Effect of hepatitis C virus core protein on modulation of cellular proliferation and apoptosis in hilar cholangiocarcinoma

BACKGROUND: Hepatitis C virus (HCV) is believed to be an important human pathogen causing carcinoma. But the effect of HCV infection on the alteration of cellular pro- liferation and apoptosis and the relationship between the effect and the development of hilar cholangiocarcinoma are largely unknown. The aim of this study was to assess the effect of HCV core protein on proliferation and apoptosis of hilar cholangiocarcinoma. METHODS: HCV core protein (HCV C protein) was de- tected by peroxidase-antiperoxidase assay in surgical speci- mens from 48 patients with hilar cholangiocarcinoma. The apoptosis index ( AI) and PCNA index ( PI) in hilar cholangiocarcinoma were detected by in situ end labeling assay and streptavidin-biotin assay respectively. RESULTS: The expression of HCV C protein was observed in 32 (67.7%) of the 48 specimens of hilar cholangiocarci- noma. The mean ± standard deviation for AI and PI was 3.52%±0.64% and 46.24%±11.46% respectively. The AI of hilar cholangiocarcinoma specimens with HCV C protein expression was significantly lower than that of HCV C pro- tein negative specimens (P<0.01), whereas the PI of HCV C protein positive specimens was significantly higher than that of HCV C protein negative specimens (P<0.01). CONCLUSION: HCV C protein may promote the cellular proliferation of hilar cholangiocarcinoma and inhibit its cel- lular apoptosis.

Identification of the epitopes on HCV core protein recognized by HLA-A2 restricted cytotoxic T lymphocytes

AIM: To identify hepatitis C virus(HCV) core protein epitopes recognized by HLA-A2 restricted cytotoxic T lymphocyte (CTL). METHODS: Utilizing the method of computer prediction followed by a 4h(51)Cr release assay confirmation. RESULTS: The results showed that peripheral blood mononuclear cells (PBMC) obtained from two HLA-A2 positive donors who were infected with HCV could lyse autologous target cells labeled with peptide &quot;ALAHGVRAL (core 150-158)&quot;. The rates of specific lysis of the cells from the two donors were 37.5% and 15.8%, respectively. Blocking of the CTL response with anti-CD4 mAb caused no significant decrease of the specific lysis. But blocking of CTL response with anti-CD8 mAb could abolish the lysis. CONCLUSION: The peptide (core 150-158) is the candidate epitope recognized by HLAA2 restricted CTL.

Genes transactivated by hepatitis C virus core protein, a microarray assay

AIM: To explore the new target genes transactivated by hepatitis C virus (HCV) core protein and to elucidate the pathogenesis of HCV infection. METHODS: Reverse transcribed cDNA was subjected to microarray assay. The coding gene transactivated by HCV core protein was cloned and analyzed with bioinformatics methods. RESULTS: The expressive vector of pcDNA3.1(-)-core was constructed and confirmed by restriction enzyme digestion and DNA sequencing and approved correct. mRNA was purified from HepGZ and HepG2 cells transfected with pcDNA3.1(-)-core, respectively. The cDNA derived was subjected to microarray assay. A new gene named HCTP4 was cloned with molecular biological method in combination with bioinformatics method. CONCLUSION: HCV core is a potential transactivator. Microarray is an efficient and convenient method for analysis of differentially expressed genes.

Proapoptotic and pronecrosis effect of different truncated hepatitis C virus core proteins

Objective: To study the roles of different truncated hepatitis C virus (HCV) core proteins (CORE) in the pathogenesis of HCV persistent infection and hepatocellular carcinoma (HCC) and to assess intracellular localization in transiently transfected cells. Methods: Seven truncated CORE-GFP (green fluorescent protein) fusion protein expression plasmids were constructed, which contained HCV CORE sequences derived from tumor tissues (BT) and non-tumor tissues (BNT) from one patient infected with HCV. Amino acid (aa) lengths were BT: 1?172 aa, 1?126 aa, 1?58 aa, 59?126 aa, 127?172 aa; BNT: 1?172 aa and C191: 1?172 aa respectively. Subcellular localization of CORE-GFP was analyzed by con-focal laser scanning microscope. Apoptosis and necrosis were quantified by flow cytometry. Results: Different truncated CORE-GFP localized mainly in the cytoplasm, but nuclear staining was also observed. HCV CORE could induce apoptosis and necrosis, and different truncated COREs could induce cell apoptosis and necrosis at different levels. Among the same length 1?172 aa of BT, BNT and C191, the cell apoptosis and necrosis percentage of BT is highest, and C191 is the lowest (BT>BNT>C191). To the different fragment COREs of BT, N-terminal of CORE induced apoptosis and necrosis higher, compared with that of C-terminal (1?172 aa>1?126 aa>1?58 aa>127?172 aa>59?126 aa). Conclusion: These results suggest HCV CORE could induce apoptosis and necrosis of cells, which might play an important role in the pathogenesis of HCV persistent infection and HCC and the different CORE domains of dif- ferent HCV quasi-species might have some difference in their pathogenesis.

Screening and identification of interacting proteins with hepatitis B virus core protein in leukocytes and cloning of new gene C1

AIM： To investigate the biological function of HBcAg in pathogenesis of HBV replication in peripheral blood mononuclear cells （PBMCs）.METHODS： HBcAg region was amplified by polymerase chain reaction （PCR） and HBV HBcAg bait plasmid pGBKT7-HBcAg was constructed by routine molecular biological methods. Then the recombinant plasmid DNA was transformed into yeast AH109. After the HBV core protein was expressed in AH109 yeast strains （Western blot analysis）, yeast-two hybrid screening was performed by mating AH109 with Y187 containing leukocyte cDNA library plasmid. Diploid yeast cells were plated on synthetic dropout nutrient medium （SD/-Trp-Leu-His- Ade） （QDO） and synthetic dropout nutrient medium （SD/-Trp-Leu-His-Ade） （TDO）. The second screening was performed with the LacZ report gene （ yeast cells were grown in QDO medium containing X-a-gal）. The interaction between HBV core protein and the protein obtained from positive colonies was further confirmed by repeating yeast-two hybrid. After plasmid DNA was extracted from blue colonies and sequenced, the results were analyzed by bioinformatic methods.RESULTS： Eighteen colonies were obtained and sequenced, including hypermethylated in cancer 2 （3 colones）, eukaryotic translation elongation factor 2 （2 colones）, acetyl-coenzyme A synthetase 3 （1 colone）, DNA polymerase gamma （1 colone）, putative translation initiation factor （1 colone）, chemokine （C-C motif） receptor 5 （1 colone）, mitochondrial ribosomal protein L41 （1 colone）, kyot binding protein genes （1 colone）, RanBPM （1 colone）, HBeAg-binding protein 3 （1 colone）, programmed cell death 2 （1 colone）. Four new genes with unknown function were identified.CONCLUSION： Successful cloning of genes of HBV core protein interacting proteins in leukocytes may provide some new clues for studying the biological functions of HBV core protein.

Hepatitis C virus core proteins derived from different quasispecies of genotype 1b inhibit the growth of Chang liver cells

AIM: To investigate the influence of different quasispecies of hepatitis C virus (HCV) genotype 1b core protein on growth of Chang liver cells. METHODS: Three eukaryotic expression plasmids (pEGFP-N1/core) that contained different quasispecies truncated core proteins of HCV genotype 1b were constructed. These were derived from tumor (T) and non- tumor (NT) tissues of a patient infected with HCV and C191 (HCV-J6). The core protein expression plasmids were transiently transfected into Chang liver cells. At different times, the cell cycle and apoptosis was assayed by flow cytometry, and cell proliferation was assayed by methyl thiazolyl tetrazolium (MTT) assay. RESULTS: The proportion of S-phase Chang liver cells transfected with pEGFP-N1/core was significantly lower than that of cells transfected with blank plasmid at three different times after transfection (all P < 0.05). The proliferation ratio of cells transfected with pEGFP-N1/corewas significantly lower than that of cells transfected with blank plasmid. Among three different quasispecies, T, NT and C191 core expression cells, there was no significant difference in the proportion of S- and G0/G1-phase cells. The percentage of apoptotic cells was highest for T (T > NT > C191), and apoptosis was increased in cells transfected with pEGFP-N1/core as the transfection time increased (72 h > 48 h > 24 h). CONCLUSION: These results suggest that HCV genotype 1b core protein induces apoptosis, and inhibits cell- cycle progression and proliferation of Chang liver cells. Different quasispecies core proteins of HCV genotype 1b might have some differences in the pathogenesis of HCV persistent infection and hepatocellular carcinoma.

Lethality in mice infected with recombinant vaccinia virus expressing hepatitis C virus core protein

OBJECTIVE: To establish a mouse model of HCV core expression and investigate the toxicity of HCV core protein or the possible pathogenic effects. METHODS: A series of vaccinia viral expression vectors were engineered to express 5' portion of HCV genes including 5' non-translated region (NTR), core protein, and portion of the E1 gene. These HCV sequences were fused to a luciferase reporter gene and inserted into a vaccinia virus expression vector (pSC11) adjacent to the vaccinia virus promoter, p7.5. The recombinant DNA constructs were packed into infectious recombinant chimeric viruses. The expression of HCV core protein was examined in cultured cells after infection with these viruses. Death of the infected mice was investigated by specific correlation to the expression of HCV core protein and its expression levels. RESULTS: The recombinant virus (VNCE-LUA) expressed HCV core protein and an envelope-luciferase fusion protein in cultured cells. When Balb/c mice were inoculated intraperitoneally with more than 10~7 pfu per mouse of VNCE-LUA, death occurred immediately. The mortality was dependent on the amount of VNCE-LUA virus inoculated. All mice inoculated with 3×10~8 pfu of VNCE-LUA died within 4 days of infection and 50% of mice inoculated with 3×10~7 pfu of VNCE-LUA died within 7 days of infection. No death occurred in mice inoculated with 3×10~8 pfu of a control recombinant vaccinia virus, which expressed luciferase but not the HCV core and envelope proteins. Deletion of core sequences from VNCE-LUA rapidly reduced the mortality of infected mice whereas deletion of envelope sequence did not. SCID mice infected with VNCE-LUA died 2-3 days after infection, suggesting that the HCV-core induced mortality is not dependent on host T-or B-cell responses to core protein. CONCLUSIONS: HCV core protein can be lethal to mice when expressed in vivo and this specific lethality is independent of T-cells or B-cells. The findings and model itself provide a useful tool for further investigation on potential pathological effects as well as the potential toxicity of the HCV core protein.

The Identification of Three Sizes of Core Proteins during the Establishment of Persistent Hepatitis C Virus Infection in vitro

Similar to Hepatitis C virus (HCV) infection in humans, HCVcc infection can also result in persistent and chronic infection. The core protein is a variable protein and exists in several sizes. Some sizes of core proteins have been reported to be related to chronic HCV infection. To study the possible role of the core protein in persistent HCV infection, a persistent HCVcc infection was established, and the expression of the core protein was analysed over the course of the infection. The results show that there are three sizes of core proteins (p24, p21 and p19) expressed during the establishment of persistent HCVcc infection. Of these, the p21 core protein is the mature form of the HCV core protein. The p24 core protein is the phosphorylated form of p21. The p19 core protein appears to be a functional by-product generated during the course of infection. These three core proteins are all localized in the cytoplasm and can be encapsidated into the HCV virion. The appearance of the p19 and p24 core proteins might be related to acute HCVcc infection and chronic infection respectively and may play an important role in the pathology of a HCV infection.

Hepatitis C virus non-structural 5A protein can enhance full-length core protein-induced nuclear factor-κB activation

AIM： To study the effects of hepatitis C virus （HCV） core and non-structural 5A （NS5A） proteins on nuclear factor- k B （NF- k B） activity for understanding their biological function on chronic hepatitis caused by HCV infection. METHODS： Luciferase assay was used to measure the activity of NF-kB in three different cell lines cotransfected with a series of deletion mutants of core protein alone or together with NS5A protein using pNF- k B-Luc as a reporter plasmid. Western blot and indirect immunofluorescence assays were used to confirm the expression of proteins and to detect their subcellular localization, respectively. Furthermore, Western blot was also used to detect the expression levels of NF- k B/p65, NF- k B/p50, and inhibitor k B-a（k B-a）. RESULTS： The wild-type core protein （C191） and its mutant segments （C173 and C158） could activate NF- k B in Huh7 cells only and activation caused by （C191） could be enhanced by NS5A protein. Moreover, the full-length core protein and its different deletion mutants alone or together with NS5A protein did not enhance the expression level of NF- k B. The NF- k B activity was augmented due to the dissociation of NF-k： B-I k： B complex and the degradation of Ik B-a. CONCLUSION：NF- k B is the key transcription factor that can activate many genes that are involved in the cellular immune response and inflammation. Coexpression of the full-length core protein along with NS5A can enhance the NF- k B activation, and this activation may play a significant role in chronic liver diseases including hepatocellular carcinoma associated with HCV infection.

MicroRNA-185-5p mediates regulation of SREBP2 expression by hepatitis C virus core protein

AIM: To investigate the molecular mechanism for regulation of cholesterol metabolism by hepatitis C virus(HCV) core protein in Hep G2 cells.METHODS: HCV genotype 1b core protein was cloned and expressed in Hep G2 cells. The cholesterol content was determined after transfection. The expression of sterol regulatory element binding protein 2(SREBP2) and the rate-limiting enzyme in cholesterol synthesis(HMGCR) was measured by quantitative real-time PCR and immunoblotting after transfection. The effects of core protein on the SREBP2 promoter and 3'-untranslated region were analyzed by luciferase assay. We used different target predictive algorithms, micro RNA(mi RNA) mimics/inhibitors, and site-directed mutation to identify a putative target of a particular mi RNA.RESULTS: HCV core protein expression in Hep G2 cells increased the total intracellular cholesterol level(4.05 ± 0.17 vs 6.47 ± 0.68, P = 0.001), and this increase corresponded to an increase in SREBP2 and HMGCR m RNA levels(P = 0.009 and 0.037, respectively) and protein expression. The molecular mechanism studyrevealed that the HCV core protein increased the expression of SREBP2 by enhancing its promoter activity(P = 0.004). In addition, mi R-185-5p expression was tightly regulated by the HCV core protein(P = 0.041). Moreover, overexpression of mi R-185-5p repressed the SREBP2 m RNA level(P = 0.022) and protein expression. In contrast, inhibition of mi R-185-5p caused upregulation of SREBP2 protein expression. mi R-185-5p was involved in the regulation of SREBP2 expression by HCV core protein. CONCLUSION: HCV core protein disturbs the cholesterol homeostasis in Hep G2 cells via the SREBP2 pathway; mi R-185-5p is involved in the regulation of SREBP2 by the core protein.

Hepatitis C virus core protein modulates several signaling pathways involved in hepatocellular carcinoma

Hepatocellular carcinoma(HCC) is the fifth most common cancer, and hepatitis C virus(HCV) infection plays a major role in HCC development. The molecular mechanisms by which HCV infection leads to HCC are varied. HCV core protein is an important risk factor in HCV-associated liver pathogenesis and can modulate several signaling pathways involved in cell cycle regulation, cell growth promotion, cell proliferation, apoptosis, oxidative stress and lipid metabolism. The dysregulation of signaling pathways such as transforming growth factor β(TGF-β), vascular endothelial growth factor(VEGF), Wnt/β-catenin(WNT), cyclooxygenase-2(COX-2) and peroxisome proliferator-activated receptor α(PPARα) by HCV core protein is implicated in the development of HCC. Therefore, it has been suggested that this protein be considered a favorable target for further studies in the development of HCC. In addition, considering the axial role of these signaling pathways in HCC, they are considered druggable targets for cancer therapy. Therefore, using strategies to limit the dysregulation effects of core protein on these signaling pathways seems necessary to prevent HCV-related HCC.

Effect of Hepatitis C Virus Core Protein on Interferon-Induced Antiviral Genes Expression and Its Mechanisms

Emerging data indicated that HCV subverts the antiviral activity of interferon(IFN);however,whether HCV core protein contributes to the process remains controversial.In the present study,we examined the effect of HCV core protein on interferon-induced antiviral gene expression and whether the effect is involved in the activation and negative regulation of the Jak/STAT signaling pathway.Our results showed that,following treatment with IFN-α,the transcription of PKR,MxA and 2'-5'OAS were down-regulated in HepG2 cells expressing the core protein.In the presence of HCV core protein,ISRE-dependent luciferase activity also decreased.Further study indicated that the core protein could inhibit the tyrosine phosphorylation of STAT1,whereas the level of STAT1 expression was unchanged.Accordingly,SOCS3,the negative regulator of the Jak/STAT pathway,was induced by HCV core protein.These results suggests that HCV core protein may interfere with the expression of some interferon-induced antiviral genes by inhibiting STAT1 phosphorylation and induction of SOCS3.

Preliminary Analysis of Gene Expression Profiles in HepG2 Cell Line Induced by Different Genotype Core Proteins of HCV

In present investigation, we constructed recombinants expressing the HCV genotypes 1b, 2a, and 4d core proteins, and established human hepatocellular carcinoma （HepG2） cell line that expressed various genotype core proteins. The gene expression profiles in the cells expressing different HCV genotype core proteins were compared with those in the control by microarray analysis. In data analysis, a threshold was set to eliminate all genes that were not increased or decreased by 2.5-fold change in a comparison between the transfected cells and control cells. The preliminary microarray analysis suggests that the gene expression profiles regulated by three kinds of genotype core proteins are mainly involved in transport, signal transduction, regulation of transcription, protease activity, etc., and that some pathogenesis/oncogenesis gene expressions are up/down- regulated simultaneously in the HepG2 cell line. The data suggest that each core protein has its gene expressions profile and that the profiles are implicated in HCV replication and pathogenesis, which may open up a novel way to understand the function of the HCV variant core proteins biological and their pathogenic mechanism. Cellular ＆ Molecular Immunology. 2006;3（3）：227-233.

Analysis of core protein clusters identifies candidate variable sites conferring metronidazole resistance in Helicobacter pylori

Background:Metronidazole is one of the first-line drugs of choice in the standard triple therapy used to eradicate Helicobacter pylori infection.Hence,the global emergence of metronidazole resistance in Hp poses a major challenge to health professionals.Inactivation of RdxA is known to be a major mechanism of conferring metronidazole resistance in H.pylori.However,metronidazole resistance can also arise in H.pylori strains expressing functional RdxA protein,suggesting that there are other mechanisms that may confer resistance to this drug.Methods:We performed whole-genome sequencing on 121 H.pylori clinical strains,among which 73 were metronidazoleresistant.Sequence-alignment analysis of core protein clusters derived from clinical strains containing full-length RdxA was performed.Variable sites in each alignment were statistically compared between the resistant and susceptible groups to determine candidate genes along with their respective amino-acid changes that may account for the development of metronidazole resistance in H.pylori.Results:Resistance due to RdxA truncation was identified in 34%of metronidazole-resistant strains.Analysis of core protein clusters derived from the remaining 48 metronidazole-resistant strains and 48 metronidazole-susceptible identified four variable sites significantly associated with metronidazole resistance.These sites included R16H/C in RdxA,D85N in the inner-membrane protein RclC(HP0565),V265I in a biotin carboxylase protein(HP0370)and A51V/T in a putative threonylcarbamoyl–AMP synthase(HP0918).Conclusions:Our approach identified new potential mechanisms for metronidazole resistance in H.pylori that merit further investigation.

Construction and expression of hepatitis B virus vector encoding TC-tagged core protein

Virus tagged with greenfluorescent protein(GFP)contributes to the visualization and study of the virus in living cells.However,the hepatitis B virus(HBV)particle,which is a compact virion with limited internal space,cannot be incorporated with GFP tag as a large fragment.It was recently reported that protein genetically inserted with a smaller size tetracysteine(TC)tag could be specially labeled by a biarsenicalfluorescent dye in living cells.In this study,we constructed a recombinant HBV vector encoding TC-tagged core protein for biarsenical labeling of HBV virion.TC tag was genetically inserted near the immunodominant c/e1 site of HBV core protein by mutagenesis.Western blot and enzyme-linked immu-nosorbent assay(ELISA)analysis showed that the TC-tagged core protein,hepatitis B surface antigen(HBsAg)and hepatitis B e antigen(HBeAg)could be expressed in cells transfected with the recombinant HBV vector,which is similar to the cells transfected with wild-type HBV vector.Reverse transcription-polymerase chain reaction(RT-PCR)and Southern blot analysis showed that HBV virion formation was affected by the genetic insertion of TC tag into core protein in some degree,but cells transfected with the HBV vector could still produce HBV virions incorporated with TC-tagged core proteins.Taken together,the recombinant HBV vector can serve as a useful tool to produce HBV virions incorporated with TC-tagged core proteins to befluorescently labeled by biarsencial dye for visualizing and studying HBV in living cells.

Technology and mechanism of a new protein-based core sand for aluminum casting

The protein based binding material is from natural products, which is nontoxic and recyclable. This kind of green binder is earnestly needed by aluminum casting products. The new protein based core possesses higher strength and easier shakeout. Its tensile strength is close to that of common resin sands. The micro mechanism of protein binder was investigated by using infrared spectrum, chemical element analysis, SEM and thermal lost mass analysis.

Expression and characterization of hepatitis C virus core protein fused to hepatitis B virus core antigen

Recombinant plasmids were constructed by fusing the gene fragments encoding the full-length (l-191aa) and the truncated (l-40aa and l-69aa) HCV core proteins (HCc) respectively to the core gene of HBV at the position of amino acid 144 and expressed in E. coli. The products were analyzed by ELISA, Western blotting as well as the immunization of the mice. The results showed that those fusion proteins (B144C191, B144C69, B144C40) possessed the dual antigenicity and immunogenicity of both hepatitis B virus core antigen (HBcAg) and hepatitis C virus core protein (HCc). Analysis by electron microscopy and CsCl density gradient ultra-centrifugation revealed that similar to the HBcAg itself, all fusion proteins were able to form particles. Comparison of the antigenicity and immunogenicity of those fusion proteins showed that the length of HCc gene fused to HBcAg had no much effect on the antigenicity and immunogenicity of HBcAg, however, B144C69 and B144C40 induced higher titres antibodies against HCc than B144C191. Using those fusion proteins, ELISA for screening of antibodies against both HBV and HCV in human sera was also established.

Molecular characterization of suppression of hepatitis B virus transcription by hepatitis C virus core protein

To further elucidate the molecular mechanisms underlying the suppression of hepatitis B virus (HBV) expression by the hepatitis C virus (HCV) core protein, five molecular clones of HCV cDNA sequence con-taining the 5' noncoding (5'NC) and the core regions have been isolated from Chinese HBV- and HCV-coinfected pa-tients. Sequence comparison and phylogenetic analysis showed that the HCV sequence cloned from coinfected individu-als is indistinguishable from that identified in other patients. Cotransfection assay confirmed that the core protein ex-pressed from one of the cloned sequence is capable of suppressing the expression of hepatitis B surface and e antigens (HBsAg and HBeAg, respectively). Deletion mapping revealed that the C-terminal hydrophobic region of the HCV core is necessary for the suppression. Results from reporter assays demonstrated that HCV core protein interacts with the HBV C promoter and enhancer II elements and down-regulates the transcription of HBV as well as other cellular and heterologous viral genes in both hepatic and non-hepatic cell lines. Taken together, the findings suggest HCV core protein as a multifunctional negative regulator of transcription critically involved in the molecular interactions between HBV and HCV, and between HCV and the cell.

丙型肝炎病毒基因分型及core蛋白区的分子演化分析

目前有多种方法对丙型肝炎病毒进行基因分型,但尚无一个"金标准".为确定丙型肝炎病毒基因分型的最佳区域,从GenBank中筛选出15条对各成熟肽区域有注释,来源于不同国家地区的丙型肝炎病毒全基因组序列,分别对5′UTR区、core区、E1区、E2区及NS5B区建系统进化树.结果发现,以5′UTR区建树基因分型不完全正确,而以core区、E1区、E2区及NS5B区建树,基因分型均完全正确,但同一基因型间的核苷酸演化距离存在差异.计算5条1a型序列的core区、E1区、E2区、NS5B区的核苷酸演化距离并和全基因组序列核苷酸演化距离比较,结果发现,NS5B蛋白区基因分型最能反映病毒株间的演化关系.同时分析各序列的core区的分子演化,为发明针对core区的新的PCR-RFLP基因分型方法提供新思路.

丙型肝炎病毒核心蛋白core与其结合蛋白HCBP6在哺乳动物细胞中的相互作用

目的探讨丙型肝炎病毒(HCV)核心蛋白core与其结合蛋白HCBP6在哺乳动物细胞中的相互作用。方法分别扩增HCV核心蛋白core及其结合蛋白HCBP6的cDNA片段,克隆入pGEM-T载体。测序正确后应用双杂交技术把目的片段分别插入哺乳动物细胞双杂交系统的表达质粒pBIND和pACT中构建重组载体。将构建的pBIND-core和pACT-HCBP6重组载体和报告质粒pG5luc共转染HepG2细胞,并设背景对照组(pBIND+pACT)、阳性对照组(pBIND-Myod+pACT-Id)、2个阴性对照组(pBIND-core+pACT、pBIND+pACT-HCBP6)和空白对照组。采用Dual-荧光检测系统和TurnerBiosystemsVeritas微孔板化学发光检测仪检测荧虫素酶活性。结果成功构建重组载体pBIND-core和pACT-HCBP6,共转染HepG2细胞之后,其荧虫素酶活性与海肾素酶活性的比值与各对照组相比有统计学差异(P≤0·05)。结论HCV核心蛋白core与其结合蛋白HCBP6在肝癌细胞系中确有一定的相互作用,为进一步阐明HCV核心蛋白在细胞凋亡及细胞恶变中的作用机制提供了新的思路。