可调控性鼠PLP-Ig嵌合体重组腺病毒载体的构建和表达

目的 :构建和表达可调控鼠蛋白脂质蛋白 (PLP) 免疫球蛋白重组腺病毒载体。方法 :采用常规分子生物学方法 ,从WT 1杂交瘤细胞中抽提总RNA ,克隆小鼠Ig重链Fc基因。将编码PLP13 9-151的DNA与信号肽设计在引物上 ,经两次克隆可形成可分泌型PLP Ig。经穿梭载体与可调控性腺病毒载体骨架连接 ,鉴定后在HEK2 93细胞中包装。获得高滴度病毒后 ,用Westernblot鉴定目的基因的表达。结果 :PLP Ig重组腺病毒经测序、限制性内切酶酶切分析及PCR等鉴定 ,同预期结果相一致。Westernblot检测病毒感染的细胞 ,发现目的基因得到特异性表达且可被四环素调节。结论 :构建了可调控的小鼠PLP Ig重组腺病毒载体并得到正确表达 ,为进一步基因治疗实验性变态反应性脑脊髓炎 (EAE)和诱导免疫耐受奠定了基础

Construction and expression of regulatable recombinant adenovirus vector for mouse PLP-Ig chimeric protein

AIM: To construct a regulatable recombinant adenovirus vector expressing mouse PLP-Ig. METHODS: Total RNA was extracted from WT-1 hybridoma cells to clone Ig heavy chain Fc fragment gene. PCR was carried out to link PLP_(139-151) gene and signal peptide with Fc fragment gene. pTRE-shuttle vector was used to ligate PLP-Ig gene and the backbone of the replication-incompetent adenoviral vector. After confirming the desired recombinant adenovirus by PCR and restriction endonuclease digestion analysis, it was packaged and propagated in HEK 293 cells. Tetracycline was used to regulate the expression of PLP-Ig, and Western blot was used to detect the gene expression. RESULTS:The expression of PLP-Ig was confirmed by Western blot and its expression could be regulated by tetracycline. CONCLUSION: The regulatable recombinant adenovirus vector for mouse PLP-Ig was constructed and expressed successfully, which lays the foundation for further study on gene therapy of EAE and induction of immune tolerance.