论文部分内容阅读
目的构建小鼠pVAX1-CIRP真核表达载体,并观察电穿孔法导入隐睾小鼠睾丸组织后CIRP基因的表达情况。方法 pBluescript SK(-)-CIRP质粒经EcoR I和Pst I双酶切后,连接到pVAX1载体对应的酶切位点,构建pVAX1-CIRP真核表达载体。经酶切及测序鉴定正确后,用电穿孔的方法把pVAX1-CIRP真核表达载体导入到隐睾小鼠睾丸组织中,对照组导入空载的pVAX1,7 d和10 d后取材,应用RT-PCR和Western Blot的方法鉴定CIRP mRNA和蛋白的表达情况。结果经酶切及测序鉴定构建的pVAX1-CIRP真核表达载体正确。将其导入隐睾小鼠睾丸组织后7d,CIRP mRNA和蛋白的表达量较对照组均明显升高,差异均有统计学意义(均<0.05)。其电穿孔后10d CIRP的水平与7 d时差异无统计学意义(>0.05)。结论成功构建小鼠pVAX1-CIRP真核表达载体,导入隐睾小鼠睾丸组织后能够正常的转录和表达,为进一步研究CIRP基因在精子发生中的作用及男性不育的发病机制奠定基础。
Objective To construct the mouse pVAX1-CIRP eukaryotic expression vector and observe the expression of CIRP gene in testis of cryptorchid mouse after electroporation. Methods The plasmid pBluescript SK (-) - CIRP was double digested with EcoR I and Pst I and ligated into the restriction site of pVAX1 vector to construct eukaryotic expression vector pVAX1 - CIRP. After digestion and sequencing, the pVAX1-CIRP eukaryotic expression vector was electroporated into the testes of cryptorchid testis mice. The control group was injected with empty vector pVAX1 for 7 days and 10 days, then RT The expression of CIRP mRNA and protein was identified by PCR and Western Blot. Results The pVAX1-CIRP eukaryotic expression vector was identified by restriction enzyme digestion and sequencing. After being introduced into the testicular tissue of cryptorchid mice, the expression of CIRP mRNA and protein were significantly higher than those of the control group at 7 days (all <0.05). There was no significant difference in the level of CIRP at 10 days after electroporation (P> 0.05). Conclusion The eukaryotic expression vector pVAX1-CIRP was successfully constructed and successfully transfected into testes of cryptorchid mice to normal transcription and expression, which laid the foundation for the further study on the role of CIRP gene in spermatogenesis and the pathogenesis of male infertility.