白细胞介素4反义基因腺相关病毒载体质粒的构建及鉴定

Construction and Identification of Adeno-associated Virus-based Plasmid Carrying Antisense IL-4

目的构建携带大鼠反义白细胞介素4(IL-4)基因并含有腺相关病毒(AAV)末端反向重复序列(ITR)结构的重组质粒载体pasIL-4。方法利用基因重组技术将大鼠IL-4 cDNA反向克隆到已酶切去除绿色荧光蛋白(GFP)基因的真核表达载体phMGFP中,构建重组中间质粒载体。通过PCR技术扩增获取重组中间质粒载体上的目的片段CMVp-asIL4-SV40E,将此目的片段定向插入已酶切处理的psub201中,构建出重组质粒pasIL-4。进一步通过阳离子脂质体介导,将pasIL-4转染哮喘大鼠CD4+T细胞,RT-PCR法和ELISA法分别检测细胞中IL-4 mRNA和细胞培养上清液IL-4蛋白的变化。结果pasIL-4经限制性酶切及部分序列测序分析证实IL-4反向插入正确。pasIL-4重组质粒转染的CD4+T细胞IL-4 mRNA水平和细胞培养上清液中IL-4水平较未转染组明显下降。结论pasIL-4重组质粒载体构建成功,为今后利用其进行哮喘基因治疗的研究奠定基础。

Objective To construct a recombinant plasmid pasIL-4 carrying rat IL-4 RNA antisense gene and adeno-associ ated viral （AAV） inverted terminal repeats （ITR）. Methods An intermediary plasmid was constructed by inserting IL-4 eD NA in reverse order to the eukaryotie expression vector phMGFP which had been cleaved with Xba 1 ＋EcoRV to remove the GFP gene. Then the sequence CMVρ-asIL-4-SV40E was amplified from the intermediary plasmid by PCR. The recombinant plasmid pasIL4 was constructed by replacing the viral replieable genes in the psub201 with the sequence CMVρ-asIL-4 SV40E. pasIL-4 was transfected into CD4^＋ T lymphoeytes, and IL-4 protein in supernatant of cells culture and IL-4 mRNA in T lymphocytes were detected by ELISA and semi-quantitative RT PCR respectively. Results pasILT4 was identified with double re striction enzymes digestion and DNA sequence analysis. The expression of IL-4 protein and mRNA in CD4^＋ T lymphoeytes treated with paslL-4 was significantly decreased. Conclusion The recombinant plasmid pasIL-4 was constructed successfully. The availability of the pasIL-4 should facilitate construction of a recombinant adeno-associated virus 2 （AAV）-based virions containing IL-4 antisense gene for potential use in asthma gene therapy.