Cloning And Expression Of Gene Encoding Elongation Factor P From Kocuria Rhizophila DC2201 And Preparation Of Its Monoclonal Antibody

EF-P?Elongation factor P?is a translation elongation factor widely present in bacteria.Its main function is to slow down the ribosome translation delay caused by the polyproline sequence on the nascent peptide chain and promote the synthesis of peptide bonds.In addition,post-translational modifcation of EF-P protein plays an important role in its function.At present,there are few reports on the post-translational modification of EF-P in the class Actinobacteria,which has high GC content.It is of great biological significance to clarify its post-translational modification method for its life activities.In this paper,we selected Kocuria rhizophila DC2201 as the research material,expressed the EF-P protein by E.coli and used the recombinant protein EF-P as the immunizing antigen to obtain the anti-EF-P recombinant protein monoclonal antibody.The monoclonal antibody can be used to obtain the natural protein EF-P from Kocuria rhizophila DC2201,which can lay the experimental basis for analyzing the style of post-translational modification.The specific content and results as follows:1 Bioinformatics analysis,expression and identification of EF-P protein from Kocuria rhizophila DC2201Firstly,the bioinformatics analysis showed that the efp gene was 564 bp in length and encoded 187 amino acids.The theoretical molecular weight of the protein was about 21 kDa,which was a hydrophilic acidic protein from cytoplasmic.Its main secondary structure is?-sheet;phylogenetic analysis shows that EF-P is highly conserved.Secondly,the recombinant cloning vector pMD19-T-efp and the recombinant expression vector pET-29a?+?-efp were successfully constructed by molecular biology.The recombinant protein EF-P about 25 kDa was obtained by expression,which purified by nickel ion affinity chromatography and identified by SDS-PAGE and MALDI-TOF-TOF.2 Optimization of induced expression conditions for recombinant protein EF-PA large amount of soluble protein was obtained by optimized the expression conditions?inducing initial cell concentration,induction temperature,the final concentration of IPTG,induction time?.Finally,the optimization conditions of expression were determined:Under OD_600nm=0.4,the strain containing the recombinant expression vector was induced with 0.7 mmol/L IPTG at 20?for 6 h.3 Preparation for the anti-EF-P recombinant protein monoclonal antibodiesThe Balb/c mice were immunized by purified EF-P recombinant protein as immunizing antigen.Then cell fusion,screening of positive hybridoma cells,subcloning to screen the single cells.At the end,5 ascites were successfully obtained by injecting cells to mices.The ascites titer is between 1:128,000 and 1:512,000.The3D3 ascites was isolated,purified and identified to obtain a monoclonal antibody with high purity and specificity against the recombinant EF-P protein.The results offer a material basis to study the post-translational modification of the natural EF-P from Kocuria rhizophila DC2201,explore the biological significance of its post-translational modification on the Kocuria rhizophila DC2201.