Expression of fluorescent proteins within the repeat long region of the Marek's disease virus genome allows direct identification of infected cells while retaining full pathogenicity.

Marek's disease virus (MDV) is a lymphotropic alphaherpesvirus and causes Marek's disease (MD) in chickens. RLORF4 is an MDV-specific gene located in the repeat long (RL) regions of the genome and is directly involved in attenuation. In this report, we generated recombinant (r)MDVs in which eGFP or mRFP was inserted in-frame of the 3' end of the RLORF4 gene. In vitro growth was unaffected and infected cells could be identified by using fluorescent microscopy. Interestingly, though inserted in-frame with RLORF4, eGFP and mRFP were expressed alone, confirming mRNA expression and splicing within the RL of MDV is complex. In vivo, rMDVs expressing mRFP or eGFP caused tumors similar to wild-type MDV. Fluorescent protein expression could be seen in spleen, tumor, and feather follicle epithelial cells. These results show that expression of fluorescent proteins within the RL region results in fluorescent rMDVs that still maintains full pathogenicity in the chicken.

Interaction network of proteins associated with human cytomegalovirus IE2-p86 protein during infection: a proteomic analysis.

Human cytomegalovirus protein IE2-p86 exerts its functions through interaction with other viral and cellular proteins. To further delineate its protein interaction network, we generated a recombinant virus expressing SG-tagged IE2-p86 and used tandem affinity purification coupled with mass spectrometry. A total of 9 viral proteins and 75 cellular proteins were found to associate with IE2-p86 protein during the first 48 hours of infection. The protein profile at 8, 24, and 48 h post infection revealed that UL84 tightly associated with IE2-p86, and more viral and cellular proteins came into association with IE2-p86 with the progression of virus infection. A computational analysis of the protein-protein interaction network indicated that all of the 9 viral proteins and most of the cellular proteins identified in the study are interconnected to varying degrees. Of the cellular proteins that were confirmed to associate with IE2-p86 by immunoprecipitation, C1QBP was further shown to be upregulated by HCMV infection and colocalized with IE2-p86, UL84 and UL44 in the virus replication compartment of the nucleus. The IE2-p86 interactome network demonstrated the temporal development of stable and abundant protein complexes that associate with IE2-p86 and provided a framework to benefit future studies of various protein complexes during HCMV infection.

Alternative splicing of the human cytomegalovirus major immediate-early genes affects infectious-virus replication and control of cellular cyclin-dependent kinase.

The major immediate-early (MIE) gene locus of human cytomegalovirus (HCMV) is the master switch that determines the outcomes of both lytic and latent infections. Here, we provide evidence that alteration in the splicing of HCMV (Towne strain) MIE genes affects infectious-virus replication, movement through the cell cycle, and cyclin-dependent kinase activity. Mutation of a conserved 24-nucleotide region in MIE exon 4 increased the abundance of IE1-p38 mRNA and decreased the abundance of IE1-p72 and IE2-p86 mRNAs. An increase in IE1-p38 protein was accompanied by a slight decrease in IE1-p72 protein and a significant decrease in IE2-p86 protein. The mutant virus had growth defects, which could not be complemented by wild-type IE1-p72 protein in trans. The phenotype of the mutant virus could not be explained by an increase in IE1-p38 protein, but prevention of the alternate splice returned the recombinant virus to the wild-type phenotype. The lower levels of IE1-p72 and IE2-p86 proteins correlated with a delay in early and late viral gene expression and movement into the S phase of the cell cycle. Mutant virus-infected cells had significantly higher levels of cdk-1 expression and enzymatic activity than cells infected with wild-type virus. The mutant virus induced a round-cell phenotype that accumulated in the G(2)/M compartment of the cell cycle with condensation and fragmentation of the chromatin. An inhibitor of viral DNA synthesis increased the round-cell phenotype. The round cells were characteristic of an abortive viral infection.

[Construction of phage antibody library with predetermined CDR3 gene and screening of humanized Fab of anti-human integrin alphanubeta3 monoclonal antibody].

AIM: To construct phage antibody library with predetermined CDR3 and to screen humanized Fab of anti-human integrin alphanubeta(3) monoclonal antibody (mAb) by epitope guided selection. METHODS: LCDR3 gene of mAb E10 was inserted into human light chain variable region gene library. Hybrid phage antibody library was constructed by cloning E10 chimeric Fd gene and human light chain variable region gene into pComb3. Humanized light chain gene was obtained by screening against human integrin alphanubeta(3). Likewise, humanized Fab were gained by panning human phage antibody library, which was constructed by cloning humanized light chain gene and human heavy chain Fd gene with E10 HCDR3 into pComb3. RESULTS: Three humanized Fab clones was obtained by screening hybrid phage antibody library and human phage antibody library, which contained 2.1x10(6), 2x10(7) colony forming units, respectively. Indirect ELISA and competitive inhibition ELISA analysis demonstrated that three humanized Fab antibody had specific binding activity with human integrin alphanubeta(3). The strongest anti-human integrin alphanubeta(3) reactive D5 clone was sequenced and sequencing analysis showed that the V(kappa) and V(H) were derived from VKIII and VHI, respectively. CONCLUSION: Humanized Fab of anti-human integrin alphanubeta(3) mAb has been successfully obtained by phage display technology which lays the foundation for further clinical research.

[Construction and expression of anti-human integrin alphavbeta3 scFv].

AIM: To construct single chain antibody (scFv) gene of mAb E10 against human integrin alphavbeta3. METHODS: The VH and VL genes were amplified from hybridoma cells secreting mAb E10 by RT-PCR and connected with the use of linker (Gly4Ser)3 to assemble scFv gene. The scFv gene was cloned into prokaryotic expression vector pTIG-TRX and expressed in E. coli BL21 (DE3). RESULTS: SDS-PAGE analysis showed the expressed recombinant protein with relative molecular mass (Mr) being 31,000. Western blot confirmed that the protein was labeled with His6. scFv protein was expressed as soluble protein under the condition of a small amount of IPTG induction and culture at lower temperature. The purity of the protein purified through Ni-NTA agarose metal affinity resin column was over 91%. The purified protein could bind to the human integrin alphavbeta3 by ELISA confirmation. CONCLUSION: scFv against human integrin alphavbeta3 has been successfully constructed and expressed,which lays the foundation for further clinical research.

[Construction of eucaryotic expression plasmid carrying the BMP7 gene and expression in mesenchymal stem cells].

OBJECTIVES: To construct an eucaryotic expression plasmid carrying the BMP7 gene and express in MSCs. METHODS: The BMP7 gene was cloned into the eucaryotic expression vector pcDNA3.1. At the same time, mesenchymal stem cells (MSCs) were isolated and cultured in vitro. The plasmid carrying the BMP7 gene was transfected into MSCs. RESULTS: PCR and digesting demonstrated that the eucaryotic expression plasmid -pcDNA-BMP7 was obtained. RT-PCR and immunohistochemical methods showed that the BMP7 gene was expressed in MSCs. CONCLUSION: Construction of an eucaryotic expression plasmid carrying BMP7 gene and expression in MSCs provide a sound basis for gene therapy using the BMP7 gene and the ideal seeds for tissue engineering.

In vitro assay for HCV serine proteinase expressed in insect cells.

AIM: To produce the recombinant NS3 protease of hepatitis C virus with enzymatic activity in insect cells. METHODS: The gene of HCV serine proteinase domain which encodes 181 amino acids was inserted into pFastBacHTc and the recombinant plasmid pFBCNS3N was transformed into DH10Bac competent cells for transposition. After the recombinant bacmids had been determined to be correct by both blue-white colonies and PCR analysis, the isolated bacmid DNAs were transfected into Sf9 insect cells. The bacmids DNA was verified to replicate in insect cells and packaged into baculovirus particles via PCR and electronic microscopic analysis. The insect cells infected with recombinant baculovirus were determined by SDS-PAGE and Western-blot assays. The recombinant protein was soluted in N-lauryl sarcosine sodium (NLS) and purified by metal-chelated-affinity chromatography, then the antigenicity of recombinant protease was determined by enzyme-linked immunoabsorbant assay and its enzymatic activity was detected. RESULTS: The HCV NS3 protease domain was expressed in insect cells at high level and it was partially solved in NLS. Totally 0.2 mg recombinant serine proteinase domain with high purity was obtained by metal-chelated-affinity chromatography from 5 x 10(7) cells, and both antigenicity and specificity of the protein were evaluated to be high when used as antigen to detect hepatitis C patients' sera in indirect ELISA format. In vitro cleavage assay corroborated its enzymatic activity. CONCLUSION: The recombinant HCV NS3 proteinase expressed by insect cells is a membrane-binding protein with good antigenicity and enzymatic activity.

Establishment of a simple assay in vitro for hepatitis C virus NS3 serine protease based on recombinant substrate and single-chain protease.

AIM: To establish a simple and convenient assay in vitro for the Hepatitis C virus NS3 serine protease based on the recombinant protease and substrate, and to evaluate its feasibility in screening the enzyme inhibitors. METHODS: Based on the crystallographic structure of hepatitis C virus (HCV) serine protease, a novel single-chain serine protease was designed, in which the central sequence of cofactor NS4A was linked to the N-terminus of NS3 serine protease domain via a flexible linker GSGS. The fusion gene was obtained by two-step PCR that was carried out with three primers and then cloned into the prokaryotic expression vector pQE30, and the recombinant clone was verified by DNA sequencing. The single-chain recombinant protease was expressed when the E.coli was induced with IPTG and the expression conditions were optimized to produce large amount of soluble protease. The recombinant substrate NS5ab that covers the cleavage point NS5A/B was also expressed in E.coli. Both of the protease and substrate were purified by using Ni-NTA agarose metal affinity resin, then they were mixed together in a specific buffer, and the mixture was analyzed by SDS-PAGE. The cleavage system was used to evaluate some compounds for their inhibitory activity on serine protease. RESULTS: The single-chain recombinant protease was over-expressed as soluble protein when the E.coli was induced at a low dosage of IPTG (0.2 mM) and cultured at a low temperature (15 degrees ). The protease was purified by using Ni-NTA agarose metal affinity resin (the purity is over 95 %). The recombinant substrate NS5ab was expressed in an insoluble form and could refold successfully after purification and dialysis. A simple and convenient assay in vitro was established, in which the purified single-chain serine protease could cleave the recombinant substrate NS5ab into two fragments that were visualized by SDS-PAGE. PMSF had an effect on inhibiting activity of serine protease, while EDTA had not. CONCLUSION: A simple and convenient assay in vitro for hepatitis C virus NS3 serine protease is based on recombinant substrate NS5ab and single-chain serine protease. This assay can be used in screening of enzyme inhibitors.

Identification of B cell epitopes of hepatitis C virus RNA dependent RNA polymerase.

The aim of this study was to identify the B cell epitopes of hepatitis C virus (HCV) NS5B RNA dependent RNA polymerase (RdRp). The truncated HCV NS5B protein NS5B-dc21 was expressed in Escherichia coli and its antigenicity was confirmed by Enzyme-Linked Immunosorbent Assay (ELISA) using 130 HCV-positive human sera and 15 negative sera. Antibodies specific to NS5B-dc21 protein were purified by affinity chromatography using sepharose-4B coupled with the recombinant protein. A 12-mer phage displayed random peptide library was screened four rounds with the purified antibodies. Three epitopes were identified from the phage library, which correspond to amino acids 2444-2452, 2521-2528, and 2915-2925 of HCV RdRp. These epitopes were then expressed in E. coli as fusion proteins with phage M13 pIII protein. ELISA demonstrated that two of these epitopes (P4 and P34, corresponding to amino acids 2443-2452 and amino acids 2512-2528, respectively) have good reactivity and sensitivity. Mutagenesis study of P4 peptide showed that this epitope, which is derived from a phage displayed library, exhibited higher affinity with HCV serum than the corresponding original HCV sequences.

[Selection and characterization of peptides that specifically binding to hepatitis C virus serine protease].

The HCV NS3 serine protease that plays important role in the processing of polyprotein and the replication of virus is a prime target for antiviral drugs and therapy research. Based on the crystallographic structure of HCV sreine protease, a single-chain protease was contstructed in which the central sequence of NS4A was fused to the N-terminus of NS3 serine protease domain via a flexible linker and it was expressed at high level in soluble form in E. coli. The purified protease could cleave the recombinant protein NS5ab into two parts. The purified protease was used as target to screen binding peptides from phage displayed peptide library. After three rounds of affinity screening, 37 out of 44 randomly selected phages could bind specifically with the single-chain serine protease and their specificity were verified by competitive ELISA. The 13 sequenced clones represents 6 kinds of sequences of which the amino acids composition is in bias and there is a consensus sequence: [H/F/W]-H-W-X-X-W.

[Study on the methods for virus epidemiology in epitope level].

A method for studying virus epidemiology in epitope level was established via phage random peptide library and thioredoxin surface display technique and the method was proved by test with core protein of HCV.