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1.
Sci Rep ; 12(1): 11022, 2022 06 30.
Article in English | MEDLINE | ID: mdl-35773319

ABSTRACT

The Chelonid herpesvirus 5 (ChHV5) infection possibly associated to the fibropapillomatosis (FP) disease in sea turtles worldwide remains largely unknown and limited studies have used serological approaches to detection of antibodies against ChHV5 in sea turtles with or without FP. We aimed to develop diagnostic platforms based on the viral glycoprotein B (gB) for ChHV5 infection. In this study, five recombinant sub-fragments of the gB protein were successfully expressed and subsequently served as antigens for both seroprevalence and antibody production. The results indicated that the five expressed proteins harbored antigenicity, shown by the results of using sera from sea turtles that were PCR-positive for ChHV5. Moreover, seropositive sea turtles were significantly associated with FP (p < 0.05). We further used the expressed protein to produce antibodies for immunohistochemical analysis, and found that the in-house-generated sera specifically stained FP lesions while normal epithelium tissues remained negative. Of major importance, the reactivity in the ballooning degeneration area was much stronger than that in other regions of the FP lesion/tumour, thus indicating ChHV5 viral activities. In summary, the developed serological test and specific anti-gB antibodies for IHC analysis could be applied for further understanding of epidemiological distributions of ChHV5 infection in sea turtles, and studies of ChHV5 pathogenesis.


Subject(s)
Alphaherpesvirinae , Herpesviridae Infections , Herpesviridae , Skin Neoplasms , Turtles , Animals , Antibody Formation , Glycoproteins , Herpesviridae/genetics , Herpesviridae Infections/diagnosis , Herpesviridae Infections/veterinary , Seroepidemiologic Studies
2.
Biomed Microdevices ; 16(5): 761-70, 2014 Oct.
Article in English | MEDLINE | ID: mdl-24951158

ABSTRACT

Magnetotactic bacteria exhibit superiority over other bacteria in fabricating microrobots because of their high motility and convenient controllability. In this study, a microrobot system is constructed using magnetotactic bacteria MO-1 and applied in pathogenic separation. The feasibility of this approach is demonstrated using Staphylococcus aureus. The MO-1 magnetotactic bacterial microrobots are fabricated by binding magnetotactic bacteria MO-1 with their rabbit anti-MO-1 polyclonal antibodies. The efficient binding of MO-1 magnetotactic bacterial microrobots to Staphylococcus aureus is corroborated by phase contrast microscopic and transmission electron microscopic analyses. Further, a microfluidic chip is designed and produced, and the MO-1 microrobots are magnetically guided toward a sample pool in the chip. In the sample pool, Staphylococcus aureus samples are loaded on the microrobots and then carried away to a detection pool in the chip, suggesting the microrobots have successfully carried and separated pathogen. This study is the first to demonstrate bacterial microrobots carrying pathogens and more importantly, it reflects the great potential of using magnetotactic bacteria to develop magnetic-guided, auto-propelled microrobots for pathogen isolation.


Subject(s)
Antibodies, Bacterial/chemistry , Magnetosomes/chemistry , Microchip Analytical Procedures , Serratia marcescens/chemistry , Staphylococcus aureus/cytology , Animals , Lab-On-A-Chip Devices , Microchip Analytical Procedures/methods , Rabbits
3.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 27(12): 1288-90, 1294, 2011 Dec.
Article in Chinese | MEDLINE | ID: mdl-22152806

ABSTRACT

AIM: To construct recombinant human CXCR3B gene expression vector and obtain L929-CXCR3B gene transfected cell line for stably expressing human CXCR3B. METHODS: Human CXCR3B gene of full length was amplified by PCR from the plasmid pMD19-T/huCXCR3A. Then, it was inserted into eukaryotic expression vector pIRES2-EGFP to construct recombinant vector pIRES2-EGFP/huCXCR3B. The recombinant vector was transfected into L929 cells with Lipofectamine(TM); 2000, and cell clones stably expressing human CXCR3B molecule were screened by G418.We used FCM and RT-PCR to verify expression of CXCR3B from protein level and gene level. The ability of proliferation of L929-huCXCR3B under the circumstance of CXCR3B ligand called Mig was analyzed via MTT methods. RESULTS: We have constructed recombinant human CXCR3B gene expression vector and obtained L929-huCXCR3B gene transfected cell line which can stably express human CXCR3B molecule. The positive expression rate of CXCR3B on L929-huCXCR3B cells was 93&. The result of MTT assay showed that the proliferation of L929-huCXCR3B cells were inhibited when the cells were cocultured with Mig after 24 h, 48 h and 72 h, and the inhibition ratio were 41.44&, 44.01& and 24.80& respectively. CONCLUSION: Construction of L929-huCXCR3B cell line has laid a good foundation on research of the CXCR3B signal pathway and preparation of the CXCR3B monoclonal antibody.


Subject(s)
Receptors, CXCR4/genetics , Transfection , Cell Line , Cell Proliferation , Chemokine CXCL9/pharmacology , Cloning, Molecular , Flow Cytometry , Genetic Vectors , Humans , Receptors, CXCR4/physiology
4.
PLoS One ; 6(12): e28309, 2011.
Article in English | MEDLINE | ID: mdl-22164266

ABSTRACT

Influenza A virus poses serious health threat to humans. Neutralizing antibodies against the highly conserved M2 ion channel is thought to offer broad protection against influenza A viruses. Here, we screened synthetic Camel single-domain antibody (VHH) libraries against native M2 ion channel protein. One of the isolated VHHs, M2-7A, specifically bound to M2-expressed cell membrane as well as influenza A virion, inhibited replication of both amantadine-sensitive and resistant influenza A viruses in vitro, and protected mice from a lethal influenza virus challenge. Moreover, M2-7A showed blocking activity for proton influx through M2 ion channel. These pieces of evidence collectively demonstrate for the first time that a neutralizing antibody against M2 with broad specificity is achievable, and M2-7A may have potential for cross protection against a number of variants and subtypes of influenza A viruses.


Subject(s)
Antibodies/chemistry , Influenza A virus/immunology , Viral Matrix Proteins/chemistry , Animals , Antibody Specificity , Camelus , Cell Line , Chromatography/methods , Dogs , Enzyme-Linked Immunosorbent Assay/methods , Female , Gene Library , HEK293 Cells , Humans , Ion Channels/chemistry , Kinetics , Mice , Mice, Inbred BALB C , Neutralization Tests , Oligonucleotides/chemistry , Polymerase Chain Reaction/methods , Protein Binding , Protein Structure, Tertiary , Surface Plasmon Resonance
5.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 26(7): 627-30, 2010 Jul.
Article in Chinese | MEDLINE | ID: mdl-20619083

ABSTRACT

AIM: To construct recombinant murine CXCR3 gene retroviral vector and obtain L929-mCXCR3 gene transfected cell line for stably expressing murine CXCR3. We further study on L929-mCXCR3 migration effect resulted from interaction by CXCR3 and its ligand IP-10. METHODS: One female BALB/c mouse (7 weeks) was injected with 0.5 mg Con A intravenously (i.v.) via a tail vein. Twelve hours later the mouse was sacrificed and the spleen was removed.The spleen was pressed through a 150 microm stainless steel mesh. The isolated splenocytes were cultured in RPMI1640 supplemented with 50 U/mL human IL-2 for 3 days.Total RNA was extracted with TRIzol. Murine CXCR3 gene of full length was amplified by RT-PCR, then, it was inserted into retrovirus vector pEGZ-term. The recombinant vector together with its helper virus vector were co-transfected into package cell 293T with Lipofectamine(TM); 2000.The supernatant of 293T was collected for infecting L929 cells (repeated three times), and cell clones stably expressing murine CXCR3 molecule were screened by zeocin(500 mg/L). We used FCM and RT-PCR to verify expression of CXCR3 from protein level and gene level, respectively. Studied migration ability of L929-mCXCR3 interacted with its ligand IP-10 by transwell system. RESULTS: We have constructed recombinant murine CXCR3 gene retroviral vector and obtained L929-mCXCR3 gene transfected cell line which can stably expressing murine CXCR3 molecule. Positive expression rate of membrane is 97.0%, and it can directly migrate induced by IP-10, the chemotatic index is 4.356%. CONCLUSION: Construction of L929-mCXCR3 cell line has laid a good foundation on research of biologic characteristics of CXCR3 signal path , establishment of tumor metastasis model and preparation of anti-murineCXCR3 monoclonal antibody.


Subject(s)
Cell Line/metabolism , Gene Expression , Receptors, CXCR3/genetics , Transfection , Animals , Cell Line/cytology , Cell Migration Assays , Chemokine CXCL10/genetics , Chemokine CXCL10/metabolism , Female , Genetic Vectors/genetics , Genetic Vectors/metabolism , Humans , Mice , Mice, Inbred BALB C , Receptors, CXCR3/metabolism
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