Prokaryotic Expression Of Phytoplasma Immunodominant Membrane Protein And Preparation Of Its Polyclonal Antibody

Phytoplasma Immunodominant membrane protein(Imp) is one of the most important member ofthe immunodominant membrane proteins（IDPs）system, that may play a key role in the spread andpathogenic process of phytoplasma. Studies of phytoplasma membrane protein groupcould explore thepathogenic mechanism of phytoplasma in plant. High-throughput protein detection chip can be madethrough obtaining phytoplasma membrane protein-specific antibodies after immunization. In this study,a series of work, the prokaryotic induced expression of Imp, affinity chromatography purified protein aswell as Imp polyclonal antibody preparation, have been carried out.The Imp gene fragments was amplified by polymerase chain reaction(PCR) from the recombinantcloning vector pMD18-T-imp.Imp gene including restriction sites Nde I and Xho I was inserted intoplasmid pET-28a(+),and the recombinant plasmid pET-28a(+)-imp was transformed into E.coliBL21(DE3). The recombinant plasmid was extracted and purified. PCR and double enzyme digestionassay confirmed that the recombinant plasmid pET-28a(+)-imp was constructed successfully.By IPTGinducing，the recombinant protein was obtained in E.coliBL21(DE3)．SDS-PAGE analysis and Westernblotting test indicated that the fusion protein was about20kDa, matched with the target protein with6×His-Tag (19.5KD). The soluble analysis showed that expression of Imp fusion protein in E. colisystem as soluble protein and inclusion bodies. Inclusion body protein was the main forms.A culture conditions orthogonal experiment was designed and results showed that E.coliBL21-pET-28a(+)-Imp should be cultured at37℃, pH7.0, medium volume20%, rotating at200r/min.Recombinant bacteria cultured for8hours could reach the growth peak. According to the analysis ofvariance, the most significant factor was culture temperature for growth of recombinant strain. Theinfluence of factors, which affected the strain culture, should be in following order: temperature>liquidvolume>pH.Inducing conditions optimized by the orthogonal experiment showed that the best induction:temperature37℃, the starting OD600about1.5, IPTG final concentration of0.1mmol/L, induced for6h.According to the analysis of variance, temperature and starting OD600value were the key factors forImp expression. The order of influence factors that affected the induce culture were as following: theinduced temperature>initial OD600>induction time>IPTG. The experimental results showed that theIPTG and the target protein affected the cell growth and the fusion protein expression. The expressionlevel of fusion Imp protein was about70mg/L.The fusion proteins with6×His-Tag could be purified by Ni-NTA His-Bind resin.One ml resin wasadd to200ml fermentation lysate, resin was washed by8ml wash buffer containing40mM imidazole andeluted by4ml elution buffer with300mM imidazole, and purified target protein was obtained. Theexperimental results showed that higher concentration and purity Imp protein could be obtained from inclusion body under denaturing conditions. Imidazole was removed from Imp protein solution bydialysis and0.8mg/ml protein solution, which purity of95%was obtained. Anti-imp polyclonalantibody was obtained from rabbit serum after immunity. The indirect ELISA titers determined the titersas1:128,000.Western-blotting analysis showed that the polyclonal antibody reacted specifically with theImp protein.