大鼠opticin表达基因小发夹RNA真核表达质粒的构建及鉴定

Construction and identification of small hairpin RNA expression plasmid targeting opticin gene in rat

目的 构建针对大鼠opticin表达基因（OPTC）的特异性小发夹RNA（shRNA）真核表达质粒.方法 用DNA重组技术将针对大鼠OPTC基因不同位点所设计的4个shRNA序列克隆到真核表达 质粒中.根据构建的4对质粒设置shRNA-1组、shRNA-2组、shRNA-3组、shRNA-4组,同时只加转染试剂不加质粒的细胞设为正常对照组,转染阴性质粒的细胞为阴性对照组.用脂质体lipofectimine 2000将4个shRNA质粒分别转染至体外培养的原代大鼠睫状体非色素上皮（NPE）细胞,转染后采用半定量逆转录-聚合酶链式反应（RT-PCR）和蛋白免疫印迹法（Western blot）分别检测OPTC基因的mRNA及opticin蛋白的相对表达量和抑制率.结果 各组质粒转染大鼠NPE细胞后,shRNA-1、shRNA-2、shRNA-3、shRNA-4组细胞OPTC mRNA相对表达较正常对照组均明显下调（F=10.239,P=0.000）,各组OPTCmRNA抑制率为85.7%、62.87%、54.87%、48.77%.各质粒干扰组细胞opticin蛋白表达较正常组均有不同程度下降（F=17.870,P=0.000）,各组opticin蛋白抑制率为78.7%、34.6%、31.1%、16.8%.结论 OPTC基因干扰质粒shRNA-1可以有效抑制大鼠睫状体NPE细胞opticin的表达.

Objective To construct small hairpin RNA （shRNA） expression plasmid targeting rat opticin gene. Methods Four pairs of opticin oligonucleotides were synthesized and inserted into the plasmid vector, resulting into four plasmids： shRNA-1, shRNA-2, shRNA-3 and shRNA-4. Then the four constructed shRNA expression vectors and empty vector were transfected into rat ciliary non-pigment epithelium （NPE） cells by lipofectmaine 2000. Non-transfected NPE cells were set as control group. The expression of opticin mRNA and protein were measured by Reverse transcriptase polymerase chain reaction （RT-PCR） and Western blot respectively. Results The opticin mRNA expression of the shRNA-1,shRNA-2, shRNA-3, shRNA-4 group were decreased compared with the control group （F = 10. 239, P = 0. 000）;the inhibitory rate were 85.7% ,62. 87% ,54.87% and 48.77% respectively. The opticin protein expression of the shRNA-1,shRNA-2, shRNA-3, shRNA-4 group were also decreased compared with the control group （F=17.870, P= 0.000）;the inhibitory rate were 78.7%, 34.6%, 31.1% and 16.8% respectively.Conclusions The shRNA-1 expression plasmid has most potent inhibitory effect on opticin expression in rat ciliary NPE cells.