| In the marine environment,arthropod barnacles have the unique ability to permanently attach to a variety of underwater matrices by producing,secreting and curing viscous polyprotein complexes(barnacle cement).At present,barnacle cement has an important application value in the field of biomedicine as a new type of high-strength bioadhesive.Although we have some understanding of the compositional characteristics,structure and physical and chemical properties of barnacle cement,the characteristics of synthesis and secretion,and the mechanism of adhesion and curing.However,due to its poor solubility and complex secretion process,the research on the structure and function of barnacle protein needs to be further studied.Cp19k protein is one of the main components in barnacle cement,and it mainly plays the role of interfacial adhesion during barnacle adhesion.In this study,the soluble expression of Cp19k protein in E.coli was achieved by genetic engineering technology.The recombinant Cp19k protein was purified and the adhesion ability was preliminary explored.The specific research content is as follows:Part Ⅰ: Expression and purification of Cp19k protein and analysis of influencing factors.Gene Bank database was used to search balcp19k,mrcp19k and bicp19k gene sequences,and three gene sequences were optimized according to E.coli codon preference and GC content,and the optimized sequences were sent to the company for synthesis.At the same time,the recombinant expression plasmids pET21a-balcp19k,pET21a-mrcp19k and pET21a-bicp19k were constructed.The recombinant plasmid was transferred into E.coli BL21(DE3)and induced expression under different conditions.The results showed that the mrcp19k and bicp19k genes had no target protein expression,and the Balcp19k protein was successfully expressed.We analyzed the factors that did not express Mrcp19k protein and Bicp19k protein.After excluding the factors affecting the codon bias and expression conditions,analysis of the secondary structure of the Mrcp19k protein and the mRNAs of the three cp19k genes revealed that the 5′-end secondary structure of the mrcp19k and bicp19k genes was too stable,which was the main factor that caused the protein not to be expressed.By optimizing the expression conditions of Balcp19k protein,it was finally determined that when the induction temperature was 27℃,the concentration of the inducer IPTG was 1.0mM,and the induction time was 4h,the expression of soluble Balcpk protein was the highest.The recombinant protein was purified by affinity chromatography and molecular sieve chromatography to obtain Balcp19k protein with a purity of about 92.5%.Part Ⅱ: Balcp19k protein identification and viscosity detection.The purified Balcp19k protein was identified by mass spectrometry.The identification results showed that the expressed and purified protein was Balcp19k protein.Circular dichroism analysis results showed that the secondary structure of Balcp19k protein was mainly β-sheet structure,which was consistent with the literature reports.CBB stained surface coating analysis experiments show that Balcp19k protein has an interfacial adhesion effect on both hydrophobic and hydrophilic materials.The adhesion ability of Balcp19k protein plus Cell-TakTM is greater than that of Cell-TakTM.The specific adhesion mechanism further research is needed.In summary,this study successfully established the Cp19k protein prokaryotic expression system,successfully prepared soluble recombinant Balcp19k protein,and conducted a preliminary exploration of its interface adhesion ability.The research results provide a reference for the research of other barnacle cement proteins,and also provide a reference for the research of biomedical adhesive materials. |
