Studies On The Cloning Expression And Function Of Insulin-like Growth Factor Binding Protein-1(IGFBP-1)

Insulin-like growth factor binding protein-i (IGFBP-i) is one of the important endocrine hormones, however its bioactivity and mechanism remain unclear A series of studies on IGFBP-l were performed in the experiment with the technique of gene recombinant, yeast two-hybrid, cell cultrue, CAM and chiken embroyo dorsal root ganglion culture, which included the following three parts1 The human IGFBP-l eDNA was isolated by PCR from fetal hepatic eDNA library and confirmed by sequence analysis The recombinant plasmid was constructed to express human IGFBP-1 as a GST fusion protein The fusion protein was successfully expressed in E coli BL21, purified with affinity chromatography and cleaved by thrombin to produce mature humanIGFBP- 12 Yeast two hybrid system was firstly used on studying the biological activities and mechanism of IGFBP-1 The results showed that IGFBP-1 has transcriptional activation The relationship of the structure and function of IGFBP-1 was analysised by bioinformatics A mutant was designed by deleting 27 amino acid of the carboxyl end of ]IGFBP-l The mutant cDNA was obtained by PCR, characterized by sequencing, and inserted into BD plasmid. The transcriptional activation was deleted by constructing the mutantSeven genes, which encode proteins interacting with the mutant of the IGFBP-1, were found by screening the library using the Yeast two-hybrid system By homology comparing, these genes identified as human metallothionein-Ie, hemoglobin, autonomously replicating sequence, hepatocellular carcinoma-associated antigen 112, fibrinogen,5haptog1obln and ser1ne (or cyste1ne) prote1nase 1nh1b1 tor3 1n order to study the blo1og1ca1 act1v1ty of the human IGFBP--1 andlts effect on ce11 pro11ferat1on 1nduced by IGF--1, the MTT method wasused to determ1ne the dynam1c changes of prol1 feratlon of endo11 the1la1ce11 ECV304 The ce11 growth was 1nhlb1ted by IGFBP--1, however st1mu1atedby IGF--1 The m1xture of IGFBP--l and IGF--1 1eaded to slgn1f1cantlnhlbltlon of ce11 pro11ferat1on 1nduced by IGF--1To lnvest 1gate the effect of human IGFBP--1 and 1GF--l on the nervous t 1 ssue,chlcken embroyo dorsa1 root gang11on cu1ture was app11ed 1n the studyIGFBP--1 and 1GF--1 dld not affect the growth of dorsa1 root gang11ons l gn 1 fl cant l y.To lnvest1gate the effect of IGFBP--1 and IGF--I on the vascu1ar1zatlon,the CAM techn1que was flrst1y used on study of the act1v1ty on IGFBP--1and IGF--1 The resu1ts lnd1cated that IGF--l cou1d promote the CAMnewang1ogenes1s, the IGFBP--1 d1dn' t affect the ang1ogenes1s, but 1tcou1d lnh1blte newang1ogenes1s 1nduced by IGF--1 1n the CAMThe conc1us 1 on:Some new techn1ques and methods were app11ed to study IGFBP--1 ThecDNA of 1GFBP--1 was c1oned, successfu11y expressed 1n E co11 The prote1nlnteract 1ng w1 th IGFBP--1 may be new cues for 1 nvest 1gat 1ng new b1olog1 calact1v1ty and mechan1sm of 1GFBP--1 IGFBP--1 cou1d 1nh1b1te the growth ofendothe11a1 ce1l and be as 1nh1b1tor of IGF--l on ce1l pro11ferat1on andang 1 ogenes l s...