Expression Of New Recombinant Allophycocyanin Subunits And Primary Study On Biological Activities

In order to study the antitumor mechanism of recombinant allophycocyanin (rAPC) and screen more effective antitumor protein, recombinant allophycocyanin and its subunit genes were expressed in Pichia pastoris and Escherichia coli. Their properties and biological activities were also studied. The allophycocyanin (APC) was cloned into yeast integrated vector pPIC6αA. The Pichia pastoris X-33 strain was transformed using Lithium Chloride method. Western blot analysis of the culture supernatant of the yeast indicated that the recombinant allophycocyanin(YAPC) consisted of two kinds of subunits which had molecular weights of 19 kD and 21 kD respectively. 5 mmol/L LaCl3 increased 110% expression yield and also shortened the optimum culture time. The highest expression yield using the flask shake method was 4.4 mg/L at 48h. The recombinant allophycocyanin subunits MαAPC and MβAPC combined with mannose-binding protein (MBP) in their N-terminal and recombinant MBP (rMBP) were expressed in E. coli P2, P3 and PMBP strains. They were purified by amylose affinity chromatography. The purity was above 88% determined by SDS-PAGE and HPLC. Their molecular weights were 61kD, 61kD and 43kD and the maximum absorbant wavelengths were 275nm, 285nm and 281nm respectively. The immunogenicity of MαAPC and MβAPC was identical with natural allophycocyanin. The recombinant allophycocyanin (HAPC) was expressed in E.coli P4/pET-28a– APC. HAPC was purified by immobilized metal ion affinity chromatography and the purity reached above 90%. The molecular weights of HAPC subunits α and β were 21kD and 17kD respectively and its maximum absorbant wavelength was 235nm. The immunogenicity of HAPC was identical with natural allophycocyanin. The results from pyrogallic acid self-oxidation rate determination and 1,10-phenanthroline-Fe2+ oxidative assays showed that HAPC could scavenge both superoxide radical and hydroxide radical evidently. The results of physiological and pharmacological experiments indicated that HAPC could increase the number of white blood cells evidently and showed some antitumor activities. T-cell and B-cell epitope prediction was completed using ProPred, MAPPP and ABCpred servers. The results indicated that all kinds of universal T-cell epitopes and HLA-II-restricted T-cell epitopes and B-cell epitopes were found in HAPC and rMBP. Different degree of immunity-enhancing effects also was predicted. HAPC was shown the application foreground as a potential multiple antigen peptide vaccine. According to the predicted epitopes, the new peptides or proteins will be designed and expressed and used in pharmacological experiments in order to illustrate the antitumor mechanism and screen new antitumor protein. In this paper, the heterogeneous expression of recombinant allophycocyanin in Pichia pastoris was completed. The proper LaCl3 concentration could increase expression level of P. pastoris expression system. Thus it could decrease cost and abbreviate production process effectively. The establishment of purification methods of recombinant allophycocyanin and subunits, their properties determination and biological activities analysis were the base of elucidating the antitumor mechanism and exploiting new medicine from genetic engeneering.