Development of oral vaccine carrying GCPII gene and its role in reducing the dosage of pentobarbital in rat: a primitive research

Abstract

To develop an oral vaccine carrying glutamate carboxypeptidase II (GCP II) and to explore whether it can affect the dosage of pentobarbiturate. Polymerase chain reaction, digestion of endonuclease and ligation, blue-white selection were used to construct an expression vector pcDNA3.1-GCP II. HEK293 cells were cultured. The vector pcDNA3.1-GCP II was transfected into the HEK293 cells by Ca(3)(PO(4))(2) coprecipitation method. The transfected HEK293 cells were cultured in HEM liquid culture prepared with G418. Three weeks after, positive clones, HEK293-GCP II, were identified. Reverse-transcription PCR and immunofluorescence cell staining were used to testify positive cell line; Method of CaCl(2) was used to prepare oral vaccine of attenuated Salmonella typhimurium carrying GCP II (SL-GCP II). Expression of SL-GCP II in vitro was observed by adding SL-GCP II into the primarily cultured macrophage. Fifty male SD rats were randomly divided into 2 groups of 25 rats: group A, undergoing intragastrical infusion of SL-GCP II, 600 micro l/time, in total 4 times in 4 days; and group B, as control group, undergoing intragastrical infusion of SL3261. Fifteen days after, 5 g/L pentobarbital sodium was injected intraperitoneally with the first dosage of 1.0 ml and the response was observed in 10 minutes, then 0.1 ml was added every time. The specific dosage of pentobarbital sodium was recorded when anesthesia meeting the requirement of operation was reached. Phenobarbital sodium of this dosage was used to anesthetize the rats to observe the response of the rats. Immunofluorescence method was used to detect the titer of antibody in rat circulation with HEK293 GCP II cells as target cells. An expression vector containing GCP II, pCMV-GCP II, pCDNA3.1-GCP II was constructed. The cell line, HEK 293-GCP II was established. In vitro experiment proved that primarily cultured macrophage phagocytized SL-GCP II and effectively expressed GCP II gene. After infusion of the oral vaccine 22 of the 25 SD rats of the group A produce GCP II antibodies. The dosage of pentobarbiturate used in experimental group was 36.9 mg/kg +/- 1.6 mg/kg; significantly lower than that in the control group (40.8 mg/kg +/- 1.4 mg/kg, P = 0.00). An oral vaccine carrying GCP II gene has been developed that activates the immune response of rat to produce GCP II antibodies and lower the dosage of pentobarbiturate needed.