ABSTRACT
Objective To express and purify the glycoprotein extracellular domain (Ex-GP) of Rabies virus strain CTN in soluble form with high efficiency.Methods A recombinant expression plasmid containing the gene encoding the Ex-GP was constructed.Various expression conditions were screened to obtain an optimum prokaryotic expression system for Ex-GP in soluble form.The expressed target protein was purified using affinity chromatography and gel filtration chromatography.Results The target protein Ex-GP with high antigenicity was efficiently expressed in soluble form by using the recombinant PBCX expression system and effectively purified by using affinity and gel filtration chromatography.Conclusion The soluble form of Ex-GP is successfully expressed and purified in a simple and convenient way.This study paves the way for further researches on the biological functions of rabies virus glycoprotein,the pathogenic mechanism of rabies and the development of diagnostic reagent and vaccines for rabies virus.
ABSTRACT
Objective To prepare a neutralizing monoclonal antibody against rabies virus. Meth-ods BALB/c mice were immunized with the inactivated rabies virus CTN strains on day 0, 7, 14 and 28. Spleen cell samples were collected and then fused with SP2/0 cells to prepare the hybridoma cell line. Posi-tive hybridoma cells that were screened out with RFFIT technique were injected into BALB/c mice intraper-itoneally. Ascites samples were collected from the mice to separate neutralizing monoclonal antibodies. Affin-ity chromatography was used for the purification of neutralizing monoclonal antibodies. Subtype identification and sequencing analysis were performed for further identification. A colloidal gold strip based method for rap-id detection of rabies vaccine was established with the prepared monoclonal antibodies. Results The hybri-doma cell line, CTN-McAb1, was prepared successfully with stable secretion of neutralizing monoclonal anti-bodies against rabies virus. The purity of those antibodies was more than 95% after purification and the sub-type of them was IgG1. The colloidal gold strip for raid detection of rabies vaccine was successful prepared. Conclusion The neutralizing monoclonal antibody against rabies virus was successfully prepared and could be used for preliminary application. This study will be of great significance for the quality control of rabies vaccine.
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Objective To construct the eukaryotic expression plasmid of the rabies virus glycoprotein gene DNA,and detect the immunogenicity.Methods Using RT-PCR amplified the glycoprotein gene of rabies virus CTN strain,sequenced and cloned into pcDNA5.0 (+) vector to construct the recombinant plasmid pcDNA5.0-G plasmid.Detect glycoprotein transient expression with transfecting the plasmid into 293T cells.Intramuscular immunization of BALB/c mice by the recombinant plasmid on day 0 and 7,then challenge by rabies virus CVS strain observed the mice survived.Results The results of the transient expression of glycoprotein abundantly expressed.The survival ratio of mice with CVS challenge after routine intramuscular injection of pcDNA5.0-G plasmid is 73.3％,and 6.7％ for the control group.Conclusion Rabies virus glycoprotein eukaryotic expression plasmid pcDNA5.0-G was successfully constructed,and has been good immunogenicity.It's to be the foundation for candidate DNA vaccine research and development.
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Objective To explore the application of atomic force microscopy( AFM ) on the research of morphology of the rabies viruses. Methods To prepare the rabies virus CTN-1v strains by ultracentrifugation, and observe it with transmission electron microscopy (TEM) which negatively stained by phosphotungstic acid. Then study the morphology of rabies virus with AFM based on the result of TEM. AFM image applies the tapping mode to rabies virus without any further treatment in air at room temperature. Results The TEM image is two-dimensional image which can be seen the classical bullet-shaped structure,and the spike structure can also be seen. The AFM image showed the rabies virus morphology with three-dimensional image which can shows the characteristics of the virus surface and edge. The rabies virus particle was successfully observed by TEM or AFM methods. Conclusion It's the first time to get the three-dimensional morphological structure of rabies virus by atomic force microscopy, compared with transmission electron microscopy, AFM is a new research tool for viral morphology study with the advantages of simple sample preparing and intuitionistic and visible interface for researchers.