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Cloning and Expression of Human Thrombospondin-1 cDNA Fragments

Author: WangXiaoQiang
Tutor: TongYing
School: Sichuan University
Course: Cell Biology
Keywords: Thrombospondin 1 cDNA clones CDNA expression E. coli Pichia pastoris
CLC: R346
Type: Master's thesis
Year: 2005
Downloads: 32
Quote: 0
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Abstract


Thrombospondin 1 ( Thrombospondin - 1 , TSP -1 ) is a homotrimeric glycoprotein and platelet a major component of the particles . Or adults were injured in the process of fetal development , TSP-1 expression in the epithelial cells of mesodermal cells . TSP-1 and coagulation functions and cell adhesion , in the different cells and under different conditions , can also stimulate or inhibit cell growth and migration , and inhibition of angiogenesis . TSP-1 can be applied to the tumor anti - angiogenesis therapy . According to existing research results , TSP-1 to inhibit angiogenesis mainly rely Ⅰ procollagen homologous fragments and three properdin hormone -like (properdin) type Ⅰ repeat sequence region . First, in this research , two pairs of primers were used for RT-PCR amplification of TSP- 1 - 1 ( type I procollagen homologous fragments and three I repeat sequence region ) and TSP- 1 -2 ( Ⅰ type repeat region ) fragment , verified by sequencing , and then were cloned into E. coli and Pichia pastoris expression , and the expression product was purified . The main results are as follows : 1 using two pairs of primers , TSP-1-1 and TSP - 1 -2 cDNA fragment was cloned from human blood cells , sequence analysis showed , are 699 and 528bp . 2 the two cDNA fragment inserted into the E. coli expression vector pET-29a , to obtain the recombinant form of inclusion body after IPTG induction to express the corresponding polypeptide fragment having a molecular weight of 30kD and 20kD respectively . With urea in vitro dissolution of inclusion bodies through ion exchange (CM-Sepharose) chromatography , and TSP-1-2 polypeptide . 3 then the two cDNA fragment inserted into the Pichia pastoris secretion expression vector pICZ α A to obtain recombinants containing the TSP-1-2 of the yeast after methanol induction secretory expression the corresponding polypeptide fragment having a molecular weight for 20kD.

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