Screening And Indentification Of The Interacting Proteins Between Host And The Mycoplasma Suis α-Enolase

Eperythrozoonosis is a new zoonosis, and the main symptoms are jaundice, fever as well as anemia. E.suis is mainly adhesion to host cell, it is involved in the adhesion of E. suis to porcine host cell by the adhesion protein in small space, but there is a few factional genes which is known before the study. In this study, we constructed a peripheral blood mononuclear cells (PBMC) cDNA library, and the recombinant bait vector of E.suis a-enolase gene was constructed for screening and inentifying interactional target protein in peripheral blood mononuclear cells (PBMC) cDNA library by yeast two-hybird screening technology, it provided basic research for cell adhesion and invasion mechanism of E.suis upon protein levels.Constructing peripheral blood monoculear cell yeast two-hybrid cDNA library:In order to construct yeast two-hybrid cDNA library.Firstly,the experiment took sterile health pig blood,then separated PBMC and extracted total RNA.Synthesized two-stranded cDNA through retroviruses with the using of SMART technology.The double-sranded cDNA(dscDNA) was purified by CHROMA SPINT-400 columns,and the linearized pGADT7-REC vector were co-transformed into competent cells of yeast strain Y187.A yeast two-hybrid cDNA library was constructed in yeast cells by homologous caninum. the total RNA concentration was 1143 ng/μL,OD26o/OD28o was 1.90,the library capacity was 3.11×108 CFU/mL with insert size ranging from 400bp to 4000bp,and the average length of inserts of was approximately 2000bp,with the recombination rate of 100%.These results showed that a cDNA library was constructed,which could be used for the yeast two-hybrid screening system.Constructing the recombinant bait vector pGBKT7-enolase of a-enolase:The experiment replaced the termination codon of a-enolase.And we designed and synthesized three pairs of primers according to published E.suis whole genome sequence (NC-015153.1).The amplification of the a-enolase full length gene by Overlap PCR technology.And it was subcloned into pMD-18-T Simple vector, which connected with expresson vector pGBKT7 through the double enzyme for constructing bait vector pGBKT7-enolase.Then the sequencing action, self-activition and toxic action was tested, the results indicated that a-enolase gene foll length by over lap PCR technology was 1623bp contrasting results of the sequencing,we can conclude that there are 23 base mutations and 1 amino acid point mutation. The homology of nucleotide sequence was 98.6% and that of amino acid sequence was 99.8%.The pGBKT7-enolase gene was transformed into yeast cells Y2H, Colony PCR showed that pGBKT7-enolase into Y2H successfully. Toxic effect proved pGBKT7-enolase non-toxic to yeast cells growth and the self-activation test proved that no effection to the reporter gene. So the bait vector pGBKT7-enolase can be used for screening in yeast two-hybrid system.Screening of the PBMC library and selection of E.suis a-enolase interactive clones:The experiment transformed the constructed bait vector pGBKT7-enolase to yeast cell Y2H for screening the interaction protein with transformed PBMC cDNA of Y187 yeast cells in 50mL 2xYPDA nutrition liquid medium. Results showed that there were 162 white, edge neat, uplift single clones in the defected solid medium SD/-4.After marking the clones on SD/4 plates,there were 33 clones transformed to blue on SD/4/X/A plates.The library universal primers amplified blue clones by PCR. 1.0% gel electrophoresis showed the strips were mainly concentrated in between 250-2000 bp.The PCR product sequencing results was alignmented with nucleotide sequence and amino acid of NCBI.The results showed consistent with the experiment, and there were four proteins:number 24 protein endonuclease/reverse transcriptase, number 25 protein beta actin, partial, number 26 protein 60s ribosomal protein L11, partial and number 28 protein clusterin precursor, the number of clones respectively was 3,4,3, and 1.Co-transformation verifying interactional protein:Yeast plasmid transformed into competence escherichia coli, after purification it devided into two groups:the control group and experimental group, and groups co-transformation into in Y2H yeast competent respectively, Applied to 150 mm SD/-2, SD/-4, SD/-4-/X/A plate (Kan) 30℃ constant temperature incubator inversion training 3-4d.According to the results of pGADT7-24, pGADT7-25, pGADT7-28+pGBKT7-enolase in three groups respectively, SD/-4 and SD/4/X/A grow the white and blue bacterial colonies on plates respectively, the results consistent with library screening results. On the contrary, pGADT7-24+pGBKT7 group in SD/-4 and SD/-4/X/A grow the white and blue bacterial colonies on plates respectively, it do not tally with the expected results. So it was the false positive protein. It indicated that No.24 Endonuclease/reverse transcriptase existed self-activition.Namely under the condition of no a-enolase genes couid self-activate the yeast two hybrid reporter gene. Thus, in subsequent experiences we will no longer research on it And No.26 protein 60 s ribosomal protein L11 in all the groups did not appear any clone, may be due to the yeast plasmid extraction is incomplete or transformation into competence escherichia coli unseccessfully. No.25 protein beta actin had high repeatability. According to data analysis,β-actin was beta actin protein,the protein had highly conserved gene sequence. The high number of mRNA expression. It commonly was used in mRNA internal standard, nucieotide protection analysis also could be used as an external standard. We guessd β-actin plays a very important role between E.suis and the host. It will be served as the focus of the further research.This test for the in-depth study of interaction mechanism between E.suis and host cells,which will lay a foundation for molecule and protein. And it provide a precondition for screening drug target function point of Eperythrozoonosis.