N-甲基-D-天冬氨酸受体NR1亚基口服基因疫苗的制备和激活SD大鼠产生循环抗体的初步报告

A Preliminary Report of the Production of Oral Vaccine Carrying the NR1 Gene and Its Role on Producing NR1 Antibodies in SD Rats

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 produce the oral vaccine carrying NMDAR NR1,and explore whether it can induce circular antibodies in SD rats. Methods Polymerase chain reaction,digestion of endonucelease and ligation,blue-white selection were used to construct the expression vector;Ca 3(PO 4) 2 method was used to transfect HEK293 cell,G418 to select positive cell clone;reverse-transcription PCR and immunofluresene cell staining were used to testify positive cell strain;the method of CaCl 2 toproduceoralvaccineofattenuatedSalmonella typhimurium carrying NR1(SL-NR1);when D(λ) 260 nm equal 0.6,600 μL of SL-NR1 was given to the SD rat intragastrically,4 times per 2 weeks;the positive cell strain as target cell,use immunofluresence method to detect the level of antibody in rat blood circulation. Results NR1 was amplified by PCR method; the expression vector containing NR1: pCDNA3.1-NR1 was constructed;the cell line——HEK 293-NR1 was established;the oral vaccine could elicite 23/25 SD rats to produce NR1 antibodies. Conclusions The attenuated Salmonella oral vaccine carrying NR1 gene is produced successfully and it can activate the immune response of rat to produce NR1 antibodies.