CAJ | 학술논문

目的：构建含变异链球菌表面蛋白SpaP的P区编码基因spap-P和葡聚糖结合蛋白GbpA的葡聚糖结合区编码基因gbd的嵌合体真核表达质粒pVAX1-SPG,并观察其在哺乳动物细胞293T中的表达。方法：通过基因工程技术构建嵌合体真核表达质粒pVAX1-SPG,并采用脂质体转染法将其转染至293T细胞中；然后分别采用免疫组化SABC法和Western-blot检测嵌合蛋白SpaP/P-GBD在真核细胞中的表达。结果：重组真核表达质粒pVAX1-SPG经酶切、测序鉴定证实,其所携带的外源基因片段为2.4 kb的目的基因片段,序列同源性为99%；免疫组化染色结果显示,经pVAX1-SPG转染的293T细胞胞质呈棕褐色染色；Western-blot检测结果显示,分子量为72 kDa的嵌合蛋白SpaP/P-GBD能够被正确表达。结论：构建成功的嵌合体真核表达质粒pVAX1-SPG能在真核细胞293T中正确表达目的蛋白。
목적：구건함변이련구균표면단백SpaP적P구편마기인spap-P화포취당결합단백GbpA적포취당결합구편마기인gbd적감합체진핵표체질립pVAX1-SPG,병관찰기재포유동물세포293T중적표체。방법：통과기인공정기술구건감합체진핵표체질립pVAX1-SPG,병채용지질체전염법장기전염지293T세포중；연후분별채용면역조화SABC법화Western-blot검측감합단백SpaP/P-GBD재진핵세포중적표체。결과：중조진핵표체질립pVAX1-SPG경매절、측서감정증실,기소휴대적외원기인편단위2.4 kb적목적기인편단,서렬동원성위99%；면역조화염색결과현시,경pVAX1-SPG전염적293T세포포질정종갈색염색；Western-blot검측결과현시,분자량위72 kDa적감합단백SpaP/P-GBD능구피정학표체。결론：구건성공적감합체진핵표체질립pVAX1-SPG능재진핵세포293T중정학표체목적단백。
AIM::To construct the eukaryotic plasmid including surface protein antigen ( SpaP) P domain (SpaP-P) and glucan binding domain (GBD) of glucan binding protein A (GbpA) of Streptococcus mutans, and to observe the expression of recombinant plasmid pVAX1-SPG in mammalian cells 293T. METHODS: The eukaryotic plasmid pVAX1-SPG carrying encoding gene of GBD of GbpA and SpaP-P of SpaP of Streptococcus mutans was con-structed by recombinant DNA technology. The eukaryotic plasmid was transfected into 293T cells by lipofectamine rea-gent. The transient expression of the protein in 293T cells was detected by immunochemistry technique and Western-blot. RESULTS:The recombinant plasmidp VAX1-SPG was obtained and identified by restriction endonuclease anal-ysis. Immunohistological staining showed that positive expression of SpaP/P-GBD was detected in the plasma of 293T cells transfected with recombinant plasmid pVAX1-SPG. Western-blot confirmed the expression of the fusion protein SpaP/P-GBD in 293T cells. CONCLUSION:The eukaryotic plasmid pVAX1-SPG can be constructed. The target protein SpaP/P-GBD can be expressed correctively in 293T cells.