Construction of Cox7a2 fluorescent vector and its effect on cytochrome C oxidase activity in mouse Sertoli cell line TM4
10.3760/cma.j.issn.1000-6702.2011.07.016
- VernacularTitle:Cox7a2荧光载体构建及其对小鼠支持细胞细胞色素C氧化酶活性的影响
- Author:
Baoxing LIU
;
Shengjie PENG
;
Gang LIU
;
Shengqiang ZHANG
;
Liang CHEN
;
Chuanhang WANG
- Publication Type:Journal Article
- Keywords:
Electron transport complex Ⅳ;
Cox7a2;
pEYFP-C1 vector;
Sertoli cell line TM4;
COX activity
- From:
Chinese Journal of Urology
2011;32(7):490-493
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct Cox7a2 fluorescent vector and study its effect on cytochrome C oxidase (COX) activity in mouse Sertoli cell line TM4. Methods The coding region of Cox7a2 was amplified from mouse Sertoli cell line TM4 by RT-PCR. The PCR product was inserted into pEYFP-C1 vector with BamH I and EcoR I restriction site, and confirmed by DNA sequencing. The recombinant fusion protein vector was amplified by transforming into DH5a and transfected into TM4 cells. The protein expression was identified by Western blot. COX activity was measured by spectrophotometer 6, 12, 24 and 48 h after the transfection of recombinant vector into the TM4 cell line. Results The entire coding sequence of Cox7a2 was cloned with 252 bp length. Plasmid pEYFP-C1-Cox7a2 vector was constructed and the positive clones were verified by restriction enzymes digestion and DNA sequencing. The transfection efficiency of the TM4 cell line was about 70% and 37000 D fusion protein was obtained. The COX activities were (0.642±0.051), (0.542±0.049), (0.311±0.021) and (0.216±0.010) U/mg 6, 12, 24 and 48 h after the transfection of recombinant vector in the TM4 cell line. Meanwhile, the COX activities were (0.714±0.064) and (0.653±0.031) U/mg in non-tranfected and naked vector group respectively. Compared with the non-tranfected group, COX activity decreased significantly 12, 24 and 48 h after the transfection. Conclusions The recombint plasmid vector was successfully constructed. Cox7a2 gene has an inhibiting effect on COX activity and may play an important role in the regulation of COX activity in mouse Sertoli cell line TM4.
