Screening of nucleic acids affinity to recombinant human TGF-? RⅡ
- VernacularTitle:人重组TGF-? RⅡ亲和核酸筛选方法的建立
- Author:
Lin XIE
;
Ren LIU
;
Xudong ZHU
;
Xiangge HE
;
Caiyu CHEN
- Publication Type:Journal Article
- Keywords:
systematic evolution of ligants by exponential enrichment;
recombined human TGF-? RⅡ;
candidate sequence
- From:Journal of Third Military Medical University
2003;0(23):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective To select the aptamer to an extracellular soluble fragment of recombinant human TGF-? receptor Ⅱ (TGF-? RⅡ) in order to antagonize TGF-? effectively by using systematic evolution of ligants by exponential enrichment (SELEX). Methods Initial random RNA library was transcripted in vitro from ssDNA 5′-TAATACGACTCACTATAGGGAGGACGATGCGG-N60-CAGACGACTCCCCGA-3′; rhTGF-? RⅡ was used as target protein. Totally,selection of 8 times was carried out in SELEX experiment. Membrane binding assay was performed to detect the evolution of enriched RNA library; Electrophoretic mobility shift assay (EMSA) was done to determine the affinity between the selected nucleic acid sequence and TGF-? RⅡ. Results Evolution of the enriched RNA library along the increased affinity to TGF-? RⅡ was observed with the development of selection. Two types of dominant sequences were isolated and named as sequence A and sequence B. In membrane binding assay,both sequences A and B showed obvious affinity to TGF-? RⅡ. However,no retarded bands were seen in EMSA. Conclusion The affinity of sequences A and B to TGF-? RⅡ is beyond satisfaction. However,possible sequences with improved affinity to TGF-? RⅡ can be selected by post-SELEX on the basis of candidate sequences A and B.
