Cloning,Expression And Detection Of Trichomonas Vaginalis RRas

Trichomonas vaginalis(Tv) is a sexually transmitted protozoan parasite,pathogenic mechanism is not clear. RRas protein has been demonstrated to play an important role in cell growth, differentiation, cell proliferation, cytoskeleton, cell adhesion and migration. Trichomonas vaginalis RRAs protein and the human have similar domains, whether the Trichomonas vaginalis pathogenicity related needs further study.This study one step cloning and expressed Trichomonas vaginalis RRas gene from Trichomonas vaginalis by genetic engineering technology. After purification of recombinant proteins, immune BALB/c mice, measuring its titer recombinant protein immune serum and by SDS-PAGE and Western blot analysis of the immunogenicity of the recombinant protein, and in the organization of Trichomonas vaginalis position preparation. Using CCK8 kit for detection of vaginal Trichomonas vaginalis RRAs protein on the proliferation of human tumor cells.For preparing to investigate trichomonas vaginalis RRas protein effect on the occurrence of human tumor development.Materials and Methods1.Trichomonas vaginals, plasmid、strain、Experimental animals and cell linesThe Trichomonas vaginals(Trichomonas vaginalis isolates from zhengzhou university third hospital clinical laboratory outpatient women); plasmid and strain( kept in our laboratory); BALB/c mice were bought from Experimental animal center of henan province Female mice aged 4 weeks. Cell lines used in human cervical cancer(HeLa) cell line(kept by our laboratory), Esophageal cancer cell line(Eca- 109)(saved by pathology teaching and research section).2.One step Cloning and expression of Trichomonas vaginalis RRas geneSearch the mRNA sequence of Trichomonas vaginalis RRas gene in the Gen Bank. Combined with PQE-80 L plasmid cloning sites and homologous recombination sequence design and downstream primers. Extracting total RNA of trichomonas vaginalis, reverse transcription of cDNA.The PCR products after purification through the PCR products purification kit were cloned into the expression vector PQE-80 L with the sites of BamHI and HindIII. Colony PCR was carried out on the recombinant plasmid, double enzyme digestion and sequencing identification. After the right identification,Then the recombinant plasmid was transformed into E.coli BL21. After appraisal, with suitable concentration of IPTG induced positive BL21, collecting bacteria precipitation, By one-step bacterial activity protein extraction kit to extract protein, sds-page, observe whether the targeted protein expression, and analyze the expression of product soluble.3. Trichomonas vaginalis recombinant protein PQE- 80 L-TvRRas purified and identifiedUsing 0.5mmol/ml isopropyl- beta- D- galactose and glycosides(0.5mmol/ml IPTG) induced expression, Sangon biological active protein extraction kit was used to extract protein, use Ni Agarose his label protein purification kits affinity chromatography purification.For Western blot analysis and identification whether the trichomonas vaginalis protein have Immunogenicity and antigenicity.4. Trichomonas vaginalis recombinant protein of human tumor cell proliferationCo-culture With hela and Eca- 109 cell from different concentrations of purified recombinant TvRRas protein a cell cycle, with CCK8 detection kits of its effect on tumor cell proliferation.Results1. The recombinant plasmid PQE-80L-TvRRas which has been constructed successfully through the double enzyme digestion and sequencing identification.After using 0.5mM IPTG induces the positive bacteria(BL21with PQE-80L-TvRRas) expressed about a 26 kDa fusion protein. Cloning of trichomonas vaginalis RRas gene to the length of 664 bp, coding protein with 204 amino acids, isoelectric point is 4.81, about 23.6 kd protein size, The solubility of fusion protein was identified and the result indicated that expressed protein existed as soluble protein.2. Western blot showed that the TvRRas recombinant protein can be recognized with His label mouse monoclonal antibody, and trichomonas vaginalis sloblue protein immune serum, but it was not immunostained with normal serum. And a trichomonas vaginalis sloblue protein antigens of approximately 22-25 kDa was recognized with the recombinant TvRRas immune serum, but it was not immunostained with normal serum.3. CCK8 cell proliferation experiment results prove that Trichomonas vaginal RRas recombinant proteins incubated th human tumor cell hela and Eca- 109 had a similar effect to promote cell proliferation as the same function with the human RRas protein and the difference was statistically significant(P<0.05).Conclusions1.The RRas gene of Trichomonas vaginalis was successfully one step cloning and expressed..2.Recombinant protein has good antigenicity and immunogenicity, for further study on its orientation in the tissue and pathogenic mechanism of trichomonas vaginalis is of great significance.3.Trichomonas vaginal RRas recombinant proteins incubated th human tumor cell hela and Eca- 109 had a similar effect to promote cell proliferation as the same function with the human RRas protein.