Experimental study of dual promoter baculovirus-mediated tumor-targeting radioiodine therapy / 中华核医学与分子影像杂志

Objective To construct a recombinant baculovirus dual expression vector containing NIS gene under the control of human telomerase reverse transcriptase (hTERT) promoter and plasminogen kringle 5 (K5) gene driven by early growth response 1 (Egr1) promoter,and to explore the feasibility of targeting both tumor and tumor vessel with combination of radioiodide and antiangiogenic therapy.Methods The hTERT-NIS gene and Egr1-K5 gene fragments were subcloned into baculovirus vector,then packaged and amplified in the sf9 cells to obtain recombinant baculovirus Bac-hTERT-NIS-Egr1-K5.Bac-CMV-NISEgr1-K5,Bac-hTERT-0-Egr1-K5 and Bac-hTERT-NIS-Egr1-0 were constructed as controls.The expression of NIS and K5 genes in human cervix cancers cells (HeLa) was examined by Western blot and quantitative real-time PCR.Functional NIS activity was confirmed by the uptake of 125I,the inhibition of NaClO4 and the cytotoxicity of 131I.The apoptotic effect of 131I-inducedK5 on human umbilical veins endothelial cells (HUVEC)was analyzed by an apoptosis assay using flow cytometry.Statistical analysis was performed using the analysis of variance.Results The recombinant baculovirus Bac-hTERT-NIS-Egr1-K5 was successfully constructed.The NIS gene under the control of hTERT promoter was specifically expressed in HeLa cells.The baculovirusinfected HeLa cells showed a significant increase of 125I uptake,which was significantly inhibited by NaClO4(F199.296,P＜0.05).Furthermore,a notable decreased cell survival rate (38.3％) was found after 131I treatment.The expression of K5 gene induced by 131I was elevated in a dose or time dependent manner and resulted in obvious inhibition with cell survival rate of 30.8％ in baculovirus-infected HUVEC cells,which was significantly higher than that in the control groups (11.2％ and 10.9％ respectively,F=19.926,45.409;both P＜0.05).Conclusions A recombinant baculovirus dual expression vector containing the NIS and K5 genes has been successfully constructed.This study suggests the feasibility of a synergistic strategy of NISbased raidoiodide therapy and K5-based antiangiogenic therapy in vitro,and make it possible to perform in vivo study in the near future.

Virucidal evaluation of sodium dichloroisocyanurate formulation against nucleopolyhedroviruses of silkworm / Avaliação antiviral do formulado de sódio dicloroisocianurato contra o nucleopoliedrovírus do bicho-da-seda

This study evaluated the effectiveness of a disinfectant with low corrosive action and which is not toxic to the environment, the sodium dichloroisocyanurate formulation, on the Bombyx mori nucleopolyhedrovirus (BmNPV). For this, 5th-instar B. mori silkworm larvae were divided into four experimental groups of 4 replicates with 15 larvae each, totalling 60 larvae per group. The groups were fed with mulberry (Morus sp.) leaf discs containing: BmNPV treated with the disinfectant, untreated BmNPV, only the disinfectant, and water (control). The results showed that the disinfectant does not inactivate the BmNPV and also exerts a negative effect on the insect"s resistance.

O estudo avaliou a eficiência de um desinfetante que apresenta baixa ação corrosiva e que não é tóxico ao meio ambiente, o formulado de sódio dicloroisocianurato, sobre o nucleopoliedrovírus Bombyx mori (BmNPV). Para tanto, lagartas do bicho-da-seda, B. mori, de 5º instar foram divididas em quatro grupos experimentais, 4 repetições com 15 lagartas cada, totalizando 60 lagartas por grupo. Os grupos foram alimentados com discos foliares de amoreira (Morus sp.) contendo: o BmNPV tratado com o desinfetante (solução 1); o BmNPV não tratado (solução 2); apenas o desinfetante (solução 3); e água (solução 4, controle). Os resultados mostraram que o desinfetante não inativa o BmNPV e também exerce efeito negativo na resistência do inseto.

Fusion of human orphan G protein-coupled receptors GPR45, GPR85 or GPR174 with Gi1α and their expression in insect Sf9 cells / 军事医学科学院院刊

Objective To obtain the fusion genes of several human orphan G protein coupled receptors (oGPCRs) with Gi1α subtype of G protein and their expression system. Methods The whole open reading frames of GPR45, GPR85, GPR174 and Gilα were cloned by RT-PCR from HepG2 cDNA separately,and the corresponding fusion genes were amplified by overlap extension PCR. Then, the fusion genes-containing pBacmids were successfully constructed with the Bac-to-Bac baculovirus expression system indicated by specific transposition and virus recombination. The insect Sf9 cells were transfected with pBacmid-oGPCRs-Gi1α, and the supernatant containing recombinant virus was harvested. With the supernatant, insect Sf9 cells were infected under an optimized condition (MOI=5, infection time=72 h) and the fusion proteins were prepared and detected by Western blotting.Results The three fusion genes of GPCR45, GPR85 or GPR174 with Gi1α were obtained. The corresponding fusion proteins could be properly prepared in Sf9 cells.Conclusion Human oGPCRs could be fused with Gilα, and the fusion genes could be expressed using the Bac-to-Bac baculovirus expression system in insect Sf9 cells.

Construction and Transduction of SARS-3CL Protease Gene in Baculovirus Vector / 天津医药

Objective: To construct severe acute respiratory syndrome (SARS)coronavirus 3CL protease gene into transforming vector prepare recombinant baculovirus and transduct it into infect insect cells to express SARS-3CL protease. Mothods: The 3cl-Teasy and pFastBac HTb bacmida were amplified. The 3cl gene was cloned into baculovirus transforming vector pFastBac HTb by enzyme-digest- and-ligase method,which was named recombinant pFB HTb-3cl. The pFB HTB-3cl was transformed into E.coli DH10Bac competent cells.The positive colonies were screened by three antibiotics and blue-white patch method. The bacmid-HTb-3cl recombinant baculovirus bacmid was obtained and purified to transfect St9 insect cells.The protease expressed in Sf9 insect cells were identified by SDS-PAGE. Results: Recombinant expression vector was obtained successfully. The 3CL protease expressed in insect cells were identified by SDS-PAGE. Conclusion: The expression of 3CL protease in insect cells provided foundation for detecting protein activities and screening inhibitor against SARS-3CL protease.

Expression.purification and antigenicity of a recombinant respiratory syncyttal virus fusion gene encoded protein(amino acids 168-289)in the insect cells / 中华检验医学杂志

Objective To explore the antigenicity of the recombinant respiration syncytial virus (RSV)fusion protein (amino acids 168-289) encoded by 546-881 bases of the fusion gene expressed in insect baculoviruses expression system.Methods The fragment F' of fusion gene 546-881 bases was amplified from viral RNA( Long strain ) by the reverse transcription-polymerase chain reaction(RT-PCR).F' was inserted into transfer vector pBacPAK9 and the recombinant plasmid pBacRSV F' was constructed.Sf9 insect cells were then co-transfeeted with a mixture of recombinant plasmids pBacRSV F' and linearized BacPAK6 viral DNA( Bsu36 Ⅰ -digested).The recombinant baculoviruses BacPAK F' was constructed and was able to express the recombinant protein in Sf9 insect cells.The recombinant protein was purified by Ni2+ NTA chromatography and its antigenicity was identified by Western blot(WB) analysis.Specimens including the nasopharyngeal aspirates(NPAs) and sera were collected from 33 infants and young children with acute lower respiration tract infection. Indirect immunofluorecenee assay (IFA) and WB were used to detect the RSV antigen in NPAs and the anti-RSV antibody in sera respectively.Results The recombinant protein (molecular weight 13 000) was expressed in Sf9 insect cells.WB analysis demonstrated that the purified recombinant protein had a specific RSV antibody-binding activity. The recombinant protein could be recognized by positive serum infected by RSV.The positive rate was 27.3% and 33.3% respectively.There was no significant difference between them(X2 = 0.29 ,P ＞ 0.05).Conclusion The recombinant respiratory syncytial virus fusion gene (546-881 bp) encoding protein is expressed in Sf9 insect cells and it has strong antigenicity and could have clinical application value for detection of RSV infection.

Genetic Variation in Field Populations of Baculoviruses: Mechanisms for Generating Variation and Its Potential Role in Baculovirus Epizootiology / 中国病毒学·英文版

Baculoviridae is a family of insect-specific DNA viruses that have been used as biological control agents for insect pest control. In most cases these baculovirus control agents are natural field isolates that have been selected based on their infectivity and virulence. The advent of molecular tools such as restriction endonucleases, targeted polymerase chain reaction and new DNA sequencing strategies have allowed for efficient detection and characterization of genotypic variants within and among geographic and temporal isolates of baculovirus species. It has become evident that multiple genotypic variants occur even within individual infected larvae. Clonal strains of baculovirus species derived either by in vitro or in vivo approaches have been shown to vary with respect to infectivity and virulence. Many of the cell culture derived plague-purified strains have deletions that interrupt egt expression leading to virus strains that kill infected hosts more quickly. As well, in vitro clones often involve larger genomic deletions with the loss of pif gene function, resulting in strains deficient for oral infectivity. There are an increasing number of baculovirus species for which complete genome sequences are available for more than one strain or field isolate. Results of comparative analysis of these strains indicated that hr regions and bro genes often mark "hot spots" of genetic variability between strains and of potential recombination events. In addition, the degree of nucleotide polymorphisms between and within strains and their role in amino acid substitutions within ORFs and changes in promoter motifs is also beginning to be appreciated. In this short review the potential mechanisms that generate and maintain this genetic diversity within baculovirus populations is discussed, as is the potential role of genetic variation in host-pathogen interactions.

Heterogenous expression and activity analysis of human cytochrome P450 3A4 mutants CYP3A4.3, CYP3A4.4, CYP3A4.5 and CYP3A4.18 / 中国药理学与毒理学杂志

AIM To express recombinant human cytochrome P450 3A4 mutants CYP3A4.3, CYP3A4.4, CYP3A4.5 and CYP3A4.18, and to employ them for in vitro metabolism studies of CYP3A4. METHODS Use Bac-to-Bac baculovirus expression system to recombinant baculovirus carrying cDNA of CYP3A4 mutants CYP3A4.3, CYP3A4.4, CYP3A4.5 and CYP3A4.18. Spodoptera frugiperda 9 (Sf9), cells were co-infected by recombinant viruses of CYP3A4 mutants, human NADPH-P450 oxidoreductase and cytochrome b5 to obtain recombinant proteins CYP3A4.3, CYP3A4.4, CYP3A4.5 and CYP3A4.18 with metabolic activity. RESULTS The mRNA transcription of CYP3A4 mutants in Sf9 cells were validated by RT-PCR. Testosterone and 7-benzyloxy-4-(trifluoromethyl) coumarin were metabolized by the lysates of Sf9 cells infected by the recombinant viruses. CONCLUSION CYP3A4 mutants CYP3A4.3, CYP3A4.4, CYP3A4.5 and CYP3A4.18 with metabolic activity were successfully expressed by baculovirus-insect cell expression system. The results indicated that recombinant CYP3A4. 5 showed lower activity comparing to the wild type protein towards testosterone, while CYP3A4. 18 with higher activity, and for CYP3A4.3 and CYP3A4.4 showing similar activity to the wild type protein.

Expression of Hepatitis C Virus Core Protein in Hepatocytes Does Not Modulate Proliferation or Apoptosis of CD8+ T Cells

Hepatocytes are the primary targets of the hepatitis C virus (HCV). While immunosuppressive roles of HCV core protein have been found in several studies, it remains uncertain whether core protein expressed in hepatocytes rather than in immune cells affects the CD8+ T cell response. In order to transduce genes selectively into hepatocytes, we developed a baculoviral vector system that enabled primary hepatocytes to express a target epitope for CD8+ T cells, derived from ovalbumin (OVA), with or without HCV core protein. Culture of OVA-specific CD8+ T cells with hepatocytes infected with these baculoviral vectors revealed that core protein has no effect on proliferation or apoptosis of CD8+ T cells. Our results suggest that HCV core protein does not exert its suppressive role on the CD8+ T cell immune response through expression in hepatocytes.

Construction and expression of recombinant cecropin B-binding site of luteinizing hormone releasing hormone gene and its anticancer function / 中华妇产科杂志

Objective To construct and express recombinant cecropin B-binding site of luteinizing hormone releasing hormone(CB-LHRH')gene,and to evaluate the anticancer function of CB-LHRH' on human ovarian cancer cell line SKOV3 and human endometrial cancer cell line HEC-1B.Methods The sequence of the cDNA encoding CB-LHRH' was designed,artificially synthesized,verified by DNA sequence analysis and expressed by Bac-to-Bac baculovirus expression system.The expression of CB-LHRH' proteins were identified by western dot blot using rabbit polyclonal antibody against LHRH as the primary antibody.To determine the anticancer effects of the CB-LHRH' protein,ovarian cancer cell line SKOV3 and endometrial adenocarcinoma cell line HEC-1B were treated by different doses of the CB-LHRH' protein.Cell growth inhibition assay was performed using the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)5[(phenylamino) carbonyl]-2H-tetrazolium hydroxide(XTT)kit at different times,and cell morphologic changes were observed under the inverted microscope.Results The inhibitory rate of proliferation by CB-LHRH' increased with the increase of dose and time respectively:SKOV3 cell,from(5.03?0.08)% to(53.24 ?1.22)%;HEC-1B cell,from(5.13?0.37)% to(56.16?1.08)%.The inhibitory effect on HEC-1B cell was stronger than that on SKOV3 cell(P

Construction and identification of Hepatitis B Virus (genotype C) recombinant baculovirus / 中华传染病杂志

Objective To construct Hepatitis B Virus (HBV) Chinese-strain wild type and Lamivudine-resistant variant recombinant baculovirus. Methods 1.2-unit length HBV construct (adr serotype, C genotype) was cloned into the multiple cloning region of the pFastBac Ⅰ vector and then transformed into DH10Bac, in which homologous recombination occurred between the pFastBac vector and the inside bacmid with the help of helper plasmid. So HBV recombinant bacmid was produced and transfected into sf9 cells to generate a Recombinant HBV baculovirus called vAcHBVc. The viral titers were detected by immune plaque assay and TCID 50. Lamivudine resistance mutation (YVDD) was introduced by site-directed mutagenesis using continuous PCR. HBV YVDD recombinant baculovirus (vAcHBVcYVDD) was generated and titered as above. Meanwhile, the recombinant HBV baculovirus containing GFP reporter was constructed for evaluating the efficacy of transfection and titration of virus. Results Recombinant wild and YMDD variant HBV baculovirus were successfully developed and the titer was between 10~6～10~8pfu/ml through plaque assay. Conclusion Different kinds of recombinant HBV baculovirus could be generated rapidly and efficiently with Bac to Bac expression system. The developed recombinant HBV baculovirus could be further used in the study of anti-viral resistances.