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目的 研制携带 N-甲基 -D-天冬氨酸受体 1 ( N-methyl,D-aspartate receptor subunit1 ,NR1 )的口服疫苗 ,探讨疫苗激活 SD大鼠产生循环抗体的可能性。方法 采用聚合酶链反应、酶切连接和蓝白筛选的方法构建NR1的表达载体 ;以磷酸钙共沉淀的方法转染 HEK2 93细胞 ,并用 G41 8筛选阳性细胞克隆 ;应用逆转录聚合酶链反应和免疫荧光细胞染色方法鉴定阳性细胞株 ;经氯化钙法制备携带 NR1表达载体的减毒鼠伤寒沙门 (氏 )菌口服疫苗 ( SL-NR1 ) ;胃内灌注 60 0 μL、D( λ) 2 6 0 nm为 0 .6的 S L-NR1 ,2周内 4次给药 ;以阳性细胞株为靶细胞 ,用免疫荧光方法检测大鼠循环中 NR1抗体滴度。结果 扩增出 NR1基因并构建了含有 NR1基因的表达载体—— p CDNA3 .1 -NR1 ;建立了细胞株 HEK 2 93 -NR1 ;证实口服疫苗可激活 SD大鼠 ( 2 3 /2 5只 )产生 NR1抗体。结论 成功研制了携带 NR1基因的口服减毒鼠伤寒沙门 (氏 )菌活疫苗 ,其可激活机体免疫反应产生 NR1抗体。
OBJECTIVE: To develop an oral vaccine containing N-methyl-D-aspartate receptor 1 (NR1) and investigate the possibility of circulating antibodies in SD rats. Methods NR1 expression vector was constructed by polymerase chain reaction, restriction enzyme digestion and blue-white screening. HEK2 93 cells were transfected by calcium phosphate co-precipitation, and positive cells were screened by G41 8. Reverse transcription-polymerase chain reaction (SL-NR1). The intragastric instillation of 60 0 μL, D (λ) was performed by using calcium chloride method to prepare an attenuated S. typhimurium oral vaccine with NR1 expression vector. 2 260 nm to 0.6 S L-NR1, 4 doses within 2 weeks; Positive cell lines as target cells, immunofluorescence assay NR1 antibody titers in rat circulation. Results The NR1 gene was amplified and the expression vector containing NR1 gene was constructed - pCDNA3 .1 - NR1. The cell line HEK2 93 - NR1 was established. The oral vaccine can activate SD rats (23/25) The NR1 antibody is produced. Conclusion The live attenuated Salmonella typhimurium vaccine carrying NR1 gene has been successfully developed, which can activate the immune response to produce NR1 antibody.