Mass spectrometry based proteomics profiling of human monocytes

Human monocyte is an important cell type which is involved in various complex human diseases. To better understand the biology of human monocytes and facilitate further studies, we developed the first comprehensive proteome knowledge base specifically for human monocytes by integrating both in vivo and in vitro datasets. The top 2000 expressed genes from in vitro datasets and 779 genes from in vivo experiments were integrated into this study. Altogether, a total of 2237 unique monocyte-expressed genes were cataloged. Biological functions of these monocyte-expressed genes were annotated and classified via Gene Ontology (GO) analysis. Furthermore, by extracting the overlapped genes from in vivo and in vitro datasets, a core gene list including 541 unique genes was generated. Based on the core gene list, further gene-disease associations, pathway and network analyses were performed. Data analyses based on multiple bioinformatics tools produced a large body of biologically meaningful information, and revealed a number of genes such as SAMHD1, G6PD, GPD2 and ENO1, which have been reported to be related to immune response, blood biology, bone remodeling, and cancer respectively. As a unique resource, this study can serve as a reference map for future in-depth research on monocytes biology and monocyte-involved human diseases.

Construction of recombinant yellow fever virus 17D containing 2A fragment as a vaccine vector / 生物工程学报

The Yellow Fever (YF) vaccine, an attenuated yellow fever 17D (YF-17D) live vaccine, is one of the most effective and safest vaccines in the world and is regarded as one of the best candidates for viral expression vector. We here first reported in China the construction and characterization of the recombinant expression vector of yellow fever 17D which contained the proteinase 2A fragment of foot-and-mouth disease virus (FMDV). Three cDNA fragments representing the full-length YF-17D genome, named 5'-end cDNA (A), 3'-end cDNA (B) and middle cDNA (C), were obtained by reverse transcription polymerase chain reaction (RT-PCR), together with the introduction of SP6 enhancer, necessary restriction sites and overlaps for homologous recombination in yeast. Fragment A and B were then introduced into pRS424 in turn by DNA recombination, followed by transfection of fragment C and the recombinant pRS424 containing A and B (pRS-A-B) into yeast. A recombinant vector containing full length cDNA of YF-17D (pRS-YF) was obtained by screening on medium lack of tryptophan and uracil. A recombinant YF-17D expression vector containing FMDV-2A gene fragment (pRS-YF-2A1) was then constructed by methods of DNA recombination and homologous recombination in yeast described above. In vitro transcription of the recombinant vector pRS-YF-2A1 was then carried out and introduced into BHK-21 cells by electroporation. Results of indirect immunofluorescence assay (IFA) and titer determination showed a stable infectious recombinant virus was gotten, whose features such as growth curve were similar to those of the parental YF-17D. The results suggest that the recombinant vector pRS-YF-2A1, by introduction of heterogenous genes via 2A region, is potential to be an effective live vaccine expression vector.

Selection of recombinant fowlpox virus coexpressing HIV-1 gag-gp120 and IL-6 / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To construct the recombinant fowlpox virus (rFPV) coexpressing HIV-1 gag-gp120 and hIL-6.</p><p><b>METHODS</b>The recombinant expressing plasmid pUTA-GE-IL6 was successfully constructed by inserting gag-gp120 gene and hIL-6 gene into the downstream of the combined promoter ATI-p7.5 and p7.5 tandem promoter respectively. After transfecting the plasmid into chicken embryonic fibroblast (CEF) cells preinfected with FPV 282E4 strain and selecting the recombinant virus under the pressure of BUdR. The recombinant virus was analyzed by nucleic acid probe hybridization and immunoblotting. In addition, the formation of virus-like particle and the expression of interested proteins in the recombinant virus-infected p815 cells were observed, and the immunogenicity of the recombinant virus was also analyzed.</p><p><b>RESULTS</b>There was colorable dot for the positive recombinant virus, immunoblotting analysis showed that the recombinant virus could expressed both gag-gp120 and IL-6. Virus-like particles (VLP) were formed in virus-infected cells, and the interested proteins could be expressed in mammalian cells infected by the recombinant virus. The immunity index from the immunized mice showed that the recombinant virus had good immunogenicity.</p><p><b>CONCLUSION</b>The recombinant fowlpox virus coexpressing gag-gp120 and IL-6 was successfully constructed, which may provide basis for the preparation of live vector genetic engineering vaccine and macromolecule particle vaccine against HIV-1.</p>

Expression and detection of recombinant directing toxin SL120 in Pichia pastoris / 生物工程学报

Anti-HIV-1 gp120 single chain antibody(scFv) gene and staphylococcus extoxin A(SEA) gene were inserted into vector pPIC9K. The recombinant plasmid was integrated into Pichia pastoris by electroporation. High level expression was performed by determining the Muts phenotype and screening muti-copy integrants. The recombinant protein was about 57kD and the production was 50.1 mg/L. It was shown that the two kinds of protein affected the conformation of each other by antibody affinity assay, but the recombinant targeting toxins could highly mediate CTLs to kill HIV-1 target cells.

Construction of recombinant fowlpox virus coexpressing gp120 of Chinese HIV-1 strain and IL-18 and its immunogenicity in mice / 生物工程学报

To screening out Chinese vaccine candidate against HIV-1, Chinese vaccine strain 282E4 of fowlpox virus was used as the vector to construct the recombinant fowlpox virus (rFPV) coexpressing gp120 of Chinese HIV-1 strain and IL-18, and the recombinant virus was indentified by PCR and Western blot. The specific DNA fragment could be amplified by PCR from the genome of rFPV. Western blot analysis showed that gp120 and IL-18 could be expressed not only in chicken embryo fibroblast (CEF) cells infected by rFPV, but also in mammalian cells infected by rFPV. After the recombinant fowlpox virus was inoculated into BALB/c mice, the spleen specific CTL activities and serum antibodies in the immunized mice were detected, which demonstrated that the rFPV had good immunogenicity and could induce BALB/c mice to produce specific humoral and cellular immunity. IL-18 palyed the role of immunoadjuvant. The study lays the basis on the preparation of genetic engineering live vector vaccine against HIV-1.

Amplification, cloning and sequence analysis of spider dragline silk cDNA / 生物工程学报

Spider dragline silk is synthesized in special gland named major ampulate (MA) gland. The MA glands were dissected from the abdomen of the spiders Nephila clavata and the total RNA was extracted by the TRIZOL. The cDNA of dragline silk was amplificated by RT-PCR (reverse transcription polymerase chain reaction), multiplex PCR and cloned. PCR identification, restriction analysis and DNA sequence analysis were carried out to verify the recombinant plasmids. The codon usage frequencies of the cloned cDNA were added up, and the predicted amino acid sequence was compared with Spidroin2 of Nephila clavipes. Predicted secondary structure of the predicted amino-acid sequence was analysized by DNAStar software. All results showed that the cloned cDNA we got (GenBank Accession No. AF441245) was the very fragment of spider dragline silk Spidroin2 cDNA.

Construction of HIV-2 gag recombinant fowlpox virus and its immune reactivity in mice / 中华传染病杂志

Objective To provide experimental evidences for developing a safe and effective re- combinant fowlpox virus which can prevent the infection of HIV-2.Methods A fowlpox virus(FPV) transferring vector was constructed by inserting HIV-2 gag gene to the downstream of a synthetic complex promoter ATI-p7.5?20 of vector pUTA2.Transfection was then carried out,and recombi- nant FPV(rFPV)was screened by 5'-bromo-deoxyuridine(BrdU),genome PCR and western blot detection.Balb/c mice were immunized with rFPV by muscular injection.Anti-HIV-2 antibody, CD4~+ and CD8~+ T-cell count and specific target-killing activity of spleen CTL in immunized mice were analyzed by ELISA,FACS and LDH release assay,respectively.Results A transferring vector pA- gag was constructed and confirmed by amplifying a fragment of 766 bp from the rFPV genome.Mean- while,HIV-2 multi-antibody-specific protein blot(55 000)was detected from the recombinant virus and the HIV-2 specific antibody was detected from the immunized Balb/c mice.HIV-2 specific target- killing activity of spleen CTL was observed in immunized mice.Conclusion A recombinant fowlpox virus expressing HIV-2 structural protein Gag has been obtained,and it can stimulate HIV-2-specific eelluar and humoral immune reactions in mice.