Objective To investigate the interactions between melanoma-derived exosomes and the microenvironment.Methods The exosomes were isolated from the culture medium of mouse melanoma cells and then co-cuhured with mesenchymal stromal cells (MSC) after identification.Immunofluorescence assay was performed to observe the exosomes engulfed by MSC.CCK-8 and transwell assays were used to evaluate the proliferation and migration of MSC.Effects of the exosomes on the expression of α-smooth muscle actin (α-SMA) in MSC were analyzed by Western blot.Results Co-culture of MSC with melanoma cell-derived exosomes enhanced the proliferation and migration of MSC as well as the expression of α-SMA.All of the changes mediated by the exosomes could be blocked by using the inhibitor of TGF-β receptor.Conclusion Melanoma cell-derived exosomes enhanced the proliferation and migration of MSC as well as the expression of α-SMA through TGF-β signaling pathway,which provided an advantageous microenvironment for melanoma progression.

To obtain human antibodies against the Gn protein of Severe fever with thrombocytopenia syndrome virus (SFTSV) with phage display technology, this study aimed to screen anti-Gn protein antibodies from an anti-SFTSV Fab human phage display library. Antibody genes were identified by sequence analysis and the specificity of antibodies was confirmed by ELISA. The Fab antibody genes were cloned into the HL51-14 vector and expressed in a mammalian cell expression system. IgG antibodies were then purified by protein A affinity chromatography,and the results were further confirmed by ELISA,IFA,western blotting assays and micro-neutralization tests. The results showed that, after three rounds of panning, there were 390 human Fab antibodies against SFTSV particles, of which 364 were specific for nucleoprotein. Coated with the Gn protein, eight different Fab antibodies specific for Gn protein were obtained after the determination of the subtype and subclass of antibodies by gene sequencing; five of these antibodies were from the Lambda library and three were from the Kappa library. The eight IgG antibodies could specifically bind to Gn protein according to the ELISA, IFA and Western blotting assays. The micro-neutralization test showed that these eight antibodies had no neutralizing activity,but they could still provide a reference for research in human monoclonal antibodies against SFTSV.
Antibodies ; genetics ; immunology ; Bunyaviridae Infections ; genetics ; immunology ; virology ; Cell Line ; Cloning, Molecular ; Humans ; Immunoglobulin Fab Fragments ; genetics ; immunology ; Immunoglobulin G ; genetics ; immunology ; Neutralization Tests ; Phlebovirus ; genetics ; immunology ; Viral Proteins ; genetics ; immunology

The Ebola virus is highly infectious and can result in death in ≤ 90% of infected subjects. Detection of the Ebola virus and diagnosis of infection are extremely important for epidemic control. Presently, Chinese laboratories detect the nucleic acids of the Ebola virus by real-time reverse transcription-polymerase chain reaction (RT-PCR). However, such detection takes a relatively long time and necessitates skilled personnel and expensive equipment. Enzyme-linked immunosorbent assay (ELISA) of serum is simple, easy to operate, and can be used to ascertain if a patient is infected with the Ebola virus as well as the degree of infection. Hence, ELISA can be used in epidemiological investigations and is a strong complement to detection of nucleic acids. Cases of Ebola hemorrhagic fever have not been documented in China, so quality-control material for positive serology is needed. Construction and expression of human-mouse chimeric antibodies against the nucleoprotein of the Ebola virus was carried out. Genes encoding variable heavy (VH) and variable light (VL) chains were extracted and amplified from murine hybridoma cells. Genes encoding the VH and VL chains of monoclonal antibodies were amplified by RT-PCR. According to sequence analyses, a primer was designed to amplify functional sequences relative to VH and VL chain. The eukaryotic expression vector HL51-14 carrying some human antibody heavy chain- and light chain-constant regions was used. IgG antibodies were obtained by transient transfection of 293T cells. Subsequently, immunological detection and immunological identification were identified by ELISA, immunofluorescence assay, and western blotting. These results showed that we constructed and purified two human- mouse chimeric antibodies.
Animals ; Antibodies, Monoclonal ; genetics ; immunology ; Cloning, Molecular ; Ebolavirus ; genetics ; immunology ; Hemorrhagic Fever, Ebola ; immunology ; virology ; Humans ; Immunoglobulin Heavy Chains ; genetics ; immunology ; Mice ; Nucleoproteins ; genetics ; immunology ; Viral Proteins ; genetics ; immunology

Objective: To establish a multiplexed immunoassay for detection of IgG antibodies against viral hemorrhagic fever epidemic in Africa. Methods: The recombinant antigens of hemorrhagic fever viruses (HFVs) epidemic in Africa were expressed and purified, and then coupled with the fluorescent microspheres. The coupling effects were evaluated by monoplexed detection of rabbit immune sera. Blood specimens were collected from people from Africa with fever, and multiplexed detection of IgG antibodies to HFVs was performed. Comparison of multiplexed assay and ELISA was performed by paired χ² test using SPSS software. Results: Both the purity and concentration of HFVs recombinant antigen met the standards for coupling and detection, and the antigens were successfully coupled with fluorescent microspheres. Seventy-eight sera samples of people from Africa with fever were multiplex detected. Among these, one was tested positive for LASV-specific IgG, one was tested positive for LUJV-specific IgG, 4 were tested positive for DENV-specific IgG and 6 tested positive for YFV-specific IgG. There was no statistically significant difference compared with ELISA, and the two method were highly correlated. Conclusions A multiplexed luminex-based immunoassay for detection of IgG antibodies to viral hemorrhagic fever epidemic in Africa was established, which laid the foundation for the differential diagnosis.

Objective To obtain a human single-chain antibody against rabies virus (RV) with high affinity by error-prone PCR technology.Methods A combinatorial scFv antibody mutant library was constructed by error-prone PCR amplifying VH and VL of scFv RV08.After package by hyperphage,the scFv phage mutant library was panned by ELISA and IFA with purified RV AG.The selected mutant antibodies were converted to full human IgG antibodies with the VH and VK Express cassettes.The affinity of mutant antibodies was measured by non-competitive enzyme immunoassay.Results The mutant antibody library with capacity of 6 × 106 cfu/ml contained target gene insertion rate of 100％.Heavy chain and light chain were sequenced with mutation frequency of about 0.95％ and 1.2％ respectively.Finally 5 mutant antibodies with high affinity were obtained,of which HL46,HL2 and HL37 had higher affinity with 1.79 times,1.47 times and 1.23 times of RV08 respectively.Conclusions In this study,error-prone PCR technology and phage display technology combination enables to obtain significantly improved affinity of antibody.

Objective: To obtain the optimum of lentiviral library packaging based on CRISPR/cas9 (Clustered regularly interspaced short palindromic repeat sequences/CRISPR-associated protein 9). Methods: Reverse transcription polymerase chain reaction (RT-PCR), immunofluorescence antibody (IFA) and enzyme linked immunosorbent assay (ELISA) were used to detect the lentivirus titers in condition of different ratio of packaging plasmids, different addition of lipofectamine 3000 reagent and different time points post-transfection. Then, high-throughput sequencing was performed to evaluate the representation and distribution of single guide (sg)RNAs in the library. Results: The lentivirus titer was the highest when the molar ratio of psPAX2∶pMD2.0G∶Lentivirus library was 2∶1∶1, and the optimum addition of Lipofectamine 3000 reagent was 10 μl, while the result of ELISA were correspondent to that of RT-PCR. The IFA result showed that the lentivirus titer was the highest at 60 h post-transfecion. The coverage of sgRNAs in the lentivirus library packaged with the optimum we obtained was 99.3%, and the read counts of sgRNAs was observed in a normal distribution. Conclusions The optimal lentivirus library packaging was obtained, and this can provide basis for CRISPR/cas9-based screening.

Objective To obtain recombinant human anti-RVP genetically engineered antibodies from rabiator-derived antibody phage library.Metbods A combinatorial human Fab library against rabies virus was constructed with antibody genes harvested from peripheral blood of rabiators with high titer of antibody.The library was panned with phage display and selected by purified CTN-1 virus particles.After that the selected antibody was converted to full human IgG antibody with recombinant mammal cell antibodyexpression system.Results Unique 1 human Fab antibodies specific for RVP were obtained and conformed by competitive ELISA,Western Blotting and IFA.IFA,competitive ELISA and Western blotting showed the panned antibody 1A2 bound specifically to RVP.Conclusion Human antibody against RVP was firstly obtained through phage antibody library,and could be used for RVP functional mechanism research and rabies diagnosis.

Objective:To develop a rapid nucleic acid detection method for Zika virus (ZIKV), Dengue virus (DENV), Yellow fever virus (YFV), Chikungunya virus (CHIKV) based on microfluidic fluorescence quantitative RT-PCR technologies, in order to achieve rapid diagnosis of these four viral infections.Methods:Four sets of specific primers and probes were designed targeting the NS1 gene of ZIKV, the NS5 gene of DENV, and YFV, the E1 gene of CHIKV, respectively. The sensitivity was evaluated using in vitro transcribed RNA of ZIKV, DENV, YFV and CHIKV, and the specificity were evaluated using other viral nucleic acid. ZIKV, YFV and CHIKV detection method were verified using simulated positive samples, and DENV detection method was verified using clinical patient samples, the result of which were also compared with the quantitative RT-PCR detection method . Results:The limit of detection (LOD) of ZIKV, DENV, YFV, and CHIKV microfluidic qRT-PCR method were 14.57 copies/μl, 94.27 copies/μl, 8.25 copies/μl, and 223.19 copies/μl, respectively, and the four detection method showed no cross-reactivity with other viral nucleic acids. The prepared ZIKV, YFV and CHIKV simulated positive samples were 100% detected, and the variation coefficient of Ct value measured at each concentration were all around 2%; the 20 clinical patient specimens of DENV infection were 100% detected, which is consistent with the result of fluorescent quantitative RT-PCR detection.Conclusions:The ZIKV, DENV, YFV, and CHIKV microfluidic quantitative RT-PCR detection method showed good sensitivity, specificity, and stability. The detection could be completed within 25 minutes, which could be used for laboratory detection and early diagnosis.

Objective:To establish real-time fluorescent quantitative RT-PCR method for detection of Avalon virus (AVAV) and Hughes virus (HUGV), two Nairoviruses in the family of Nairoviridae. Methods:The genomic sequences of the two viruses published in the international public database were collected, collated, compared and analyzed to define the detection targets, and the viral specific primers and probes were designed accordingly. Real-time fluorescent quantitative RT-PCR detection method were established, and the operating detection procedures were optimized using simulated samples prepared using in vitro transcription assay, other virus infected samples, virus strains and normal human blood samples. The detection limit, specificity and repeatability of the method were evaluated.Results:The real-time fluorescent quantitative RT-PCR method could effectively amplify and detect AVAV and HUGV viral target RNA with detection limits of about 20 copies/μl and 70 copies/μl, respectively. No nonspecific amplification was found in the samples of Kyasanur forest disease virus, influenza B virus BV and BY, influenza A virus H3N2, yellow fever virus, Japanese encephalitis virus, Crimean-Congo hemorrhagic fever virus, SFTS virus, nairobi sheep disease virus and Tahyna virus. There was no cross reaction between the two nairoviruses. The coefficient of variation was within 2% by repeated comparative analysis.Conclusions:The real-time fluorescent quantitative RT-PCR method for detection of AVAV and HUGV might be used for screening of humans, vectors and host animal samples for rapid detection of related pathogens.

Objective: To isolate, purify and culture fibroblastic reticular cells (FRCs) of mouse in spleen, to develop a reliable and robust method to immortalize primary mouse FRCs, to filter stable FRCs cell lines, to prove that the clones can be infected by SFTSV in vitro. Methods: After purifying FRCs by fluorescence activated cell sorting (FACS) from autoMACS-enriched stroma cells of mouse spleen, we infected FRCs by simian virus 40 large T antigen in vitro, screened the FRCs clones with puromycin, compared primary and immortalized FRCs by RNA sequencing(RNA-seq) technology, infected the clones with severe fever with thrombocytopenia syndrome virus (SFTSV) in vitro. Results: We succeed in culturing purified FRCs from spleen, isolated four stable FRCs clones, two of which have a purity of 99%, survived for more than 50 passages, express the key FRCs marker podoplanin and do not express CD31 and CD45. Clone 01 lost the typical FRCs-like morphology, the rate of expansion of which is quite different from that of primary FRCs and Clone 02. Clone 02 can be infected with SFTSV, which has the same gene expression pattern and immunophenotype with primary FRCs. Conclusions The stable FRCs clone Clone 02 has FRCs-like morphology and express key FRCs surface markers podoplanin (GP38 or PDPN) and do not express endothelial cell markers CD31 and leukocyte common antigen CD45. The RNA expression profiles identified by RNA-seq are also characteristic of FRCs. Infected with SFTSV in vitro, Clone 02 will be a new platform to study SFTSV.