二型谷氨酸羧肽酶口服基因疫苗的制备及其降低大鼠麻醉药用量的初步研究

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

目的 研制携带二型谷氨酸羧肽酶 (GCPⅡ )的口服疫苗 ,探讨该疫苗对大鼠麻醉药用量的影响。方法 采用聚合酶链反应、酶切连接和蓝白筛选的方法 ,构建GCPⅡ的表达载体 ;用磷酸钙共沉淀的方法转染HEK2 93细胞 ,G4 18筛选阳性细胞克隆 ;逆转录聚合酶链反应和免疫荧光细胞染色的方法鉴定阳性细胞株 ;氯化钙法制备携带GCPⅡ表达载体的减毒鼠伤寒沙门氏菌口服疫苗(SL GCPⅡ ) ;采用原代培养的巨噬细胞吞噬SL GCPⅡ的方法 ,观察疫苗的体外表达情况 ;将 5 0只雄性SD大鼠随机分为A、B 2组 ,每组 2 5只 ,2组鼠的体重分别为 ( 174 g±13g)和 ( 172 g± 11g) ,差异无显著意义 (P =0 0 72 )。A组胃内灌注 6 0 0 μL、OD2 60 0 6SL GCPⅡ ,两周内 4次给药 ;B组为对照组 ,给予SL32 6 1,剂量和方法与A组相同。采用免疫荧光的方法检测大鼠循环中GCPⅡ抗体滴度 ,观察麻醉药戊巴比妥钠的用量。结果 扩增GCPⅡ基因并构建了含有GCPⅡ基因的表达载体———pCDNA3 1 GCPⅡ ;建立了细胞株HEK 2 93 GCPⅡ ;体外实验证明培养的巨噬细胞吞噬SL GCPⅡ后 ,能够有效表达GCPⅡ基因。体内实验发现口服疫苗能够激活 2 2 / 2 5只SD大鼠产生GCPⅡ抗体 ,实验组麻醉药戊巴比妥钠的用量 ( 36 9mg/kg± 1 6mg/kg)显著低于对照组 ( 4 0

Objective To develop an oral vaccine carrying glutamate carboxypeptidase Ⅱ (GCP Ⅱ) and to explore whether it can affect the dosage of pentobarbiturate. Methods Polymerase chain reaction, digestion of endonucelease 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 immunofluresene 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 μl/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. Immunofluoresence method was used to detect the titer of antibody in rat circulation with HEK293 GCP Ⅱ cells as target cells. Results 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). Conclusion 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.