Construction and expression of the recombinant plasmids of truncated and normal selenoprotein S genes
10.7652/jdyxb201802024
- VernacularTitle:硒蛋白S截短和全长重组质粒的构建与表达
- Author:
Fu-Meng HUANG
1
;
Li ZHAO
;
Fang-Xia MA
;
Zhao CHEN
;
Li WANG
;
Li-Fang TIAN
Author Information
1. 西安交通大学第二附属医院肾病内科
- Keywords:
selenoprotein S;
truncated protein;
gene recombination;
plasmid construction
- From:
Journal of Xi'an Jiaotong University(Medical Sciences)
2018;39(2):276-280
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct the recombinant plasmids of normal and truncated selenoprotein S genes so as to observe their biological function in vitro or in vivo.Methods We constructed the recombinant plasmids of normal and truncated selenoprotein genes by gene recombinant technology.The gene of truncated selenoprotein was coding domain sequence(CDS)fragment of mRNA;the gene of normal selenoprotein was CDS and 3'untranslated region(including Sec insertion sequence)fragment of mRNA.We confirmed the sequence of recombinant genes by sending them to a company for comparison.The recombinant plasmids of normal and truncated genes of SelS were transfected into cells by Lipofectamine 2000.After 24 hours,the expression of green fluorescent protein was observed and transfection efficiency was detected by FACS analysis.We collected the cells to isolate the total RNA by TRIzol method,and then cDNA was obtained by mRNA reverse transcription and amplified by PCR.Results The sequencing results showed that the recombinant genes were completely the same as the target genes,indicating that we constructed the plasmids successfully.The expression of green fluorescent protein could be observed and transfection efficiency was detected up to 40% by FACS analysis.PCR results showed that the target selenoprotein gene was highly expressed in the experimental group than in control group.Conclusion The truncated and normal selenoprotein S genes were successfully constructed and transfected into cells where they were highly expressed.It lays foundation for observing the biological effect of truncated and normal selenoprotein in cell line or animal body.
