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.

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.

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.

[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.

Inhibition of HBV targeted ribonuclease enhanced by introduction of linker.

AIM: To construct human eosinophil-derived neurotoxin(hEDN) and HBV core protein (HBVc) eukaryotic fusion expression vector with a linker (Gly(4)Ser) (3) between them to optimize the molecule folding, which will be used to inhibit HBV replication in vitro. METHODS: Previously constructed pcDNA3.1(-)/TR was used as a template. Linker sequence was synthesized and annealed to form dslinker, and cloned into pcDNA3.1(-)/TR to produce plasmid pcDNA3.1(-)/HBc-linker. Then the hEDN fragment was PCR amplified and inserted into pcDNA3.1(-)/HBc-linker to form pcDNA3.1(-)/TNL in which the effector molecule and the target molecule were separated by a linker sequence. pcDNA3.1(-)/TNL expression was identified by indirect immunofluorescence staining. Radioimmunoassay was used to analyse anti-HBV activity of pcDNA3.1(-)/TNL. Meanwhile, metabolism of cells was evaluated by MTT colorimetry. RESULTS: hEDN and HBVc eukaryotic fusion expression vector with a linker (Gly(4)Ser)(3) between them was successfully constructed. pcDNA3.1(-)/TNL was expressed in HepG2.2.15 cells efficiently. A significant decrease of HBsAg concentration from pcDNA3.1(-)/TNL transfectant was observed compared to pcDNA3.1(-)/TR (P=0.036, P<0.05). MTT assay suggested that there were no significant differences between groups (P=0.08, P>0.05). CONCLUSION: Linker introduction enhances the inhibitory effect of HBV targeted ribonuclease significantly.

Anti-HBV effect of TAT- HBV targeted ribonuclease.

AIM: To prepare and purify TAT-HBV targeted ribonuclease fusion protein, evaluate its transduction activity and investigate its effect on HBV replication in 2.2.15 cells. METHODS: The prokaryotic expression vector pTAT containing TR gene was used in transforming E.coli BL21 (DE3) LysS and TR was expressed with the induction of IPTG. The TAT-TR fusion protein was purified using Ni-NTA-agrose and PD-10 desalting columns, and analyzed by SDS-PAGE. Transduction efficiency of TAT-TR was detected with immunofluorescence assay and the concentration of HBeAg in the supernatant of the 2.2.15 cells was determined via solid-phase radioimmunoassay (spRIA). MTT assay was used to detect the cytotoxicity of TAT-TR. RESULTS: The SDS-PAGE showed that the TAT-TR fusion protein was purified successfully, and the purity of TAT-TR was 90 %. The visualization of TAT-TR by immunofluorescence assay indicated its high efficiency in transducing 2.2.15 cells. RIA result suggests that TAT-TR could inhibit the replication of HBV effectively, it didn't affect cell growth and had no cytotoxicity. CONCLUSION: TAT-TR possesses a significant anti-HBV activity and the preparation of TAT-TR fusion protein has laid the foundation for the use of TR in the therapeutic trial of HBV infection.

Effect of vector-expressed siRNA on HBV replication in hepatoblastoma cells.

AIM: To study the effect of siRNA expressed from DNA vector on HBV replication. METHODS: Human U6 promoter was amplified from genomic DNA and cloned into plasmid pUC18 to construct a mammalian siRNA expression vector pUC18U6. Then oligonucleotides coding for a short hairpin RNA against HBV were cloned into pUC18U6 to form pUC18U6HBVsir which was introduced into 2.2.15 cells by using liposome-mediated transfection. 2.2.15 cells transfected by pUC18U6 and pUC18U6GFPsir which expressed siRNA against green fluorescent protein and mock-transfected 2.2.15 cells were used as controls. Concentration of HBsAg in the supernatant of the transfected cells was measured by using solid-phase radioimmunoassay. RESULTS: A mammalian siRNA expression vector pUC18U6 was constructed successfully. Compared with controls, pUC18U6HBVsir which expressed siRNA against HBV decreased concentration of HBsAg significantly by 44%(P<0.05). CONCLUSION: HBV replication in 2.2.15 cells is inhibited by siRNA expressed from the DNA vector.

Targeted ribonuclease can inhibit replication of hepatitis B virus.

AIM: To study the effect of a novel targeted ribonuclease (TN), the fusion protein of HBVc and human eosinophil-derived neurotoxin (hEDN), on the HBV replication in vitro. METHODS: The gene encoding the targeted ribonuclease was cloned into pcDNA3.1 (-) to form recombinant eukaryotic expression vector p/TN. Control plasmids, including p/hEDN, p/HBVc, and p/TNmut in which a Lys113-Arg mutation was introduced by sequential PCR to eliminate the ribonuclease activity of hEDN, were also constructed. Liposome-mediated transfection of 2.2.15 cells by p/TN, p/TNmut, p/hEDN, p/HBVc, and pcDNA3.1 (-), or mock transfection was performed. After that, RT-PCR was used to verify the transgene expression. Morphology of the transfected cells was observed and MTT assay was performed to detect the cytotoxicity of transgene expression. Concentration of HBsAg in the supernatant of the transfected cells was measured using solid-phase radioimmunoassay. RESULTS: Transgenes were successfully expressed in 2.2.15 cells. No obvious cytotoxic effect of transgene expression on 2.2.15 cells was found. The HBsAg concentration in the p/TN transfected cells was reduced by 58 % compared with that of mock transfected cells. No such an effect was found in all other controls. CONCLUSION: The targeted ribonuclease can inhibit HBV replication in vitro while it has no cytotoxicity on host cells. The targeted ribonuclease may be used as a novel antiviral agent for human HBV infection.

Quantifying anti-HBV effect of targeted ribonuclease by real-time fluorescent PCR.

AIM: To quantify the inhibition of HBV replication by targeted ribonuclease by using real-time fluorescent PCR. METHODS: Targeted ribonuclease was introduced into 2.2.15 cells by liposome-mediated transfection or HIV-TAT mediated protein transduction. Forty-eight hours after the transfection and 24 h after the transduction, supernatants of 2.2.15 cells were collected and HBV DNA in the supernatants was quantified by real-time fluorescent PCR with a commercial kit. RESULTS: HBV DNA concentrations in the supernatants of 2.2.15 cells transfected or transducted with targeted ribonuclease were 4.9+/-2.4 x 10(8) copies/L and 8.3+/-4.0 x 10(8) copies/L, respectively. Compared with controls, transfection or transduction of targeted ribonuclease reduced HBV DNA concentration in the supernatants of 2.2.15 cells by 90.4% and 90.1%, respectively (P<0.05). CONCLUSION: Targeted ribonuclease can inhibit HBV replication in 2.2.15 cells.

Functional characterization of the 5' proximal flanking fragment of Plasmodium falciparum GBP130 gene.

OBJECTIVE: To identify the putative regulation elements with strength- and stage-specificity in 5' proximal flanking sequence of P. falciparum GBP130 gene. METHODS: Plasmids containing different deletions of upstream of the GBP130 promoter were constructed. For strength-specific analysis, pGBPCAT delta 2, pGBP delta 2/400 and pGBP delta 2/800 were electroporated into ring-stage P. f. respectively, and the expression level of CAT in each plasmid was detected by liquid scintillation counts(LSC). For stage-specific analysis, transfectants with pGBP delta 2/400 and pGBP delta 2/800 were harvested at 5 hours post-transfection(h), 15 h and 46 h respectively, and the CAT expression levels were detected. RESULTS: In strength-specific analysis, the expression level of CAT in pGBP delta 2/800 and pGBPCAT delta 2 was similar, down-regulated significantly in pGBP delta 2/400. The CAT level showed significant difference between pGBP delta 2/400 and the control. In stage-specific analysis, the CAT level of pGBP delta 2/400 was higher than that of pGBP delta 2/800 at the time point of 5 h, and lower at 15 h and 46 h. CONCLUSION: This strength-specific promoter activity was due to the difference of 5'UTR length: the longer the 5'UTR the higher the promoter strength, and two poly (dA:dT) tracts in the proximal sequence could enhance the promoter activity. The length of 5'UTR regulated the promoter activity in a stage-specific manner. The shorter 5'UTR was functional at ring stage, while the longer one prompted transcription at trophozoite and schizont stage. The functional role of poly (dA:dT) tracts in stage-specific regulation of GBP130 remains unclear.

Effect of sodium citrate based anticoagulants on the growth activity of malaria parasites.

OBJECTIVE: To study the effect of anticoagulants based on sodium citrate on the growth activity of malaria parasites. METHODS: The parasites were treated with 3 anticoagulants (ACD, CD and SC), respectively, and the parasitemia was determined to measure the effect of the anticoagulants on the growth of the parasites. Unsynchronized Plasmodium falciparum was treated with the anticoagulants at different concentrations for 3 h at 37 degrees C. Treated erythrocytes were mixed with normal parasites or treated parasites with normal erythrocytes, which was followed by parasitemia determination of the two cultures to determine the cell target of the anticoagulants. Stage-synchronized parasites (ring, trophozoite and schizont) were treated as above to investigate the stage target. P. berghei was also treated with anticoagulants and inoculated in mice to detect the effect of anticoagulants on the animal malaria parasite by counting the parasitemia. RESULTS: All 3 anticoagulants inhibited falciparum parasite growth and ACD had the strongest potency. The treatments of the erythrocyte and the parasite with anticoagulants respectively showed that the anticoagulants targeted the parasites rather than normal erythrocytes. Stage-synchronized parasite treatment suggested anticoagulants primarily inhibited schizonts. The effect of anticoagulants on P. berghei was similar to that on P. falciparum. CONCLUSION: ACD showed the most significant inhibitive effect on the growth of malaria parasites and SC was the best anticoagulant based on sodium citrate for malarial experiments.

[Protective antibody response to recombinant fragments of Plasmodium falciparum apical membrane antigen 1].

OBJECTIVE: To determine the role of putative apical membrane antigen (AMA)1 domains in inducing protective immunity and to provide basis for selection of vaccine applicable segments. METHODS: Encoding gene segments of AMA1 were amplified and cloned into pET prokaryotic expression vectors. Recombinant proteins were expressed and purified. Groups of BALB/c mice were immunized by using recombinant protein in Freund's adjuvant, and the IgG titer and specificity of the immune sera were analyzed by IFA and Western blotting. Efficiency of the immune sera in inhibiting Plasmodium falciparum in vitro growth was evaluated. RESULTS: Recombinant AMA1 fragments including the entire ectodomain E and subdomain I + II, I, II and III were successfully expressed and purified. Different levels of antibody were induced in mice by individual proteins and all the immune sera recognized native antigen in the parasites. Sera from protein E and I + II immunized mice inhibited the growth of parasites. CONCLUSION: The integrality of the ectodomain of AMA1 determines the conformation of the protective antibody epitopes, and these protective epitopes distribute mainly in the subdomain I.

[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.

Effects of tetracycline operator element insertion on Plasmodium falciparum glycophorin binding protein 130 gene promoter activity.

OBJECTIVE: To construct tetracycline operator (TetO) modified glycophorin binding protein 130 gene (GBP130) promoter of Plasmodium falciparum and investigate the position effect of insertion of TetO on the promoter activity. METHODS: Cloning of 7-copy of TetO (7cot) sequence into 4 points relative to transcriptional initiation site of GBP130 promoter in pGBPCATdelta2 plasmid (2 upstream and 2 downstream), respectively, produced 4 derivative plasmids, pG/7T (-5), pG/7T (-2), pG/7T (+2) and pG/7T (+5). After transient transfection, the expression level of reporter gene CAT in both pGBPCATdelta2 and its derivative plasmids was detected and analysed by CAT ELISA. RESULTS: Identification by enzyme restrictions and PCR amplifications, as well as DNA sequencing confirmed that the plasmids were successfully constructed. Transfection of these plasmids and CAT detection suggested that insertion of 7cot into each point of GBP130 promoter enhanced the promoter activity, and downstream location of 7cot in the promoter showed higher promoter activity than those located upstream, with the strongest effect in Ins 4 (point +5). CONCLUSION: Plasmid pG/7T (+5), in which 7cot was inserted into +5 point of GBP130 promoter, can be chosen as the responsive plasmid in establishing tetracycline-controlled transgenic expression system of malarial parasites.

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.

[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.

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.