Construction and Immunogenicity of A Retrovirus Containing the Glycoprotein Gene of Rabies Virus
|School||Gansu Agricultural University|
|Course||Preventive Veterinary Medicine|
|Keywords||Rabies Glycoprotein Recombinant retrovirus Immune effect|
Retroviral vector is a replication -defective viral vectors , recombinant retrovirus can be constructed in the carrier in the packaging cell lines proliferate after the viral infection may integrate its genome into the host cell in order to achieve a foreign gene sustained and stable expression, the immunized animal is obtained lasting immune protection reaction. Retroviral vector pLNCX , the rabies virus glycoprotein gene was cloned into the pLNCX vector of the two long terminal repeat (LTR) , between the transfected packaging cells , to obtain recombinant virus after immunizing an animal , detection of animals tested specific antibodies in the level of protection . RT-PCR amplification the rabies virus SRV9 glycoprotein gene was cloned into pMD18-T vector and sequenced , sequencing analysis correctly glycoprotein gene was cloned into the eukaryotic expression vector pVAX1 digested and recycled by gel glycoprotein gene expression cassette inserted the retroviral vector pLNCL two long terminal repeat sequences middle, while deletion of the CMV promoter in the vector , to obtain a recombinant plasmid containing the glycoprotein genes pLNC - G . Preparation the recombinant plasmid pLNC - G to a large number of alkaline lysis method , liposome -mediated transfection of retrovirus packaging cells PA317 400μg/mL G418 pressure screening , a stably transfected cell lines . , Cell passaging extracted the cell genome using the PCR method , confirmed glycoprotein gene integrated into the the PA317 cell genome , with the host cell can be stably passaged . Extraction of total cellular RNA , RT - PCR method confirmed the G gene can be stable transcription . The virus titer of toxin-producing cell supernatants on NIH3T3 cells to 0.8 × 103 ~ 1.2 × 104CFU/mL. Higher titer of toxin-producing cell lines , expanding culture harvest recombinant virus , immune Kunming mice . 1.2 × 104CFU/mL recombinant virus to the abdominal cavity, mice immunized intramuscularly in two ways , every 2 weeks after immunization blood was collected , the serum was separated to the FAVN test detection serum anti-rabies virus neutralizing antibody titers . The results showed that the recombinant virus can induce the body to produce a good humoral immune response, muscle and intraperitoneal injection can make mice produce anti-rabies neutralizing antibody seroconversion rates were 6.7% and 86.7 % .