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.

Objective： To construct the recombinant adenovirus vectors of calretinin （CALB2） gene and small interfering RNA （siRNA）, for over-expression or knock-down of CALB2, as the basis of functional investigation of CALB2 in testicular Leydig cells. Methods： The cDNA sequence of CALB2 was cloned by the reverse transcriptive polymerase chain reaction （RT-PCR）. A CALB2 gene fragment was sub-cloned into adenovirus shuttle plasmid pAdTrack-CMV to construct the shuttle plasmid pAdTrack-CALB2. Then it was transformed into BJ5183 cells with the adenoviral backbone pAdEasy-1 to obtain the homologous recombinant AdCMV-CALB2. The recombinant AdCMV-CALB2 was further packaged and amplificated in AD293 cells. The expression of CALB2 protein in AD293 cells was detected by Western blotting. CALB2 protein was over-expressed in mouse Leydig cell line （MLTC-1 cells） by the constructed AdCMV- CALB2. CALB2 gene siRNA recombinant adenovirus vector （Ad-HI-siRNA/CALB2 was also constructed simultaneously. Its efficacy was detected in AD293 cells by Western blotting. Results： The CALB2 gene recombinant adenovirus vector AdCMV-CALB2 and the CALB2 gene siRNA recombinant adenovirus vector Ad-H1-siRNA/CALB2 were constructed successfully by endonulease digestion and sequencing. AD293 cells infected with AdCMV-CALB2 or Ad-H1-SiRNA/CALB2 significantly expressed GFP protein. The expression of CALBz protein was significantly up-regulated in AD293 cells infected with AdCMV-CALB2 plasmids, while the expression of CALB2 protein was down-regulated by 60% in the CALB2 cells infected with Ad-H1- SiRNA/CALB2. MLTC-1 cells did not markedly express CALB2 protein, while MLTC-1 cells infected with AdCMV-CALB2 expressed CALB2 protein at a high level. Conclusions： The recombinant adenovirus vectors of AdCMV-CALB2 and Ad-H1-SiRNA/CALB2 were successfully constructed. Both vectors effectively expressed in AD293. CALB2 protein was over-expressed in the cultured MLTC-1 cells by AdCMV-CALB2. These vectors of CALB2 gene and Leydig cell line are useful tools for investiga- ting the testicular function.