CAJ | 학술논문

目的：构建猕猴B病毒囊膜蛋白gD的真核表达载体，并且检测其在293T细胞内的表达情况。方法首先通过基因合成手段获得含B病毒gD蛋白的基因片段，在经由PstⅠ和NotⅠ双酶切后连接到pEGFP-N3载体，随后将构建的pEGFP-N3-GD重组质粒转染到人胚胎肾上皮细胞系293T细胞。再用Western blot检测所提蛋白其在细胞内的表达情况，并用激光共聚焦分析其在细胞内的表达定位情况。结果成功获得携带gD基因的阳性重组质粒pEGFP-N3-GD，且pEGFP-N3-GD重组质粒能在293T细胞的表面正常表达。结论利用真核表达系统，既能够在细胞表面产生B病毒gD蛋白的特异性重组抗原，而且可用于B检测抗原的制备。
목적：구건미후B병독낭막단백gD적진핵표체재체，병차검측기재293T세포내적표체정황。방법수선통과기인합성수단획득함B병독gD단백적기인편단，재경유PstⅠ화NotⅠ쌍매절후련접도pEGFP-N3재체，수후장구건적pEGFP-N3-GD중조질립전염도인배태신상피세포계293T세포。재용Western blot검측소제단백기재세포내적표체정황，병용격광공취초분석기재세포내적표체정위정황。결과성공획득휴대gD기인적양성중조질립pEGFP-N3-GD，차pEGFP-N3-GD중조질립능재293T세포적표면정상표체。결론이용진핵표체계통，기능구재세포표면산생B병독gD단백적특이성중조항원，이차가용우B검측항원적제비。
Objective To establish an eukaryotic vector of monkey B virus glycoprotein D gene and analyze the expression of gD gene in human embryonic kidney 293T cells.Method First, the protein of monkey B virus glycoprotein D was obtained by gene synthesis.The gene fragments were digested with Pst I and Not I, and ligated to pEGPF-N3. Then, the recombinant plasmid pEGPF-N3-GD was transfected into 293T cells.The expression of gD protein in the cells was detected by Western blot, and the expression localization was investigated using laser scanning confocal microscopy. Results The recombinant plasmid pEGPF-N3 carrying gD gene was successfully constructed, and normally expressed in the 293T cells.Conclusions Glycoprotein D of monkey B virus is expressed successfully in the 293T cells and the protein is located on the cell surface.It may be useful for the preparation of specific recombinant antigen to the glycoprotein D of monkey B virus on cell surface, and can be also used for preparation of antigen slide for detection of monkey B virus.