| IntroductionAfter the completion of human Genome Project, scientific community has entered the post - genomic era. The study of these 40000 or so human genes, their functions and their association with diseases is perhaps the more difficult and urgent steps in life scientific community. Human a - 1B glycoprotein precursor gene (a - 1BGP) was i-dentified in 2001 by our laboratory, which is 1694bp in full length, encoding a protein of 495 amino acids. Its function is still not clear. Based on the homology comparison, gene expression pattern and the domain analysis, we presumed that a - 1B glycoprotein might play a role in the proliferation process of the cell. On the base of previous research , we used human recombinant growth hormone, estrogen, LPS to treat hepatoma cells ( BEL - 7402) to explore the regulation of a -1BGP expression. We also investigate the possible proliferation function of a - 1BGP by means of pcDNA3.1 - a - 1BGP transfection into cells; finally we construct pPICZaA - a - 1 BGP vector, then transform it into GS115 in order to acquire the secretory recombinant a -1B glycoprotein.Materials1. Reagents for cell culture and calcium - phosphate coprecipita-tion2. Reagents for Northern Blot and Western Blot3. Marathon - ReadyTM cDNA Kit and EasySelectTM Pichia Expression Kit4. Molecular Biology Reagents for pcDNA3. 1 - a - 1BGPvector and PIczaA - a - 1BGP vector constructed5. Reagents for Flow CytometryMethodsHuman hepatoma cells ( BEL - 7402 ) were treated with recombi-nant growth hormone (6nmol/l) , estrogen ( 10nmol/l) , LPS ( 10mg/ ml) ; then we explored the regulation of a - 1 BGP expression using Northern Blot; we also analyzed the 5 ' region of a - 1BGP by TRANSFAC 4. 0. pc DNA3. 1 - a - 1BGP vector was constructed and transfected into hepatoma cells ( BEL - 7402 ) using calcium - phosphate coprecipitation , then cell DNA content was analyzed by Flow Cytometry. We also constructed pPIczaA - a - 1BGP vector , and transformed it into GS115,then confirmed the Mut phenotype, examined the presence of insert using PCR, conducted small - scale expression , analyzed the expression by SDS - PAGE and Western Blot.Resulta -1BGP expression is up - regulated in hepatoma cells treated with GH by Northern blot. GH increased a -1 BGP expression after 3 hr, reaching maximal levels at 9hr. Using TRANSFAC 4.0 and TSSG TSSH NSITE of softbeny, we can see that a TATA box lie in - 188bp - 198bp from transcriptional initiation site, there are several API, C/ EBP binding motifs near it or in other 5' flanking region, a c - Fos response element is in - 1133bp - 1143bp from transcriptional initiationsite. The Estrogen - stimulated increase in a - IBGP mRNA transcripts were observed by Northern blot analysis. The increase was detectable within 3 hour and peaked within 9 hour, after 15 hour there was no increase. LPS can also stimulated increase in the levels of the a - 1BGP transcripts in hepatoma cells. pcDNAS. 1 - a -1BGP vector was successfully constructed. Northern analysis result indicated that a - 1BGP could be stably expressed in transfected cell. The transfected cells showed increase in S phase and PI index and displayed decrease in G0/G1 phase by Flow Cytometry analysis. We also constructed pPIczaA - a - 1BGP vector successfully. Pme I - digested pPIczaA -a - 1BGP and pPIczaA was used to transform GS115. The expression cassette was correctly integrated into GS115 chromosome . We used SDS - PAGE and Western blot to detect a -1B glyco-protein moleculers in culture medium. One bind was detected at mole-culer mass between 66 - 43 KDa, which show a maximal level at 7 day.DiscussionThe function of human a - 1B glycoprotein precursor gene is still not known. In our study, we found that a - 1BGP expression is up -regulated in hepatoma cells treated with GH. It suggests that GH is capable of stimulating the expression of a -1BGPin hepatoma cells. So a - 1 BGP may play a role in postnatal growth and intermediary metabolism which are major physiological actions of GH. GH bingi...
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