Segmented cloning and expression of the pigment epithelium derived factor and its effect on the proliferation of human SCL-1 cells
10.16571/j.cnki.1008-8199.2019.10.002
- VernacularTitle: 色素上皮衍生因子的分段克隆表达对人皮肤鳞状细胞癌细胞增殖的影响
- Author:
Zhi-bin ZHANG
1
;
Sheng-nan PENG
2
;
Ou-gen LIU
1
;
Ying-peng ZHANG
1
;
Chun-ming LI
1
;
Ya-ting PENG
1
;
Shu-lan ZHANG
1
Author Information
1. Department of Dermatology, the Second Affiliated Hospital of Nanchang University, Nanchang 330006, Jiangxi, China
2. Science and Technology College of Jiangxi Traditional Chinese Medicine University, Nanchang 330004, Jiangxi, China
- Publication Type:Journal Article
- Keywords:
pigment epithelium derived factor;
human cutaneous squamous cell carcinoma;
segmental cloning;
cell proliferation
- From:
Journal of Medical Postgraduates
2019;32(10):1014-1018
- CountryChina
- Language:Chinese
-
Abstract:
Objective Studies are rarely reported on the effect of short peptides of the pigment epithelium derived factor (PEDF) on the proliferation of human cutaneous squamous (SCL-1) cells. The purpose of this study is to investigate segmented cloning and expression of the PEDF protein and observe its effect on the proliferation of human SCL-1 cells. Methods The target genes of PEDF1, PEDF2 and PEDF3 were amplified by PCR and the recovered fragments subjected to double digestion of NheⅠ and Hind Ⅲ and inserted into the pET28a(+) plasmid. The product was transformed into human E coli BL21 and induced to express, followed by isolation and purification of the fusion protein. CCK-8 assay was used to detect the proliferation of the SCL-1 cells with PEDF1, PEDF2 and PEDF3 at 100, 400, 800 and 1000 nmol/L at 24, 48 and 72 hours. Results The prokaryotic expression vectors of PEDF1, PEDF2 and PEDF3 were successfully constructed, and their fusion proteins prepared, with the molecular weight of 18 000, 17 000 and 13 000, respectively. The proliferation of the SCL-1 cells was significantly decreased in the 800 and 1000 nmol/L PEDF3 groups compared with that in the 0 nmol/L PEDF3 group at 24 hours (0.16 ± 0.03 and 0.78 ± 0.07 vs 1.00 ± 0.00, P < 0.05), inhibited in a concentration-dependent manner in the 400, 800 and 1000 nmol/L PEDF3 groups at 48 hours (P < 0.05), markedly lower in the 800 and 1000 nmol/L PEDF3 groups at 72 hours (0.53 ± 0.05 and 0.51 ± 0.05) than in the in the 400 and 0 nmol/L PEDF3 groups (0.60 ± 0.05 and 1.00 ± 0.00) (P < 0.05). Conclusion The PEDF fusion proteins were successfully segmentally cloned and expressed and PEDF3 inhibited the proliferation of SCL-1 cells, which has paved the ground for further screening of active functional short peptides of PEDF.
