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Establishment and Characterization of Transformed Mammalian Cell Lines Stably Expressing the Rabies Virus Glycoprotein

Author: YuZhiFeng
Tutor: ZuoRongLiang
School: Jilin University
Course: Preventive Veterinary Medicine
Keywords: rabies virus glycoprotein transfection stable expression mammalian cell immunization viral replication
CLC: S852.65
Type: Master's thesis
Year: 2006
Downloads: 133
Quote: 2
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Abstract


Genetically engineered rabies vaccine is one example of the mostsuccessful genetically engineered vaccine. Recently, more and more studiesfor rabies vaccine focus on the recombinant vaccine. The recombinantcanine adenovirus expressing the rabies glycoprotein constructed in ourlaboratory can induce good immune response, but the neutralizing antibodylevel is not as high as that induced by the conventional vaccines, i.e.,inactivated or attenuated live vaccines. To increase the immunogenecity ofthe recombinant vaccine or the live rabies vaccine, we established severallines of transformed cell expressing the rabies virus glycoprotein andstudied their characteristics.Firstly, the glycoprotein of rabies virus strain CVS-24 was amplifiedby reverse transcription-polymerase chain reaction and cloned it and itsfusion gene with green fluorescent protein into eukaryotic expression vectorpIRES1neo. The resultant plasmid pICG and pIEGFP-CG were transfectedinto BHK-21 cell line by liposome transfection. After screening with G418,G418-resistant BHK-21 colonies were obtained and amplified. Withwestern-blot analysis, there appeared a reaction band between the specificantibody and the expressed protein, which indicated that the protein hasbeen expressed. By screening with indirect ELISA, three cell clones of eachexpression vector with high level expression were established and used forfurther identification, named respectively ICG1, ICG2, ICG3 andIEGFP-CG1, IEGFP-CG2, IEGFP-CG3.The expressed protein was used to inject mice for immunologicalevaluation. Totally 80 mice were randomly divided into eight groups, allwere intramuscularly immunized with 300μl the supernatant of cell clones,inactivated vaccine, and the supernatant of BHK-21 cells at day 0, 7 and21, respectively. Indirect ELISA and RFFIT were used to detect thespecific antibody and the rabies virus neutralizing antibody. Resultsshowed that the antibody was produced after vaccination. The more thevaccination was given, the higher the antibody level increased. After 3times of vaccination, the neutralizing antibody could completely neutralize60.25 TCID50 of rabies virus.The IEGFP-CG recombinant cells and BHK-21 cells were separatelyinoculated with Rabies virus CVS-11. Infection with the virus showed thatthe recombinant cell produced a lower level of virus that theuntransformed BHK-21 cells. We also found that the more theglycoprotein expressed, the lower the rabies virus produced.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Basic Veterinary Science > Animal Microbiology ( Veterinary Microbiology, ) > Livestock Virology
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