CAJ | 학술논문

DNA疫苗是将编码抗原的基因插入载体质粒中构成重组体，直接接种机体，在接种部位摄取表达并达到抗感染目的的一种疫苗。 pCMV-Taq2B载体具有人巨细胞病毒（ Human cytomegalovirus， CMV）高效启动子／增强子序列，可使目的基因在真核细胞中高水平稳定表达成为DNA疫苗载体。构建pCMV-Taq2B-Surface antigen 2（表面膜抗原2）（ pSAG2）、 pCMV-Taq2B-Dense granule protein 2（致密颗粒蛋白2）（pGRA2）及 pCMV-Taq2B-SAG2-GRA2（ pSAG2-GRA2） DNA 疫苗并研究其免疫保护效果。将 pSAG2、pGRA2、 pSAG2-GRA2 DNA疫苗（实验组）、磷酸盐缓冲液（Phosphate buffered saline， PBS）、 pCMV-Taq2B空质粒（对照组）分别肌肉注射BALB／c小鼠， ELISA法检测血清IgG抗体水平。末次免疫后第28 d，各组每只小鼠经腹腔注射弓形虫速殖子1000个，观察小鼠的生存时间。结果显示单基因疫苗pSAG2、 pGRA2和双基因疫苗pSAG2-GRA2免疫组均能诱导产生特异性IgG抗体，且于末次免疫后第28 d达到最高值，此时单基因疫苗pGRA2（0.382±0.66）和双基因疫苗pSAG2-GRA2（0.548±0.137）免疫组吸光度值显著高于PBS （0.137±0.016）或pCMV-Taq2B （0.135±0.010）对照组吸光度值。免疫单基因疫苗pSAG2（14.3±1.8）、 pGRA2组（13.5±2.1）小鼠存活时间（d）明显长于PBS （4.3±1.6）及pCMV-Taq2B组（4.1±1.3），且双基因疫苗 pSAG2-GRA2组（19.6±2.4）小鼠存活时间明显长于单基因疫苗 pSAG2组及pGRA2组。弓形虫双基因疫苗pSAG2-GRA2能够诱导小鼠产生抗弓形虫感染保护性免疫，其免疫保护性优于pSAG2、 pGRA2单基因疫苗。
DNA역묘시장편마항원적기인삽입재체질립중구성중조체，직접접충궤체，재접충부위섭취표체병체도항감염목적적일충역묘。 pCMV-Taq2B재체구유인거세포병독（ Human cytomegalovirus， CMV）고효계동자／증강자서렬，가사목적기인재진핵세포중고수평은정표체성위DNA역묘재체。구건pCMV-Taq2B-Surface antigen 2（표면막항원2）（ pSAG2）、 pCMV-Taq2B-Dense granule protein 2（치밀과립단백2）（pGRA2）급 pCMV-Taq2B-SAG2-GRA2（ pSAG2-GRA2） DNA 역묘병연구기면역보호효과。장 pSAG2、pGRA2、 pSAG2-GRA2 DNA역묘（실험조）、린산염완충액（Phosphate buffered saline， PBS）、 pCMV-Taq2B공질립（대조조）분별기육주사BALB／c소서， ELISA법검측혈청IgG항체수평。말차면역후제28 d，각조매지소서경복강주사궁형충속식자1000개，관찰소서적생존시간。결과현시단기인역묘pSAG2、 pGRA2화쌍기인역묘pSAG2-GRA2면역조균능유도산생특이성IgG항체，차우말차면역후제28 d체도최고치，차시단기인역묘pGRA2（0.382±0.66）화쌍기인역묘pSAG2-GRA2（0.548±0.137）면역조흡광도치현저고우PBS （0.137±0.016）혹pCMV-Taq2B （0.135±0.010）대조조흡광도치。면역단기인역묘pSAG2（14.3±1.8）、 pGRA2조（13.5±2.1）소서존활시간（d）명현장우PBS （4.3±1.6）급pCMV-Taq2B조（4.1±1.3），차쌍기인역묘 pSAG2-GRA2조（19.6±2.4）소서존활시간명현장우단기인역묘 pSAG2조급pGRA2조。궁형충쌍기인역묘pSAG2-GRA2능구유도소서산생항궁형충감염보호성면역，기면역보호성우우pSAG2、 pGRA2단기인역묘。
DNA vaccine is a recombinant plasmid vector with antigen encoding gene inserted, when directly inoculated into the body, it was uptake at the inoculation site and expressed, finally achieve the purpose of anti infection.pCMV-Taq2B vector contains human cytomegalovirus ( CMV ) promoter/enhancer sequence and make it possible to objective gene expressed high and stable in eukaryotic cells and used as DNA vaccine vector.We constructed the pCMV-Taq2B-Surface antigen 2 (pSAG2), pCMV-Taq2B-Dense granule protein 2 (pGRA2) and pCMV-Taq2B-SAG2-GRA2 (pSAG2-GRA2) DNA vaccine and studied their protective efficacy of immune responses.BALB/c mice were intramuscular injected with pSAG2, pGRA2, pSAG2-GRA2 DNA vaccine ( experimental group) or Phosphate Buffered Saline ( PBS) , pCMV-Taq2B empty vector ( control group) and the serum IgG antibody levels were detected by ELISA method.Twenty-eight days after the final immunization, mice were intraperitoneal injected with Toxoplasma gondii RH strain (1 000 tachyzoites per mouse) and survival days were observed.Results showed that mice immunized with single gene vaccine of pSAG2, pGRA2 or double gene vaccine pSAG2-GRA2 showed significantly increased serum IgG levels and these levels were peaked twenty-eight days after the last immunization; Moreover, mice immunized with single gene vaccine pGRA2 ( 0.382 ±0.66 ) or double gene vaccine pSAG2-GRA2 (0.548 ±0.137) had significantly higher optical density compared with PBS (0.137 ±0.016) or pCMV-Taq2B (0.135 ±0.010) control group.The mice immunized with pSAG2 (14.3 ±1.8) or pGRA2 (13.5 ±2.1) single gene vaccine survived significantly longer days than the control group mice immunized with PBS ( 13.5 ±2.1 ) or pCMV-Taq2B ( 4.1 ±1.3 ).And the pSAG2-GRA2 ( 19.6 ±2.4 ) double gene vaccine immunized mice survived significantly longer than the pSAG2 or pGRA2 immunized groups.The pSAG2-GRA2 double gene DNA vaccine was more effective in stimulating host protective immune responses than separately injected pSAG2 or pGRA2 single gene vaccine.