inhibitive effect of hepatitis B virus (HBV)-TRL on HBV replication. METHODS: Based on previously constructed pcDNA3.1(-)/TRL, TR, TRmut, HBV core protein (HBVc) and hEDN, interest gene sequences TRL, TR, HBVc and hEDN were inserted into adenovirus shuttle plasmid pDC316 respectively and co-transfected HEK293 cells with rescue plasmid pBHGloxdeltaE1,3Cre to acquire RAd/TRL, TR, HBVc and hEDN. And then RAds were identified, amplified and the titers in HEK293 cells were determined. RAd/TRL and TR were named as the experimental groups, and others were control ones. After HepG2.2.15 cells were infected, RAd/TRL expression was identified by indirect immunofluorescence staining. Supernatant HBV-DNA content was determined by fluorescent quantification PCR. Meanwhile, metabolism of HepG2.2.15 cells was evaluated by MTT colorimetry. RESULTS: RAd vectors with distinct interest gene sequence were successfully constructed. Effective expression of RAd/TRL in HepG2.2.15 cells resulted in a significant decrease of supernatant HBV-DNA content compared to RAd/TR (0.63+/-0.14 vs 1.60+/-0.47, P = 0.0266, <0.05) and other control groups (0.63+/-0.14 vs 8.50+/-2.78, 8.25+/-2.26, 8.25+/-2.29, 8.50+/-1.51, 8.57+/-1.63, P<0.01). MTT assay suggested that there were no significant differences in cell metabolic activity between groups (P>0.05). CONCLUSION: The construction and expression of RAd/TRL has been achieved and it could inhibit HBV replication successfully, which has laid the foundation for further research on anti-HBV activity in vivo.

[Modulation of immune response to Plasmodium falciparum apical membrane antigen 1 DNA vaccine by cytokine plasmids].

OBJECTIVE: To explore the effect of cytokine encoding plasmids on DNA immunization in mice. METHODS: Prototype DNA vaccine VR1020/E which contain Plasmodium falciparum apical membrane antigen 1 (AMA1) ectodomain was constructed, and eukaryotic expression vectors pcDNA3/GM-CSF, pcDNA3.1(-)/IL-4, pIL-12 and pGM-CSF/pTPA-E were also built. BALB/c mice were immunized with VR1020/E alone or with VR1020/E plus different cytokine plasmids. Serum IgG and its subtype were determined by ELISA and in vitro splenocyte proliferation assay was done. RESULTS: GM-CSF, IL-4 and IL-12 encoding plasmids all promoted mice immune response to VR1020/E, the antibody level increased 7 to 10 times and splenocyte proliferation was enhanced too. Plasmid pcDNA3/GM-CSF induced much more IgG1 whereas plasmid pIL-12 induced much more IgG2a. CONCLUSION: Cytokine encoding plasmids might be used as adjuvant in AMA1 DNA immunization.

[Quantitation of human telomerase reverse transcriptase mRNA with real-time fluorescent RT-PCR].

AIM: To establish a real-time fluorescent RT-PCR assay to quantify human telomerase reverse transcriptase (hTERT) mRNA. METHODS: Total cellular RNA was isolated from HepG2 cells using Trizol reagent, which was then reverse-transcribed into cDNA. By using a pair of gene-specific primers and a TaqMan MGB probe, cDNA of hTERT was quantified with real-time fluorescent PCR. Human beta-actin (hBA) mRNA was quantified at the same time as an endogenous control. Serial dilutions of cloned human beta-actin gene were used to construct a standard curve. RESULTS: The correlation coefficient of the standard curve was 1.00. The mean coefficient of variation of the assay was 7.1%. The ratio of hTERT mRNA to hBA mRNA of HepG2 cells was (1.3+/-0.3)x10(-4). CONCLUSION: A real-time fluorescent RT-PCR assay to quantify hTERT mRNA was established, which will facilitate the study on the biological function of telomerase.