Screening And Identification Of The Peptides Specifically Binding Tothe Adhesion Protein Of Mycoplasma Genitalium And Construction Of CDNA Library Of SV-HUC-1 Cells

Mycoplasma genitalium(M. genitalium) is one of prokaryotic microorganism. It is between viruses and bacteria, lacks cell wall, is highly polymorphic, propagates in cell-free culture medium. It can cause urogenital system infection and is classified into one of the sexually transmitted disease pathogens.In recent years, the infection rate of M. genitalium shows a rising trend due to the abuse of antibiotics. However, there is still no drug or vaccine available to meet the clinical need. The most critical M. genitalium outer membrane adhesion protein is Mycoplasma genitalium adhesin protein(Mg Pa), and its C terminal has strong immunogenicity. The mutant strain of M. genitalium lacks Mg Pa and has no cell adhesion activity. Therefore, Mg Pa plays an important role in the pathogenicity of M. genitalium. In this study, the constructed prokaryotic expression vector PET-30a(+) / Mg Pa was used to express the recombinant protein(r Mg Pa) in E.co1 i. r Mg Pa-specific binding peptides were screened and identified by phage display peptide library technology and identified by ELISA, competive ELISA etc. Objectives of this research: 1、To express and purify the r Mg Pa through constructing recombinant vector containing the dominant epitope(1075~1364 aa) of Mycoplasma genitalium adhesin protein, to provide the experimental foundation for studying the biological function of Mg Pa. 2、To provide the experimental basis for the study on the pathogenic mechanism of M. genitalium by screening and identifying the r Mg Pa-specific binding peptides using phage display random peptide library. 3、To study the interaction between M. genitalium and human urothelium cells by constructing T7 phage display c DNA library of SV-HUC-1 cells. Methods of this research: 1、The recombinant vector containing the dominant epitope(1075~1364 aa) of Mycoplasma genitalium adhesin protein was constructed and the r Mg Pa was expressed in IPTG-induced E.co1 i RosettaTM2(DE3). The r Mg Pa was identified by SDA-PAGE analysis and Western blot. And the recombinant protein was then purified by Ni-NTA purification column. Finally the concentration of r Mg Pa was detected by BCA method. 2、The phage display random peptide library was used for 3 rounds of biopanning target on r Mg Pa. The phages were collected for amplification after biopanning. The single strand DNA of phage clones were extracted and purified using Sodium iodide method. And DNA sequencing analysis was then performed. The exogenous amino acid sequences displayed on the surface of phages were deduced.The combination specificity of positive phage and r Mg Pa was detected by ELISA, competitive binding assay and dot immunobinding assay. 3、The total RNA of SV-HUC-1 cells was extracted using Trizol reagent, m RNA was separated and purified by m RNA isolation kit and the ds-c DNA were synthesized by reverse transcription, The end of ds-c DNA were ligated with Eco R I / Hind III sticky ends. The ds-c DNA fragments that were longer than 200 bp were collected and purified and then ligated into the T7 phage vector. After packaging in vitro, the recombinant T7 phage vectors were transformed into BLT5403 to construct the T7 phage display library. Results of this research: 1 、 SDS-PAGE displayed that the r Mg Pa(containing 6×His) with about 37 k D was successfully expressed from Mg Pa recombinant strain under the induction of IPTG. The target protein with higher purity was obtained after purified by Ni-NTA purification column. The purified target protein was detected with nucleic acid protein instrument by BCA method. The concentration was about 1 900μg /m L. 2、The yields from the first round to the third round were 1.78×10-6, 8×10-6 and 7.3×10-5 respectively, showing the phage clones were obviously enriched. 38 phages were randomly picked to clone and amplify. The sequencing analysis of single stranded DNA was performed. The results showed that the two groups of consistent core sequences for 37 phage displayed exogenous peptide sequences were V-H-W-D-F-R-QW-W-Q-P-S and D-W-S-S-W-V-Y/H-R-D-P-Q-T/S. A specific peptide sequence screenned from the phage display random 7 peptide library was H-Y-I-D-F-R-W. 3 、 ELISA results showed that 10 representive phage clones(P1 ~ P10) containing different peptide sequences were positive and the binding capacity was higher. The results of competitive binding experiment indicated that the specific combination of these phages and r Mg Pa could be partially inhibited by anti-r Mg Pa antibody with different dilution rates. And with the increase of dilution rate, the inhibitory effect was correspondingly decreased. The dot immunobinding assay showed that the positive phages also could specifically combine with r Mg Pa. 4、The SV-HUC-1 cells T7 phage display c DNA library was successfully constructed, the titer of amplified library was 1.2×106pfu/ cm3 by plaque assay. The results of PCR identification for selected plaques showed that the recombination ratio was about 93.75%, and the inserts fragments were longer than 200 bp. Conclusion of this research: 1、The recombinant protein r Mg Pa with molecular weight about 37 k D and concentration about 1 900μg/m L was successfully expressed and purified. 2 、 Two peptides coming from phage display 12 peptides library specifically combined with r Mg Pa were successfully screened: V-H-W-D-F-R-Q-W-W-Q-P-S and D-W-S-S-WV-Y/H-R-D-P-Q-T/S: One r Mg Pa-specifific binding peptides screened from 7 peptides library was H-Y-I-D-F-R-W. 3、The SV-HUC-1 cells T7 phage display c DNA library was successfully constructed,which will contribute to the study of the interaction between Mycoplasma genitalium and human urothelium cells.