小鼠Calb_2基因重组腺病毒载体的构建与鉴定

目的:为了体外研究Calretinin(Calb2)基因在生殖系统、神经系统、视觉传导中的作用,构建小鼠Calb2基因表达重组腺病毒载体。方法:用反转录聚合酶链反应(RT-PCR)方法,以小鼠Calb2cDNA为模板,扩增Calb2基因。亚克隆后,将Calb2基因片段克隆至腺病毒穿梭质粒pAdTrack-CMV上,构建穿梭质粒pAdTrack-Calb2。将穿梭质粒pAdtrack-Calb2转化至BJ-5183感受态细菌中,与腺病毒骨架质粒pAdEasy-1同源重组,获得重组质粒AdCMV-Calb2。重组质粒AdCMV-Calb2转染AD-293细胞,进行病毒包装和扩增,通过绿色荧光蛋白(GFP)报告基因观察重组病毒的产生。同时,构建对照腺病毒载体AdCMV-GFP。用获得的重组腺病毒感染AD-293细胞,通过观察GFP检测感染效率、蛋白质印迹方法检测Calb2基因的表达。结果:测序和酶切鉴定重组腺病毒质粒构建正确;经AD-293细胞包装后可观察到GFP表达;获得的重组腺病毒载体体外感染AD-293细胞,观察到GFP表达;经蛋白质印迹方法检测,与未感染腺病毒载体组(Mock)和感染对照腺病毒载体组(AdCMV)相比,Calb2基因腺病毒载体(AdCMV-Calb2)感染组CALB2蛋白水平显著增高(P<0.05)。结论:成功构建了小鼠Calb2基因重组腺病毒载体,并在AD-293细胞超表达,为课题组研究Calb2基因在生殖相关疾病的生理与病理生理作用奠定了基础。

亚砷酸钠致早期鸡胚胚胎发育毒性与基因甲基化的关系

目的:研究亚砷酸钠致鸡胚胚胎毒性与基因组DNA整体甲基化的关系以及补充甲基供体胆碱对无机砷致胚胎毒性的影响。方法:以鸡胚为模式动物检测亚砷酸钠及其与胆碱联合处理对鸡胚生存能力、体重及形态的影响;采用whole-mount免疫荧光技术检测鸡胚基因组DNA整体甲基化水平;应用荧光定量PCR技术,研究受甲基化调节的bcl2、c-myc、cdkn2a,calb2基因在不同处理组鸡胚中的表达模式。结果:砷处理组鸡胚存活率下降(P<0.01),体重降低(P<0.01),并诱导鸡胚发育畸形,基因组DNA整体甲基化水平降低(P<0.01),受甲基化调节的基因bcl2(P<0.05)、c-myc(P<0.01)表达下调;补充甲基供体胆碱,未明显恢复鸡胚的生存力和改变鸡胚发育异常,对鸡胚基因组DNA整体甲基化水平亦无明显影响,但c-myc(P<0.01)、cdkn2a(P<0.01)、calb2(P<0.05)基因的表达均显著下降。结论:亚砷酸钠能够诱导鸡胚发育异常,其作用可能是通过降低基因组DNA整体甲基化水平实现。补充甲基供体胆碱不能拮抗其致畸效应。

Construction of the recombinant adenovirus vectors of CALB_2 gene and small interfering RNA,and application in testicular Leydig cells

Objective:To construct the recombinant adenovirus vectors of calretinin(CALB_2) gene and small interfering RNA(siRNA),for over-expression or knock-down of CALB_2,as the basis of functional investigation of CALB_2 in testicular Leydig cells. Methods:The cDNA sequence of CALB_2 was cloned by the reverse transcriptive polymerase chain reaction (RT-PCR).A CALB_2 gene fragment was sub-cloned into adenovirus shuttle plasmid pAdTrack-CMV to construct the shuttle plasmid pAdTrack-CALB_2.Then it was transformed into BJ5183 cells with the adenoviral backbone pAdEasy-1 to obtain the homologous recombinant AdCMV-CALB_2.The recombinant AdCMV-CALB_2 was further packaged and amplificated in AD293 cells.The expression of CALB_2 protein in AD293 cells was detected by Western blotting.CALB_2 protein was over-expressed in mouse Leydig cell line(MLTC-1 cells) by the constructed AdCMV-CALB_2. CALB_2 gene siRNA recombinant adenovirus vector(Ad-H1-siRNA/CALB_2 was also constructed simultaneously. Its efficacy was detected in AD293 cells by Western blotting. Results:The CALB_2 gene recombinant adenovirus vector AdCMV-CALB_2 and the CALB_2 gene siRNA recombinant adenovirus vector Ad-H1-siRNA/CALB_2 were constructed successfully by endonulease digestion and sequencing. AD293 cells infected with AdCMV-CALB_2 or Ad-H1-SiRNA/CALB_2 significantly expressed GFP protein. The expression of CALB_2 protein was significantly up-regulated in AD293 cells infected with AdCMV-CALB_2 plasmids, while the expression of CALB_2 protein was down-regulated by 60%in the CALB_2 cells infected with Ad-H1-SiRNA/CALB_2. MLTC-1 cells did not markedly express CALB_2 protein,while MLTC-1 cells infected with AdCMV-CALB_2 expressed CALB_2 protein at a high level. Conclusions:The recombinant adenovirus vectors of AdCMV-CALB_2 and Ad-H1-SiRNA/CALB_2 were successfully constructed.Both vectors effectively expressed in AD293.CALB_2 protein was over-expressed in the cultured MLTC-1 cells by AdCMV-CALB_2.These vectors of CALB_2 gene and Leydig cell line are useful tools for investigating the testicular function.