Expression, Purification And Antibody Preparation Of Recombinant Human SM22α

In the development of atherosclerotic plaques and restenosis lesions, vascular smooth muscle cells (VSMCs) undergo a phenotypic change from the contractile (differentiated) to the synthetic (dedifferentiated) phenotype and get the ability to reproliferate. SM22αis the marker of differentiated VSMCs, and is commonly used to identify the phenotype of VSMCs. However, SM22αprotein could not be directly detected because of absence of SM22αantibody. Thus, it is necessary to prepare anti-SM22α antibody. Considering the facts that content of SM22α is very low in vascular cells and difficult to be purified, in the present study we expressed human SM22α (hSM22α) in Pichia pastoris, and prepared the anti-hSM22αpolyclonal antibody by immunizing rabbit with the purified hSM22α. Then we detected the expression of SM22αin phenotypic change of VSMCs with the prepared anti-hSM22α antibody by Western blot analysis. The present study establishes the groundwork for future studies to explore the relationship between expression of SM22αand phenotypic change of VSMCs.Methods1. Construction of yeast expression vector for hSM22α: Using pGEM3z-hSM22αwith full-length cDNA of hSM22αas the template, hSM22αcDNA was amplified by PCR. After the PCR products proved correct by analysis of restriction enzymes and DNA sequencing, they were directly inserted into the expression vector pPIC9, then the recombinant plasmid pPIC9-hSM22αwas further identified by restriction enzyme and DNA sequencing.2. Transformation of yeast and selection:The recombinant plasmid pPIC9-hSM22αwas linearized by restriction enzyme Sac I and transformed into yeast competent cell GS115 prepared by chlorination lithium. The positive clone of GS115 integrated by hSM22αwas selected by MD and MM medium plates without histidine.3. Expression of hSM22α: Methanol was added into the cultures to induce the expression of hSM22α. At different time points, the culture was sampled and was used to analyze expression levels and determine the optimal time when hSM22α expression was induced at maximal level.4. Purification of hSM22α:The culture was collected at 84 h after induction and precipitated by 35%～70% ammonium sulfate. The precipitate was dialysed against equilibrium buffer, then CM-Cellulose chromatographic purification was performed for hSM22α. The purity of hSM22αwas detected by SDS-PAGE.5. Preparation and identification of anti-hSM22αantibody:Polyclonal antibody against hSM22αwas produced by immunizing a rabbit with purified hSM22α. The titer was analyzed by bi-direction immuno-diffusion experiment.6. Expression of SM22α in vascular neointima: The expression of SM22αin the neointima after de-endothelialization was detected by Western blot analysis.Results1. Construction of yeast expression vector for hSM22α: The analysis of DNA sequence showed that hSM22αcDNA amplified by PCR was consistent with ORF of hSM22α. The recombinant plasmid pPIC9-hSM22αproved correct by restriction enzyme analysis and DNA sequencing.2. Transformation of yeast and selection: After selected by MM and MD medium plates, the two clones with His+Mut+ phenotype were identified, and an expected fragment was obtained in these clones when the genome DNA was amplified by PCR.3. Expression of hSM22 in Pichia pastosis: The results of SDS-PAGE showed that a 22 kD protein band corresponding to hSM22α appeared at 24 h induced by methanol. As times were prolonged, the expression level of hSM22α was progressively increased and reached a peak at 84 h. The negative control had no protein band at the site corresponding to 22 kD.4. Purification of hSM22α: The hSM22αprepared by ammonium sulfate fractionation and chromatographic separation showed a single band whose apparent molecular weight was 22 kD on SDS-PAGE. 5. Preparation and identification of anti-hSM22αantibody: Bi-direction immuno-diffusion experiment demonstrated that the titer of anti-hSM22αantibody was 1:4. The antibody could recognize the different forms of SM22α.6. Exp...