目的:构建人TGF-β2特异性siRNA质粒。方法:从NCBI中查找人TGF-β2的mRNA序列,并利用生物信息学对序列进行分析;根据siRNA的设计原则,选择最佳的siRNA序列;利用限制性内切酶BamHⅠ和BbsⅠ将相应的双链DNA插入到GPU6/GFP/Neo载体中;重组质粒经限制性内切酶BamHⅠ和PstⅠ单酶切进行鉴定,鉴定正确的质粒进一步通过基因测序的方法进行验证。结果:根据生物信息学选择TGF-β2mRNA的1016～1034区域作为干扰位点;重组质粒酶切结果表明双链DNA序列成功插入到了GPU6/GFP/Neo载体中;测序分析结果进一步证明插入序列与设计完全一致。结论:成功构建了人TGF-β2特异性siRNA干扰质粒,为研究TGF-β2基因功能和利用基因治疗的方法提高青光眼滤过手术成功率奠定了实验基础。

AIM:To construct specific siRNA expression vector targeting human TGF-β2.METHODS:The mRNA sequences of human TGF-β2 in NCBI were analysed in silico.The best sequence of siRNA were designed according to the siRNA designing principle.The double-stranded DNA was inserted into the GPU6/GFP/Neo vector by the BamH Ⅰ and Bbs Ⅰ.The recombinant plasmid was identified by the BamH Ⅰ and PstⅠ single enzyme digestion and verified by DNA sequencing further.RESULTS:The positions of 1016-1034 in TGF-β2 mRNA were chosen as the RNAi target.The result of the enzyme digestion to the recombinant plasmid showed that the double-stranded DNA was successfully inserted into the GPU6/GFP/Neo vector.The inserted sequence was the same as the designed sequences and was proved by the DNA sequencing.CONCLUSION:The human TGF-β2 specified siRNA interference plasmid was successfully constructed,and this plasmid can be used for the study of the function of TGF-β2 gene and improve the success rate of glaucoma filtration surgery by gene therapy.

R775

中国深圳市科技计划资助项目(No.2009036)~~