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Published on: February 13, 2013
[Cloning and sequence analysis of active fragments of human thrombospondin-1]
1Department of Anatomy, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China.
Objective:
To lay a foundation for researches on the expression of gene engineering anti-tumor drugs.
Methods:
On the basis of mRNA sequence of human thrombospondin-1(TSP-1), two pairs of primers were designed and synthesized. Two active fragments TSP-1, called TSP-1-I and TSP-1-III, were obtained by using reverse transcription-polymerase chain reaction(RT-PCR). The sizes of those two fragments were found to be 728 bp and 257 bp respectively. They were purified and then cloned into pMDT vector after transformation into Escherichia coli with recombined DNAs.
Results:
Plasmid DNAs were isolated from transformants and digested with restriction enzyme. The results from agarose gel electrophorasis showed that these two fragments had been inserted into vector and their sizes were in accord with the expected values. Sequence analysis demonstrated that TSP-1-I is 728 bp in size and encodes a peptide containing 212 amino acid residues with relative molecular mass of 23.6 x 10(3) and the TSP-I III is 257 bp in size and encodes a peptide containing 86 amino acid residues with relative molecular mass of 9.5 x 10(3).
Conclusion:
The results from homology analysis indicate that the sequence of these two fragments is identified with that of TSP-1 of Genbank.
