Objective To analyze the immunogenicity of selected B-cell epitopes of Epstein-Barr virus (EBV) latent membrane protein-2 (LMP2). Methods Three potential dominant B-cell epitopes of LMP2199-209, LMP2318-322 and LMP2381-391 from EBV LMP2 had been predicted using bioinformaties methods. The gene fragments of three epitopes were cloned respectively into pET32a(+) vector and transformed into E. coli strain BL21 (DE3). After identification by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting, the expression products were purified by Ni-NTA agarose affinity chromatography. BALB/c mice in immunized groups were immunized by multi-point intracutaneous injection with the three purified epitope proteins,respectively; and mice in control groups were injected with pET32a (+) protein or phosphate buffered saline(PBS), respectively. The sera from mice at week O, week 3 and week 6 of injection were collected for determination of epitope-specific antibody IgG by enzyme linked immunosorbent assay (ELISA) using epitope proteins as coating antigens. The ability of serum antibody recognizing nature EBV antigen was determined at week 6 of immunization. Results Three epitope proteins of LMP2199-209 ,LMP2318-322 and LMP2381-391 were successfully expressed in prokaryotic system. Epitopespecific antibodies IgG could be detected respectively in the sera of all immunized mice, and the levels of antibodies increased with immunized time increasing. The antibody levels in LMP2318-322 immunized group at week 3 and week 6 were significantly higher than that of pET32a (+) protein control group (F= 493.85 and 773.99, respectively; both P＜0. 05), and the antibody levels in LMP2381-391 immunized group at week 3 and week 6 were also significantly higher than that of pET32a (+) protein control group (F= 926.33 and 309.14, respectively; both P＜0.05). Antibody level in LMP2199-209 immunized group at week 6 was significantly higher than that of pET32a ( + ) protein control group (F=87.27, P＜0.05). The antibody IgG in serum from immunized mice with three epitope proteins could all recognize nature EBV antigens, especially LMP2199-209 and LMP2381-391 immunized groups.Conclusions Three possible dominant epitopes of LMP2199-209, LMP2318-322 and LMP2381-391 from EBV LMP2 are prepared by prokaryotic expression system and exhibit obvious immunogenicity, which could be used for further research of EBV infection and related tumor vaccine.

Objective To explore the HLA-A2 restriction and immunogenicity of 5 previously identified HCV-speeific CTL epitopes. Methods Based on T2 cell, to explore the HLA-A2 restriction of previously identified HCV-specific CTL epitopes by MHC-peptide complex stabilization assay;To detect pep-tide-specific CTL in HLA-A2~+ PBMC stimulated by HLA-A2-restricted peptides by intracellular cytokine staining(ICS) and ELISPOT; To explore the cytotoxicity of peptide-specific CTL to same peptide-loaded T2 cells (target cells) by CTL cytotoxicity test. Results Among 5 previously identified CTL epitopes NS4b_78 (SMMAFSAAL) and NS5a_367 (TVSSALAEL) have high-affinity for HLA-A2 molecules(FI 1) ;ELISPOT results shown that NS4b_78(SMMAFSAAL) and NSSa_367(TVSSALAEL) induced high levels of IFN-γ-se-creting cells [(60±6) SFC/10~4 PBMC vs (4±1 ) SFC/10~4 PBMC, P < 0.01 ; (10 ± 3 ) SFC/10~4 PBMC vs (2±1 ) SFC/10~4 PBMC, P <0.01, respectively] ;ICS results indicated that there were high percentages of CD8~+ IFN-γ~+ T cells in total CD8~+T cells stimulated by these peptides [(2.33 ±0.22 ) % vs (0.05±0.01)%, P <0.001 ; (0.36±0.06)% vs (0.03±0.01)%, P <0.001, respectively]. Furthermore,peptide-specific CTL could effectively kill same peptide-loadcd T2 cells. Conclusion NS4b_78 (SMMAF-SAAL) and NSSa_367 (TVSSALAEL) were identified as HLA-A2-restricted CTL epitopes which could in-duce immune response in vitro.

Objective To analyze the incidence and risk factors of de novo hepatitis B virus (HBV) infection from anti-HBc-positive donors in pediatric living donor liver transplantation and to explore the diagnosis and treatment.Method A retrospective analysis was conducted on 105 cases of pediatric living donor liver transplantaions (LT) perfomed during September 2006 to December 2013.HBV markers,including hepatitis B surface antigen (HBsAg) and antibody (anti-HBs),anti-HBc,hepatitis B e antigen (HBeAg) and antibody (anti-HBe) were determined in both donors and recipients before LT and in recipients after LT.HBV DNA titer was measured if the recipients were strongly suspected of de novo HBV infection.Result After 4 perioperative deaths were excluded,101 cases were studied.The median follow-up period of all the patients was 20.5 months (2.7-97.7 months).de novo HBV infection occurred in 6 of 101 recipients (5.9％) 3.5 18 months after LT.Forty-four (43.6％) of the children received HBcAb-positive allografts,and 11.4％ (5/44) of the children were had de novo hepatitis B infection.All five of the HBV-infected children received HBcAb-positive allografts without preventive treatment in 11 cases (5/11,45.5 ％),57 (56.4％) of the children received HBcAb-negtive allografts,and 1.7％ (1/57) of the children had de novo hepatitis B infection.Conclusion Anti-HBc-positive donors can significantly increase the incidence of de novo HBV infection in HBsAg-negative recipients without preventive treatment.with the appropriate treatment strategy,HBcAb allografts can safely used in pediatric recipients.

Objective To study on the specific cellular immune response produced in BALB/c mice immunized with human papillomavirus (HPV) type 6b capsid protein L1 and Chlamydia trachomatis (Ct) major outer membrane protein(MOMP) multi-epitope chimeric DNA (HPV6b L1/Ct MOMP multiepitope) , and the enhancement of the specific cellular immune response to Ct MOMP multi-epitope by HPV6b L1. Methods The Ct MOMP multi-epitope gene was connected to the C terminal of HPV6b L1,the gene of HPV6b L1 had been optimized according to the codon usage of eukaryotic system, and then the HPV6b L1/Ct MOMP multi-epitope chimeric gene was cloned to pcDNA3.1 ( + ) vector. After identification by restriction enzyme digestion and DNA sequencing, the recombinant plasmid was transfected into COS-7 cells, Indirect Immunofluorescence (IIF) was used to confirm the expression of proteins. Then, BALB/c mice were randomly assigned to receive (intramuscular injection) either pcDNA3.1 ( + )/HPV6b L1/Ct MOMP or pcDNA3.1 ( + )/Ct MOMP or pcDNA3.1 ( + ) or PBS ( n = 12, 150 μg/time), and the same immunization schedule was repeated third times at 2 week intervals. The level of cytokine( IFN-γ, IL-4, IL-10) -producing CD3+ T cells in spleen, the cytotoxicity of Ct MOMP-specific and HPV6b L1-specific cytotoxic T lymphocyte (CTL) in spleen were detected by intracellular cytokine staining-fluorescence activated cell sorter (ICS-FACS) and LDH release assays, respectively. Results After immunization, when the efCTL (44.56%±4.02%, 35.35% ±2.89% ) and HPV6b L1 specific cytotoxicity of CTL (27.08% ±2.04%, 21.68% ±4.06% ) in pcDNA3.1 ( + )/HPV6b L1/Ct MOMP multi-epitope chimeric DNA immunized mice, were significantly higher than that in pcDNA3.1 ( + )/Ct MOMP multi-epitope DNA (35.50%±2.68%, 30.24% ±1.75%; 12.27% ±3.36%, 9.32% ±3.07%) and other control groups(F=72.87, F=114.55, P＜0.05; F=30.04, F=10.47, P＜0.05), and Ct MOMP multi-epitope specific cytotoxicity of CTL in pcDNA3.1 ( + )/Ct MOMP multi-epitope DNA immunized mice were significantly higher than that in control groups( F = 58.85, F = 120.21; P<0.05). The level of intracellular cytokine IFN-γ in pcDNA3.1 ( + )/HPV6b L1/Ct MOMP multi-epitope DNA immunized mice(4.34% ±0.06%)was higher significantly than that in pcDNA3.1 ( + )/Ct MOMP multi-epitope DNA immunized mice(3.14% ± 0.18%, P<0.05 ) and other control groups ( F = 473.83, P<0.05 ), while, the levels of IL-4 ( F =0.97, P ＞ 0.05 ) and IL-10 ( F = 2.25, P ＞ 0.05 ) had no significant difference between groups. Conclusion Both Ct MOMP and HPV6b L1 protein specific cellular immune response could be induced in BALB/c mice immunized with HPV6b L1/Ct MOMP multi-epitope chimeric plasmid, and the HPV6b L1 gene optimized by eukaryotic codon could significantly enhance the cellular immune response induced by Ct MOMP multi-epitope gene in BALB/c mice.

Objective To research the homology and cross reaction characteristics of human papillomavirus(HPV)16 type L2 N-terminal(1-200)protein in clinical common HPV infection types.Methods The amino acid sequences of the common HPV infection types(6,11,16,18 ,etc.)were blasted and it was found that 1-200 N-terminal sequence of L2 protein was highly homologous.The gene of HPV16 L2(1-200)was amplificated from tissue sample of cervical cancer patient and inserted into the prokaryotic expression vector PGEX-4T-1 to construct the recombinant plasmid PGEX-4T-1-HPV16 L2(1-200).After sequencing identification,the recombinant plasmid was tranformed into E.coli BL21(DE3).Induced by IPTG,the fusion protein containing HPV16 L2(1-200)was expressed and analyzed by both SDS-PAGE and Western blot.Furthermore,the specific binding capacity of the fusion protein to the HPV 6,11,16 and 18 DNA positive patient serums were analyzed by Western blot.The fusion protein was purified with Ni-NTA Agarose Kit and coated with ELISA reaction plates.The specific serum IgG of 98 condyloma acuminatum patients,135 cervix cancer patients and 96 healthy control subjects were detected respectively by indirect ELISA.Results After comparing the amino acid sequences of the common HPV infection types(HPV6,11,16,18,etc.),We found that the homology of HPV L2(1-200)reached 52.7%-74.3%.The recombinant plasmid PGEX-4T-1-HPV 16 L2(1-200)was constructed successfully.Highly expressed HPV16 L2(1-200)fusion protein was obtained and the expression level was account for up to about 22.6% of total bacterial protein.The relative molecular mass(Mr)of the fusion protein is about 49×103,which matches up to the expected Mr Meanwhile,the serums of HPV 6,11,16,18 DNA positive patients were used as the first antibody and the specific band was detected respectively at about 49 × 103 by Western blot.Indirect ELISA showed that the A490 values of the specific IgG of condyloma acuminatum group,cervical cancer group and healthy control subjects were 0.753 ± 0.262,0.756 ± 0.274 and 0.178 ± 0.157 with the positive rate were 89.8%,88.9% and 9.4% respectively.There was no significance of the specific IgG between condyloma acuminatum group and cervical cancer group(P＞0.05),but it was significant among the three groups(P＜0.001).Conclusion The N-terminal 1-200 amino acids of HPV L2 has high homology and there exits cross reaction among the most common HPV infection types.

Objective To express in prokaryotic system and to analyze the antigenic specificity of EB virus(EBV) latent membrane protein 2(LMP2) multi-epitopes gene rich of T cell and B cell epitopes.Methods Using on-line prediction service, T cell epitopes and B cell epitopes of EB virus latent membrane protein 2 were predicted. The genes rich of CTL and th cell epitopes were selected as the candidate gene sequences, while B cell epitopes around them were taken into account. The finial selected multi-epitope gene was synthesized after being optimized according to prokaryotic codon bias and inserted into prokaryotic expression vector pET32a( + ) to get the recombinant plasmid: pET32a( + )/EBV-LMP2 multi-epitopes. After transformed into E. coli BL21 (DE3) and induced by IPTG, the target multi-epitopes gene can be expressed as Trx-His fusion protein. The expression products can be identified by SDS-PAGE and Western blot. Moreover, rabbit serum antibody to EBV membrane protein and nasopharyngeal carcinoma(NPC) patient serum were used respectively to detect the antigenic specificity of the multi-epitopes. Meanwhile, 6-8 weeks female BALB/c mice were immunized with EBV-LMP2 multi-epitope at 2 week intervals, three times in all, Trx-His protein and PBS were set as the control groups. At the second week after the last immunization, the mice were sacrificed. LDH and indirect ELISA were taken to detect the specific spleen CTL activityand specific IgG in serum, which reflected the immunogenicity of the EBV-LMP2 multi-epitope. Results Two amino acid sequences which locate at the LMP2 (aa195 -232 ) and LMP2 (aa419-436 ) were selected and connected in series to be the target gene. The recombinant plasmid containing EBV-LMP2 multi-epitope gene successfully constructed and the target protein was expressed in E. coli BL21 ( DE3 ). The relative molecular mass(Mr) of The expression products is about 27 × 103 , which matches up to the expected Mr. The antigenic specificity of the multi-epitopes protein was identified by Western blot and the multi-epitopes protein also can be detected by rabbit serum antibody to EBV membrane protein and NPC patient serum respectively. In the result of the animal experiment, EBV-LMP2 multi-epitope was able to induce the specific CTL activity in BALB/c mice. With the increasing of the effector: target ( E: T) 1: 5,1: 10, 1: 25, the CTL activity was also increased wih( 12.52% + 2.59% ), (21.80% + 1.08% ), (23.68% + 3.74% ) respectively; EBV-LMP2 multi-epitope was able to induce LMP2-specific antibody response(A490 =0.258 +0.040) as compared with Trx-His protein(A490 =0.095 +0.011) and PBS(A490 =0.068 +0.014,P＜0.05＝. Conclusion The EBV-LMP2 multi-epitopes gene was designed successfully and expressed precisely in prokaryotic expression system with good antigenicity and immunogenicity.

Objective To investigate the cross reaction characteristics of recombinant human papillomavirus 16 type L2 full-length fusion protein in HPV types of 6, 11, 18.Methods The serum samples of 108 condyloma acuminatum patients, 156 cervix cancer patients and 100 healthy control subjects were collected.The gene of full-length HPV16 L2 was amplificated from the tissue DNA of cervical cancer patient and inserted into the prokaryotic expression vector PGEX-4T-1 to construct the recombinant plasmid PGEX-4T-1-HPV16 L2.After sequencing identification, the recombinant plasmid was tranformed into E.coli BL21 (DE3).After induction by IPTG, the fusion protein containing HPV16 L2 was expressed and analyzed by both SDS-PAGE and WB.Furthermore, the specific binding capacity of the fusion protein to the HPV 6,11, 16 and 18 DNA positive patient's sera were analyzed by WB.The fusion protein was purified with NiNTA Agarose Kit and coated with ELISA reaction plates.The specific serum IgG were detected by indirect ELISA.Results The recombinant plasmid PGEX-4T-1-HPV16 L2 was constructed successfully. Highly expressed HPV16 L2 full-length fusion protein was obtained and the expression level was 27.2 %.The relative molecular mass(Mr) of the fusion protein is about 82 × 103, which matches up to the expected Mr.Meanwhile, the sera of HPV 6,11,16,18 DNA positive patients were used as the primary antibody and the Mr of the specific band was detected to be about 82 × 103 by WB.The results of indirect ELISA showed that the average levels of specific IgG in condyloma acuminatum group, cervical cancer group and healthy control subjects were 0.848 ±0.257, 0.822 ±0.247 and 0.173 ±0.143 with the positive rate of 92.6%, 94.2%and 8.0% respectively.There was no significance of the specific IgG levels between condyloma acuminatum group and cervical cancer group ( F = 0, P ＞ 0.05 ), but there was significant difference of specific IgG levels and positivity among the three groups ( F = 305.201 ,x2 = 253.178, P ＜ 0.01 ).Conclusions The HPV16 L2 full-length fusion protein has better antigenicity.However cross reactions with HPV6, 11 and 18 were found.It can be applied in serological screening reagents for HPV infection and associated cancer.

ObjectiveTo study the anti-cancer effects and mechanism of Gexia-Zhuyu decoction combined with cyclophosphamide on EAC liver cancer mice.MethodsChoose EAC liver cancer mice cell lines and production of animal models of tumor.The mice were completely randomized into blank group,model group,the Chinese medicine group,CTX group,Chinese medicine and CTX group.The blank group and model group were given normal saline 0.4 ml/d; traditional Chinese medicine group was administrated with Gexia-Zhuyu decoction (11 g/kg body weight ) 0.4 ml/d gavage; CTX group was given intraperitoneal injection of 2 mg/ml of cyclophosphamide 0.4 ml/d; and traditional Chinese medicine and CTX group was given Gexia Zhuyu decoction 0.4 ml/d orally and CTX intraperitoneal injection of 0.4 ml/d.Ten days after the administration,the body weight of mice and the general condition were tested,and morphology was observed under the light microscope electron microscope mesenchymal tumor angiogenesis situation.Results Gexia-Zhuyu decoction combined with cyclophosphamide in mice could improve appetite,gain weight,and greatly reduce the tumor stroma in the number of capillaries under the electron microscope together with significant pathological morphological changes.Conclusion Gexia-Zhuyu decoction combined with cyclophosphamide had anti-cancer effects which can improve the quality of life and inhibit the growth of tumor in interstitial capillaries.

We predict in this paper B-cell epitopes of Epstein-Barr virus nuclear antigen-1 (EBNA-1) and analyze the results matched with the related autoantigens sequence of human. We selected EBV-1 standard strain NA-1 amino acid sequence as the basis. We predicted B-cell dominant epitopes of EBNA-1 with the methods of SOPMA, GOR and HNN, combined with the multi-parameter analysis of transmembrane domain, hydrophilicity profile, surface probability, antigenicity index, polarity and average flexibility. The blastp method was adopted to analyze the matched results between the predicted B-cell epitopes of EBNA-1 and the related autoantigens sequence of human. The results have shown that the possible B-cell dominant epitopes of EBNA-1 were located in the N terminal regions of 16-23, 35-78, 332-337, 340-357, 398-404, 419-432 and 620-637, in which different regions gained higher scores when matched with small nuclear ribonucleoprotein SmB, SmD, ribonucleoprotein SSA, heterogeneous nuclear ribonucleoprotein hnRNP A1, hnRNP G, respectively. It was available to predict B-cell dominant epitopes of EBNA-1 with multiparameter methods and to analyze the same or similar autoantigens sequences of human, which laid a theory foundation for the study of pathogenesis, diagnosis and treatment of autoimmune diseases.
Amino Acid Sequence ; Autoantigens ; immunology ; Base Sequence ; Epitopes, B-Lymphocyte ; immunology ; Epstein-Barr Virus Nuclear Antigens ; immunology ; Humans ; Molecular Sequence Data ; Sequence Homology, Amino Acid

To predict the B cell epitopes for major outer membrane protein (MOMP) of Chlamydia trachomatis (CT), the secondary structure of CT MOMP was predicted by the methods of GOR based on the sequence of amino acids of E serotype CT MOMP. By combining the comprehensive analysis of transmembrane domain, hydrophilicity profile, surface probability, antigenic index and average flexibility, the B cell predominant epitopes of CT MOMP were further predicted. The N-terminal No. 73-81, 217-225, 377-386, 261-270 and 161-175 were the predominant B cell epitopes. Prediction of the B cell epitopes for the CT MOMP by the multi-parameters is helpful for the identification of B cell epitopes.
Amino Acid Sequence ; Antigens, Bacterial ; immunology ; Chlamydia trachomatis ; classification ; immunology ; Epitopes, B-Lymphocyte ; immunology ; Molecular Sequence Data ; Porins ; immunology ; Protein Conformation ; Protein Structure, Secondary ; Serotyping