CAJ | 학술논문

背景：人可诱导共刺激分子(ICOS)是迄今发现的重要共刺激分子家族成员，可促进激活T细胞的增殖和分泌、调节Th1/Th2细胞的极化、增强依赖T细胞的B细胞功能，阻断ICOS共刺激信号会导致T细胞的克隆失活或克隆无反应，从而诱导肿瘤对机体的免疫逃逸。 　　目的：构建表达ICOSIg的质粒，观察其在小鼠体内的表达。 　　方法：克隆编码ICOS的胞外片段，将其与编码小鼠免疫球蛋白IgG恒定片段(Ig)的基因融合，构建ICOSIg融合基因及其分泌型真核表达载体 pcDNA3-ICOSIg，酶切鉴定重组子，测序，利用阳性脂质体载体包被pcDNA3-ICOSIg转染小鼠右侧大腿肌肉组织，Western blot法检测血清ICOSIg水平。 　　结果与结论：经测序鉴定证实pcDNA3-ICOSIg质粒目的基因片断与Genbank上公布的ICOS序列完全一致，说明质粒构建成功。脂质体载体包被pcDNA3-ICOSIg转染小鼠7 d，在小鼠血清中检测到ICOSIg的阳性表达，说明 pcDNA3-ICOSIg 能够在小鼠肌细胞内表达。证实利用基因合成和重组技术可成功构建真核表达载体pcDNA3-ICOSIg。
배경：인가유도공자격분자(ICOS)시흘금발현적중요공자격분자가족성원，가촉진격활T세포적증식화분비、조절Th1/Th2세포적겁화、증강의뢰T세포적B세포공능，조단ICOS공자격신호회도치T세포적극륭실활혹극륭무반응，종이유도종류대궤체적면역도일。 　　목적：구건표체ICOSIg적질립，관찰기재소서체내적표체。 　　방법：극륭편마ICOS적포외편단，장기여편마소서면역구단백IgG항정편단(Ig)적기인융합，구건ICOSIg융합기인급기분비형진핵표체재체 pcDNA3-ICOSIg，매절감정중조자，측서，이용양성지질체재체포피pcDNA3-ICOSIg전염소서우측대퇴기육조직，Western blot법검측혈청ICOSIg수평。 　　결과여결론：경측서감정증실pcDNA3-ICOSIg질립목적기인편단여Genbank상공포적ICOS서렬완전일치，설명질립구건성공。지질체재체포피pcDNA3-ICOSIg전염소서7 d，재소서혈청중검측도ICOSIg적양성표체，설명 pcDNA3-ICOSIg 능구재소서기세포내표체。증실이용기인합성화중조기술가성공구건진핵표체재체pcDNA3-ICOSIg。
BACKGROUND:So far, inducible co-stimulator is the important costimulatory molecule family member. Inducible co-stimulator can promote the activation of T cel s proliferation and secretion, regulate Th1/Th2 cel polarization dependence, enhance B cel function which depend on the T cel s. So, blocking the inducible co-stimulator may result the inactivation and no reaction of cloning in T cel s, thus inducing the immune escape of tumor on the body. OBJECTIVE:To build a plasmid expression of inducible co-stimulator Ig, in order to observe the expression in rat body. METHODS:cDNA encoding the extracel ular domain of human inducible co-stimulator was prepared. The encode of the domain was fused with the gene of immunoglobulin IgG constant fragment (Ig) of encoding mouse, in order to build the inducible co-stimulator Ig fusion gene and the secreted eukaryotic expression vector pcDNA3-inducible co-stimulator Ig. Enzyme digestion of the recombinant and sequencing was performed, and then the positive liposome coated pcDNA3-inducible co-stimulator Ig was transferred into the muscle tissue of mouse right thigh. Western blot was used to detect the level of inducible co-stimulator Ig. RESULTS AND CONCLUSION:The sequencing confirmed that the size of target gene fragment pcDNA3-inducible co-stimulator Ig plasmid was exactly the same with the sequence of inducible co-stimulator published on Genebank, which indicated the successful of plasmid construction. After transferred into the mouse for 7 days, the liposome coated pcDNA3-inducible co-stimulator Ig was positively expressed in the mice serum, which showed that pcDNA3-inducible co-stimulator Ig could be expressed in the rat muscle cel s. The results suggest that gene synthesis and recombinant technology can successful y construct the eukaryotic expression vector pcDNA3-inducible co-stimulator Ig.