Characteristics analysis of hepatitis B core-related antigen in children with hepatitis B e antigen-positive chronic viral hepatitis B infection.

BACKGROUND: The objective of antiviral therapy for chronic viral hepatitis B infection (CHB) is to achieve a functional cure. An important viral marker in the serum of patients with CHB is the serum hepatitis B core-related antigen (HBcrAg). However, there is limited research on HBcrAg in juvenile patients with CHB. In this study, we aimed to investigate the correlation between serum HBcrAg and other hepatitis B virus (HBV) markers in children with CHB and its predictive significance for prognosis during antiviral therapy. METHODS: A single-center retrospective study was conducted involving 79 children with CHB, aged between 0 and 16 years. All the children were treated with interferon [or combined nucleos(t)ide analogs] for 48 weeks. HBcrAg, hepatitis B surface antigen (HBsAg), and HBV DNA were measured before treatment, and at 12 and 48 weeks after treatment. The enrolled children were classified into the seroclearance group and the nonseroclearance group based on the therapeutic outcome. RESULTS: HBsAg seroclearance was observed in 28 out of 79 patients and hepatitis B e antigen seroconversion without HBsAg seroclearance was observed in 14 out of 79 patients following the conclusion of the treatment, with baseline HBcrAg titer levels showing no statistical significance in both the seroclearance and nonseroclearance groups (P = 0.277). HBsAg and HBV DNA were positively correlated with HBcrAg in children with CHB (R2 = 0.3289, 0.4388). The area under the receiver operating characteristic curve of the decrease in HBcrAg at 12 weeks of treatment as a predictor of seroclearance at 48 weeks of treatment, exhibited a value of 0.77. CONCLUSION: A decrease in serum HBcrAg levels in children with hepatitis B serves as a prognostic indicator.

Prediction for HBsAg seroconversion in children with chronic hepatitis B.

BACKGROUND: To establish a prediction of HBsAg seroconversion in children with chronic hepatitis B (CHB), so as to help clinicians to choose therapeutic strategy. METHODS: A total of 63 children with HBeAg-positive CHB aged 1 to 17 years, who admitted to the fifth medical center of Chinese PLA general hospital and treated with interferon α (IFNα) 48 weeks were enrolled, the clinical data were measured. Based on the results of HBsAg seroconversion (HBsAg < 0.05 IU/mL and anti-HBsAg > 10 IU/L) at week 48, the patients were divided into HBsAg seroconversion (S) group and non-HBsAg seroconversion (NS) group. Multivariate COX regression was used to identify the impact factors associated with HBsAg seroconversion. A novel prediction index was established and the area under the receiver operating characteristic curve (AUROC) was used to assess the prediction for HBsAg seroconversion. RESULTS: The 63 patients were divided into S group (20.6%, 13/63) and NS group (79.4%, 50/63). Univariate and multivariate analysis identified age, baseline intrahepatic cccDNA and serum HBsAg levels were independent impact factors for HBsAg seroconversion. Intrahepatic cccDNA was positively correlated with serum HBsAg (r = 0.464, p = 0.000). AUROC of HBV cccDNA was 0.83 (95% CI 0.71 to 0.95) and AUROC of baseline HBsAg was 0.77 (95% CI 0.61 to 0.92). Intrahepatic cccDNA ≤ 0.08 log10 copies/106 cell is regarded as cutoff value, the positive predictive value(PPV) and negative predictive value(NPV) for HBsAg seroconversion were 86.8% and 60.0%, respectively, with a sensitivity of 92.0% and specificity of 56.2%. HBsAg ≤ 3.68 log10 IU/mL is used as cut off value, the PPV and NPV for HBsAg seroconversion were 91.2% and 56.3%, respectively; the sensitivity and specificity was 86.0% of 69.2%, respectively. There was no statistical difference between them for predicting HBsAg seroconversion (p = 0.146). CONCLUSIONS: HBsAg seroconversion can be predicted by the baseline serum HBsAg or intrahepatic cccDNA in children with CHB. Using the index, clinicians can choose more reasonable therapeutic strategy and reduce the waste of medical resources.

IL28B SNP rs12979860 is the Critical Predictor for Sustained Viral Response in Chinese Children Aged 1 to 6 Years with Chronic Hepatitis C.

Clinical data on children with chronic hepatitis C (CHC) remain extremely limited. This study investigated sustained virologic response (SVR) to alfa-interferon 2b plus RBV treatment in children aged 1-6 years with unsafe injection-acquired CHC. 154 children with CHC aged 1 to 6 years were enrolled, 101 of them were male (65.6%) and 53 were female (34.4%), and they were treated with alfa-interferon at a dose of 1-5 MIU/m2 3 times weekly plus oral RBV (15 mg/kg/day) for 48 weeks. 57(39.3 %) of them were genotype 1b, 73(50.3%) were genotypes 2a, 15(10.3%) were undecided type. SVR was achieved in 53 of 57(93.0%) patients with genotype 1b, in 72 (98.6%) of 73 with genotype 2a, 15(100.0%) of 15 with undecided type. There was no significant statistical difference in SVR between male and female (98.0% vs 94.3%, p=0.340), genotype 2a and those with genotype 1b(98.6% vs 93.0%, p=0.160), ALT>40U/L group and ALT≤40U/L group(96.7% vs 96.8%, p=1.000), AST>40U/L group and AST≤40U/L group(95.9% vs 98.2%, p=0.654) as well as lower baseline viral load group (<6×105 IU/ml) and higher baseline viral load group(≥6×105 IU/ml)(97.3% vs 95.3%, p=0.916). Leucopenia, neutropenia, hemoglobin concentration decrease, fever, platelet count decrease and rash were 8.4%, 69.5%, 24.0%, 50.6%, 1.9% and 4.5%, respectively. And only 12(7.8%) individuals developed thyroid autoantibodies. The SVR was higher in patients with IL-28B genotype C/C than C/T (99.0% vs 80%, p=0.002). Compared with HCV viral genotype, ALT level and baseline viral load, IL-28B rs12979860 is more suitable for predicting antiviral efficacy in children with CHC. It is inappropriate to take the increase of ALT level as an essential indicator for antiviral treatment in children aged 1-6 years.

Peripheral blood mononuclear cell traffic plays a crucial role in mother-to-infant transmission of hepatitis B virus.

The role of peripheral blood mononuclear cells (PBMCs) in HBV intrauterine infection is not fully defined. Particularly the origin of PBMCs in HBV-infected neonates remains to be addressed. We carried out a population-based nested case-control study by enrolling 312 HBsAg-positive mothers and their babies. PBMC HBV DNA as well as serum HBsAg and HBV DNA was tested in cohort entry samples. Totally, 45.5% (142/312) of the newborns were found to be infected with HBV in perinatal transmission. 119 mother-infant pairs were identified to be different in the genetic profile of maternal and fetal PBMCs by AS-PCR and hemi-nested PCR. Among them, 57.1% (68/119) of the maternal PBMCs in index cases were positive for HBV DNA while 83.8% (57/68) of the HBV DNA positive maternal PBMCs passed the placental barrier and entered the fetus. Furthermore, maternal PBMC HBV infection was significantly associated with newborn infants HBV infection. PBMC traffic from mother to fetus resulted in a 9.5-fold increased risk of HBV infection in PBMC HBV DNA positive newborn infants. These data indicate that maternal PBMCs infected with HBV contribute to HBV intrauterine infection of newborn infants via PBMC traffic from mother to fetus.

Combining Oxymatrine or Matrine with Lamivudine Increased Its Antireplication Effect against the Hepatitis B Virus In Vitro.

Some recent clinical reports have shown that the combination of oxymatrine, a phyto-derived drug, with lamivudine (3TC) could improve its curative effect against hepatitis B virus (HBV) infection. However, the experimental data in support of this combination strategy are lacking. In this study, we investigated the anti-HBV activity of the combination of 3TC and either oxymatrine or matrine on HepG2 2.2.15 in vitro. The activities of the combination and the solo compound, each in different concentrations, were compared on the 3rd, 6th, and 9th experimental days. The cytotoxicity results showed that the nontoxic concentrations of both oxymatrine and matrine to HepG2 2.2.15 cells were 800 µg/mL. We found that the single use of oxymatrine below 100 µg/ml, matrine below 200 µg/ml, and 3TC below 30 µg/ml showed weak inhibitory effects on the secretion of hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg), and HBV-DNA in culture media; the combination of 3TC (30 µg/ml) with oxymatrine (100 µg/ml) or matrine (100 µg/ml) showed significant inhibitory effects that were higher than or equivalent to the single use of 3TC at 100 µg/ml. The results provide a new impetus to develop novel, multicomponent anti-HBV drugs through the combination of natural products with nucleoside analogs to enhance their activity.

Evaluation of the liver protection and toxicity of Da-Huang-Zhe-Chong pill in rats.

CONTEXT: Da-Huang-Zhe-Chong pill (DHZCP), a classical traditional Chinese formula, consists of 12 crude drugs which have been widely used with significant therapeutic effects. Some drugs in this formula have toxicities that might result in some adverse effects of DHZCP. OBJECTIVE: The liver protection and toxicity of DHZCP were first evaluated against chronic carbon tetrachloride (CCl(4))-induced liver injury in rats. MATERIALS AND METHODS: The rats were treated by intraperitoneal injection of 10% CCl(4) for 12 weeks. At the end of week 4, DHZCP at doses of 44 g/kg (high-dose group) and 22 g/kg (low-dose group) was intragastrically administered to CCl(4)-treated rats, once a day for four weeks. At the end of weeks 8 and 12, the general status of the rats, histopathology of liver, serum alanine aminotransaminase (ALT), aspartate aminotransaminase (AST), alkaline phosphatase (ALP) and total bilirubin (TBIL) levels were observed or determined and recorded. By correspondence analysis (CA) on biochemical markers and liver histopathological score (HS), the "dose-time-response" relationship of DHZCP on the hepatic injury rats was evaluated. RESULTS: The results showed that DHZCP exhibited a significant protective effect on liver injury by reversing the biochemical parameters and histopathological changes. However, this hepatoprotective effect may be weakened, or even be transferred to toxicity with the increase of the administration dose (44 g·kg(-1)·d(-1)) and time (more than 2 months) of this formula. These results were consistent with the histopathological observation and the serum levels. DISCUSSION AND CONCLUSION: Administration of proper dose and time of DHZCP could well play its hepatoprotective effect and even treat hepatitis, but the safety on liver should be considered when large-dose (44 g·kg(-1)·d(-1)) DHZCP is used for long time (more than 2 months). We suggest that the administration dose and time of DHZCP in clinical use should not be increased and prolonged, and simultaneously liver function should be regularly monitored.

Virologic and clinical characteristics of HBV genotypes/subgenotypes in 487 Chinese pediatric patients with CHB.

BACKGROUND: The association of hepatitis B virus (HBV) genotypes/subgenotypes with clinical characteristics is increasingly recognized. However, the virologic and clinical features of HBV genotypes/subgenotypes in pediatric patients remain largely unknown. METHODS: Four hundred and eighty-seven pediatric inpatients with CHB were investigated, including 217 nucleos(t)ide analog-experienced patients. HBV genotypes/subgenotypes and reverse transcriptase (RT) mutations were determined by direct sequencing. The stage of fibrosis and degree of inflammatory activity were evaluated by the Metavir score system. RESULTS: Among 487 enrolled pediatric patients, HBV genotype C2 and B2 were the most two prevalent (73.7% and 21.1%). Comparing with HBV/B2 infected patients, no significant difference was observed in the incidence rate and mutant patterns of lamivudine- or adefovir-resistant mutations in HBV/C2 infected patients (P > 0.05). Importantly, we found that the degree of hepatic inflammation degree, fibrosis stage and ALT level were significantly higher in HBV/C2-infected HBeAg positive patients than it was in HBV/B2-infected ones. CONCLUSIONS: The pediatric patients with HBV/C2 infection might be more susceptible to develop severe liver pathogenesis.

Cloning, expression and identification by immunohistochemistry of humanized single-chain variable fragment antibody against hepatitis C virus core protein.

Expression of single-chain variable fragment (scFv) antibodies on the surface of bacteriophage is widely used to prepare antibodies with pre-defined specificities. A phage antibody library containing the gene for scFv antibody against Hepatitis C virus core protein was panned with core protein immobilized on microtiter plate wells. After five rounds of panning 60 phage clones specific to core protein were obtained and one selected clone was sequenced. It was found that the specifically detected antigen consists of 774bp and is capable of encoding 257 amino acids in the patients but not in healthy persons.

Investigation of the "dose-time-response" relationships of rhubarb on carbon tetrachloride-induced liver injury in rats.

AIM OF THE STUDY: The present study investigated the liver protection and toxicity of rhubarb against carbon tetrachloride (CCl4)-induced chronic liver injury in rats. MATERIALS AND METHODS: The rats were treated by intraperitoneal injection of 10% CCl4 for 12 weeks. At the end of week 4, rhubarb at doses of 40 g kg(-1) (high-dose group), 20 g kg(-1) (medium-dose group) and 10 g kg(-1) (low-dose group) was intragastrically administered to CCl4-treated rats once a day for three weeks. At the end of week 16, all rats were maintained for 1 month without any administration. At the end of weeks 8, 12, 16 and 20, the general status of rats, histopathology of liver, serum alanine aminotransaminase (ALT), aspartate aminotransaminase (AST), total bilirubin (TBIL) and hyaluronic acid (HA) levels were observed, respectively. Combined with clustering analysis and correspondence analysis, the "dose-time-response" relationships of rhubarb on the liver injury rats were synthetically investigated. RESULTS: High dose (40 g kg(-1)) of rhubarb exhibited a significant protective effect on injured liver by reversing the biochemical parameters and histopathological changes. But, this hepatoprotective effect will be weakened, even be transferred to toxicity with increasing the administration dose and time of rhubarb. These results were consistent with the histopathological observation and the determination of serum levels. CONCLUSIONS: The safety should be considered simultaneously in the long-term and high dose use of rhubarb, the liver function and change should be regularly detected. This study provided some useful references for the clinical rational use of rhubarb and other herbal medicinal products.

[Comparative analysis of drug resistant mutations in the reverse transcriptase domain of hepatitis B virus covalently closed circular DNA and the viral relax circle DNA].

OBJECTIVE: To establish a method for detection of reverse transcriptase region of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA), and to compare the pattern and frequency of drug-resistant mutations in the region between intrahepatic HBV cccDNA and serum HBV relax circle DNA (rcDNA). METHODS: HBV DNA were extracted from liver biopsy tissues of 20 patients with chronic hepatitis B. The RT region of HBV cccDNA was amplified by rolling circle amplification (RCA) followed by polymerase chain reaction (PCR) mediated by a pair of primers spanning across the gap region of HBV genome. The RT region of serum HBV rcDNA from the same patient was amplified by nested-PCR. The PCR products were directly sequenced and analyzed by Vector NTI Suite 8.0 and chromaslite 201 software. x2 test was used for statistical significance analysis of drug-resistant mutation occurrences between the HBV cccDNA and rcDNA. RESULTS: The RT regions of HBV cccDNA were successfully amplified from liver tissues of all enrolled patients using the RCA plus PCR assay. Simultaneously, HBV the RT regions of rcDNA were amplified from these patients serum samples. Sequence analysis showed that the drug-resistant mutations were significantly more frequently detected in HBV rcDNA (40%) than in HBV cccDNA (10%) (P<0.05). Different mutational patterns were observed between the HBV cccDNA and rcDNA in a few cases. CONCLUSION: The RCA in combination with PCR is a practical method for the detection of drug-resistant mutation in the RT region of HBV cccDNA. Drug-resistant mutational patterns could be discrepant between HBV cccDNA and rcDNA.

[Development of a high-efficient assay for amplifying Vbeta-encoding genes of human T-cell receptor].

AIM: To develop an effective assay for amplifying human T-cell receptor (TCR) variable region of beta chain (Vbeta)-encoding genes. METHODS: Based on the property of the 26 subfamilies of human TCR Vbeta-encoding gene sequence, 34 sets of outer and 37 sets of inner sense primers were divided into 8 degenerate primer groups, and a set of outer and inner antisense primers located in conserved region beta chain (Cbeta) was designed for the amplification of the TCR Vbeta-encoding genes. In addition, a sequencing primer and a sense primer for amplifying Cbeta-encoding genes were designed. CD8 T-cell RNA was extracted and subjected to reverse transcription mediated by poly A, followed by a nested polymerase chain reaction (PCR) to amplify the 26 subfamilies of human TCR Vbeta-encoding genes. Jurkat T lymphoma cells were used as control. T-easy vector was employed to detect DNA sequencing of the cloned target genes. RESULTS: All the subfamilies of the Vbeta-encoding genes were obtained from CD8 T cells of a healthy donor, except the subfamily of Vbeta8 , was obtained from Jurkat cells. The results were confirmed by DNA sequencing of the cloned genes. CONCLUSION: The nested RT-PCR assay can effectively amplify human TCR Vbeta-encoding genes with broad spectrum, which is helpful for the cloning and functional study of the TCR expressed by antigen-specific cytotoxic T lymphocytes.

[Epitope screening of influenza A (H3N2) by using phage display library].

OBJECTIVE: To screen the influenza A (H3N2) mimotopes by using phage display library. METHODS: Using influenza A (H3N2) monoclonal antibody as selective molecule, a 7 mer phage peptide library was biopanned and positive clones were selected by ELISA, competition assay and DNA sequencing. RESULTS: 21 positive clones were chosen for DNA sequencing. From the experiment and sequencing comparison results, one epitope was comfirmed as mimotope of influenza A (H3N2). CONCLUSION: Influenza A (H3N2) mimotope was obtained by phage peptide library screening. The result provide a new approach for new Influenza virals vaccine development.

[Analysis and recombinant construction of HBV the reverse transcriptase gene with drug-resistant mutations from 40 patients with chronic hepatitis B].

OBJECTIVE: To analyze genetic mutation associated with drug resistance in the reverse transcriptase (RT) domain of HBV from 40 patients with chronic hepatitis B, and to construct mutant RT gene recombinant vectors for drug-resistant phenotypic analysis. METHODS: HBV DNA was extracted from sera of the 40 patients receiving anti-HBV nucleot (5) ide analogue. The complete RT domain-encoding gene was amplified by nested PCR, and then cloned into pGEM-T-easy vector. Three to Five clones were randomly selected for DNA sequencing. Data were analyzed by UNASTAR software. The pTriEx-HBV (C) 1.1 expression vectors were constructed by replacing the 1250-hp Xho I/Nco I fragments containing complete RT domain from individual patients samples. RESULTS: All samples were detected with drug-resistant mutations associated with lamivudine, adefovir, and entacavir singly or in combination. Ninety-six mutant RT genes were cloned into pGEM-T-easy vector, from which 40 major mutant RT genes were replaced into pTriEx-HBV (C) 1.1 expression vectors. The construction was confinned to be successful by verifying mutation existence using DNA sequencing, and detectable HBsAg and HBeAg in the cell supernatant after transfecting recombinant expression vectors into Huh7 cells. CONCLUSION: The analysis of drug-resistant mutation and the construction of mutant-recombinant expression vectors were successfully implemented using the samples frum clinical patients. The work lays a foundation for drug-resistant phenotypic analysis of HBV mutants.

[Quantitative detection of hepatitis B virus covalently closed circular DNA in sera of chronic hepatitis B patients with a newly established assay].

OBJECTIVE: To quantitatively detect hepatitis B virus covalently closed circular DNA (HBV cccDNA) in sera of chronic hepatitis B patients with a newly established assay. METHODS: Primers and probe were designed in highly conservative region of HBV DNA. DNA was extracted from 175 sera samples of chronic hepatitis B patients, and was treated with plasmid-Safe-ATP-dependent Dnase(PSAD) to eliminate the relaxed circular DNA (rcDNA). The products were amplified by real-time PCR with primers spanning. RESULTS: The detection rate of serum HBV cccDNA was found to correlate directly with serum HBV DNA loading. HBeAg positive chronic hepatitis B patients had higher serum HBV cccDNA levels than HBeAg negative chronic hepatitis B patients. CONCLUSION: The method is good because of the high specificity. It can be used for detection of HBV cccDNA. DNA;

[Multiple-site analysis of HBV drug-resistant mutations in 340 patients with chronic hepatitis B].

OBJECTIVES: To analyze HBV drug-resistant mutations against nucleos(t)ide analogues at 12 reported sites in 340 patients with chronic hepatitis B. METHODS: Serum HBV DNA was extracted and a nested PCR assay was employed for the reverse transcriptase (RT) gene amplification. Direct sequencing of PCR product was performed. The significance of detected mutations was analyzed in view of clinical data of the patients. RESULTS: Drug-resistant mutations were detected in 68 patients taking lamivudine (LAM), 10 taking adefovir (ADV), 8 taking entecavir, and 1 taking telbivudine (LdT). M204V and M204I were the most common LAM-resistant mutations. The former usually emerged with L180M while the latter often emerged alone. N236T +/- A181 substitution was the most frequently seen ADV-resistant mutation. ETV-resistant mutations occurred on the basis of LAM-resistant mutations and T184 change was the most common form. LdT-resistance was observed as M204I. Interestingly, these drug-resistant mutations were detected in a few patients who had not been treated with nucleos(t)ide analogues. CONCLUSION: Detection of HBV drug-resistant mutations at multiple sites of the viral RT gene is valuable for discovering and verifying drug resistance and thus is very helpful in planning anti-HBV therapy.

Association between the frequency of class II HLA antigens and the susceptibility to intrauterine infection of hepatitis B virus.

Multiple factors determine the susceptibility to intrauterine hepatitis B virus (HBV) infection. These factors include the HBV structure, HBV mutation, HBV DNA level, placental barrier, the immune status of the mother, and the genetic make-ups of the newborn infants. Since HLA system is an integral component of the immune response, we hypothesized that the highly polymorphic HLA genes are the key determinants of intrauterine HBV infection. In this study, we selected newborn infants of HBsAg-positive mothers, and divided the infants into 2 groups: intrauterine infection group and non-intrauterine infection group according to the status whether or not they were infected at birth. Each infected infant was compared with 2 controls from the same birth cohort. HLA-DR allele typing was performed using a PCR-sequence specific primer (PCR-SSP) for 24 subjects with intrauterine infection and 48 controls without infection. We found that, among the fifteen (15) HLA-DR alleles assessed, HLA-DRB1*07 was the one, and the only one, significantly in excess (OR = 6.66, P = 0.004) in the intrauterine infection group compared to the non-intrauterine infection group. Our findings thus suggest that high frequency of HLA class II molecules, e.g. HLA-DRB1*07, is associated with the susceptibility of the infants to intrauterine HBV infection.

Identification of genes upregulated by recombinant interferon-alpha in HepG2 cells by suppressive subtractive hybridization analysis.

BACKGROUND: Interferon-alpha (IFN-alpha) is an important cytokine with multiple functions, but the target genes transactivated by IFN-alpha remain largely unknown. A study of such genes will help to understand the mechanism of function of IFN-alpha. To isolate the gene transcripts specifically upregulated by IFN-alpha in HepG2 cells, we conducted suppressive subtractive hybridization (SSH) analysis. METHODS: SSH was used to analyze the target genes transactivated by recombinant IFN-alpha protein, and a subtractive cDNA library was constructed from HepG2 cells treated with recombinant IFN-alpha (rIFN-alpha, 2000 IU/ml) for 16 hours as tester, and cells not treated with rIFN-alpha as driver. The SSH PCR products from the library were cloned into pGEM-T easy vector and with BLASTX, the positive clones were randomly selected, sequenced and compared to the database in GenBank of the 35 differentially expressed gene fragments from the library, 6 clones showed significant homology to other known proteins. RESULTS: The subtractive cDNA library of genes upregulated by IFN-alpha was constructed successfully. rIFN-alpha upregulated the expression of the RAN binding protein 5 (RANBP5), NADH dehydrogenase, exosome component 3 (EXOSC3), zinc finger RNA binding protein, Dickkopf homolog 1 (DKK1) and acetyl-coenzyme A acetyltransferase 2 (ACAT2). CONCLUSIONS: These results suggest that rIFN-alpha can upregulate the expression of important genes to exert its functions, and provide new clues for discovering the molecular mechanisms of action of IFN-alpha.

[Screening and identification of proteins interacting with HCV NS4A via yeast double hybridization in leukocytes and gene cloning of the interacting protein].

OBJECTIVE: To screen proteins interacting with HCV NS4A protein in leukocytes by yeast-double hybridization. METHODS: The bait plasmid pGBKT7-NS4A was transformed into yeast AH109 was transformed, and the expressing of the fusion protein was identified by SDS-page. The transformed yeast was mated with yeast Y187 containing leukocytes 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 selecting two times and screening. After extracting and sequencing of plasmid from blue colonies, analysis was conducted by bioinformatics. And, the gene encoding the interesting protein was cloned, and back-cross was performed. RESULTS: Forty-five colonies were sequenced, among them, 29 colonies were human calcium modulating cyclophilin ligand (CAML). The gene encoding CAML was cloned, and the interaction between NS4A and CAML was ensured. CONCLUSION: Seven kinds of proteins interacting with NS4A in leukocytes were successfully screened and the results brought some new clues for studying the pathogenesis of HCV.