Selecting, Expression And Functional Studies With Peptide Of Interacting To Human Augmenter Of Liver Regeneration

Objective: The human augmenter of liver regeneration (hALR) is known as a vital control factor in regulating the regeneration of hepatocytes. It plays the important role in the repair process of liver damage. The molecular biology mechanisms of hALR in the liver regenerative process are not clear completely. The objective of this study is to biopan the hALR interacting proteins from cDNA library of QGY hepatoma cell, to confirm and to clone hALR interacting protein,and to perform the preliminary studies of its biology function. This reasearch will be beneficial for elucidating the hALR signal conduction mechanism and offering theoretical grounds for the molecular biology mechanism of liver degeneration and regeneration.Methods:1.Biopanning the hALR interacting peptideIn experiment study, the specific phage clones interacting with target protein hALR were selected from cDNA library of hepatocarcinoma cell. The specific enrichment of the recombined phage binding to hALR was identified by ELISA and PCR. The acquired cDNA segments of specific phage clones were sequenced and analyzed by bioinformatic tools. The biological function of peptide combined with hALR which prompts QGY hepatocarcinoma cell proliferation was identified by ³H-TdR method.2. studying the correlation between hALR and citron kinase with RNA interference methodThe expression of citron kinase in QGY cells and the relationship between citron kinase and hALR were observed with immunohistochemistry using laser confocal microscopy on protein level. The expression of citron kinase in QGY cells was detected with RT-PCR on nuclei acid level. To design and to synthesize three siRNA fragments which aimed at different target position of citron kinase,then to transfect them into QGY cells by liposome transfection for 48 hours,the level of citron kinase was investigated by real-time RT-PCR on nuclei acid level,by western blot on protein level and by immunohistochemistry using laser confocal microscopy on morphology aspect. The proliferation of QGY cells treated with hALR was detected by ³H-TdR incorporation approach after transfection for 48 hours.3.Construction, expression and functional research of GST-hALRIP fusion proteinThe DNA fragment of hALRIP was amplified by PCR from the specific phage clones interacting with hALR and was subcloned into plasmids pGEX-4T-2. The recombinant plasmids pGEX-4T-2-hALRIP were transformed into host bacteria BL21(DE3)and were expressed under the induction of IPTG. The GST-hALRIP fusion protein was purified by Glutathione Sepharose 4B. The interaction between GST-hALRIP fusion protein and hALR in vitro was tested by pull-down method. The biological function of GST-hALRIP fusion protein combined with hALR effecting on QGY cells was identified by 3H-TdR method.Results:1.Biopanning the hALR interacting peptide(1)After the forth round of biopanning from cDNA library of hepatocarcinoma cell,the enriching effect of binding phage was rised from 2.9×10^-3 to 9.0×10^-1,which indicated that the special clones binding to hALR were amplified.(2)The connectivity of hALR and positive phage clones with different degr4ee of dilution was detected through the ELISA method. The result demonstrated that the binding of hALR-containing phage to hALR was specific,with a dose-dependent affinity for hALR.(3)The cDNA segments of these phages were amplified by PCR. It was indicated that all phages after forth biopanning shared the same size of cDNA segments(about 300bp). DNA sequencing revealed that different clones with 300bp cDNA segments actually shared the same cDNA sequence. (4)The homology analysis by BLAST software in Genebank showed that the sequence of hALR interacting peptide and partial mRNA sequence of citron kinase showed 100% homology. Besides,according to the software of prediction and analysis of protein transmembrane helical regions,the hALRIP sequence was located between the opening reading frame (ORF) 1846²057th basic group of citron kinase,namely 598⁶68 amino acid positions,not in the transmembrane region of citron kinase.(5)Compared with solo hALR group,the groups of phage-displayed peptide combining with hALR showed significant proliferation effects on QGY cells(F=29.28,p<0.01). Meanwhile,the control groups showed no significant proliferation effects on QGY cells(F=0.638,p>0.05). The above indicated that the phage-displayed peptide and hALR had the cooperated effects.2. studying the correlation between hALR and citron kinase by RNA interference(1)Citron kinase was expressed in QGY cells by RT-PCR. It was localized to both nucleus and cytosl during prophase but was accumulated in the cleavage furrow and midbody in the post-mitotic stage. There was relevance between hALR and citron kinase because of the expression of citron kinase increased obviously after QGY cells stimulated by hALR .(2)According to the target citron kinase mRNA,three specific short hairpin small interfering RNA were successfully constructed,in which target sequence 1 and 2 aimed at the hALRIP sequence. The real-time RT-PCR result showed that three siRNA fragments were able to suppress the expression of citron kinase gene effectively after transfection for 48 hours and the levels of citron kinase mRNA expression decreased to only 37.89%,22.69% and 26.61% compared to a non-silencing siRNA treatment. The reduction effects of citron kinase,after silencing treatment,in the protein level was confirmed using immunocytochemistry and western blot analysis. By both techniques , citron kinase protein expression was specifically more decreased in cells that were treated with citron kinase siRNA than those treated with non-silencing siRNA 48h post transfection. These results indicated that siRNA inhibited citron kinase expression at both the mRNA and protein levels.(3)Proliferation of QGY cells was decreased significantly by hALR(4639±382,3060±201 and 3576±425)after transfected with citron kinase siRNA for 48 hours,compared with negative control group(7007±156)and blank group(7566±1009),p<0.05. But there was non-significance difference in the three groups though siRNA2 inhibitory action was most obvious. The result indicated that proliferation of QGY cells stimulated by hALR could be through citron kinase which could be the downstream factor of hALR,the multiplication function of hALR could be blocked so long as blocking the expression of citron kinase .3.Construction, expression and functional research of GST-hALRIP fusion protein(1)Restriction enzyme digestion,PCR and DNA sequencing were performed to confirm the successful construction of recombinant plasmid of pGEX-4T-2-hALRIP .(2)GST-hALRIP fusion protein was successfully expressed in host bacteria BL21 ( DE3 ) and its molecular weight of 33kd was in correspondence with theoretic values. The expressed target proteins existed not only in supernatant but also in precipitation of broken bacteria mostly. Single band was watched in SDS-polyacrylamide gel electrophoresis after purification of GST-hALRIP with Glutathione Sepharose 4B. The quantitation of purified protein was 103.2μg/ml.(3)GST-hALRIP fusion protein could interact with hALR identified by GST pull-down assay,but GST protein could not do so,which indicated that hALRIP and hALR had interactions in vitro.(4)GST-hALRIP and GST fusion protein in high doses could inhibit proliferation of QGY cells stimulated by hALR obviously and there were no difference between the two proteins. The result had not been able to reflect the binding specificity between GST-hALRIP fusion protein and hALR.Conclusions:1. The hALR interacting peptide (hALRIP )was successfully biopanned from cDNA library of hepatocarcinoma cell. 2. The hALRIP fragment is one segment of citron kinase.3. Citron kinase was expressed in QGY cells and hALR could increase the expression of citron kinase.4. Successfully to constructed the short hairpin small interfering RNA which could suppress the expression of citron kinase gene effectively.5. It was through citron kinase that hALR stimulated proliferation of QGY cells.6. The expression of GST-hALRIP fusion protein with high efficiency and successful purification has laid the foundation for further research.7. hALRIP and hALR have do the interactions in vitro.