| OBJECTIVE:To obtain and purify hepatocellular carcinoma-associated antigen SMP30fusion protein with a6×HIS tag; to explore the role of molecular chaperones co-expression with two kinds of expression vectors in promoting protein’s solubility; to study SMP30antibody’s expression level within HCC patient’s serum.METHOD:PCR amplification SMP30cDNA sequence, SMP30gene was restructured to two kinds of expression plasmids pET30a and pCold III by genetic engineering technology, the two recombinant plasmids were transformed into E.c oli. BL21(DE3) pLysS host bacterium respectively. Optimization express conditions (such as induction temperature, induction time, inducer concentration and the best time to add inducers, etc.) were explored, engineering bacterium was cultured in expenditure LB-solution, bacteria was broken by ultrasound and centrifuged, His-SMP30fusion protein was purified with Ni-NTA affinity column; Five molecular chaperone plasmids (pG-KJE8, pGro7, pKJE7, pG-Tf2, pTf16) were transformed into E.c oli. BL21(DE3) host bacterium, then the two recombined plasmids (pET30a-SMP30and pCold III-SMP30) were transformed into BL21containing molecular chaperone plasmids respectively, then recombinant plasmids co-expressed with molecular chaperone plasmids, SDS-PAGE analysed,The interest protein’s expression level and its solubility were assayed between the two expression vectors; selected the best co-expression system in promoting SMP30protein to optimize (as IPTG concentration), the condition of SMP30solubility expression was detected with SDS-PAGE;175cases of HCC serum was studied with SMP30protein as antigen.RESULT:The connection product was transformed into E.coli. DH5a, selected in LB-culture medium containing Kan(pET30a) and Amp(pCold III), blue spot screening showed all the cloning were white spots, Bacterial broth PCR could amplified our purpose gene, and the extracted plasmid was digested with Nde I and Sal I, SMP30band (500bp) and a larger plasmid band were found, finally sequencing and blasted with sequence reported on NCBI, the similarity was100%; the optimal expression condition was that, cultured in37℃for2.5h before added inducer,the concentration of IPTG was0.6mM, then cultured in37℃for7h; Both the two recombined plasmids’expression level of interest protein (HIS-SMP30) was as high as60%(Quantity one4.6statisticed) when the culture conditions were optimal; inclusion body protein was up to95%after purification; Co-expression with pET30a-SMP30and four molecular chaperones, our interest protein was mainly found in insoluble fragment, there was little or none in soluble fragment; more over, the SMP30expressed level was lower in co-expressed systems than in common system, and pKJE7was the lowest; but they could improve obviously when co-expressed with pCold Ⅲ-SMP30, especially with pKJE7、pG-Tf2and pTfl6; WB suggested that the unknown protein fragment could combine with anti-His mouse monoclonal antibody and RGN antibody;22of175cases HCC serum was positive, x2two-sided test all showed P>0.05, meanless of statistic, it’s not sure there are relationship with the positive cases and clinical diagnostic datas.CONCLUSION:The high expression level of SMP30recombined plasmid was successfully constructed; Four kinds of molecular chaperone plasmids coexpressed with pET30a-SMP30cannot effectively promote the soluble protein expression, meanwhile, molecular chaperone couldn’t improve the purpose protein expressed, instead they could restrain its expression, pKJE7played the most inhibitory effect; pKJE7、pG-Tf2、pTfl6molecular chaperone plasmids could promote solubility protein expressed efficiently, the best IPTG concentration were0.1mM、0.6mM (or1.0mM) and0.6mM respectively, and obtained the soluble fusion protein; the unknown protein was HIS-SMP30degradation fragment; the positive of SMP30antibody was12.6%in HCC patients’serum, it’s not sure there are relationship with the positive cases and clinical diagnostic datas. |
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