Cloning And Expression Of Trichomonas Vaginalis Ferredoxin Gene

Background and purpose: Trichomonas vaginal is is a flagellate protozoan that parasitizes the urogenital tract of humans and is the cause of trichomoniasis. This diease is one of the most prevalent non-viral sexually transmitted diseases and has still threatened the healthy of human since Donne first described in 1836. The ferredoxins are low molecular weight iron-sulfur proteins that widely distribute in nature. The [2Fe-2S] ferredoxins are the most common ferredoxins. T. vaginalis ferredoxin is the major electron transport component of hydrogenosome. It links pyruvate:ferredoxin oxidoreductase (PFOR) which mediates oxidative decarboxylation of pyruvate with the formation of acetyl-CoA and ATP to hydrogenase. This protein is the electron donor in these reactions and is believed to be responsible for activation of the antimicrobial agent metronidazole through electron transfer. Despite decades of use, as a front-line antibiotic metronidazole has been noteworthy for relative low level of resistance developed. Recently, however, the isolates of resistant strain of T. vaginalis from clinical patients has been reported frequently, for this reason, it is especiallyimportant to study the activation mechanism of metronidazole and development of drug-resistance. Quon and Johnson discovered that intracellular levels of ferredoxin were invariably reduced in the resistant strains relative to a sensitive strain with immunoblot analysis. Similarly, Northern blot analysis showed that ferredoxin mRNA levels were reduced 50-65% in resistant strains. Ferredoxin gene transcription was reduced 40-65% in resistant strains with the unclear run-on assays. Land et al discovered that there is an approximately 90% reduction in mRNA levels encoding ferredoxin in the highly drug-resistant strain. Immunoblot analysis demonstrated undetectable levels of hydrogenosomal proteins, PFO and Fd, in the highly drug-resistant strain. In short, activation of metriodazole is reduced in resistant isolation. Our study was to redound to clarify the mechanism for resistance.Our study includes two parts.The first part: Objective: Cloning of ferredoxin gene. Methods: We isolated T. vaginalis from patient in Sichuan of China and extracted total DNA from protozoa with Chelex-100 method. Based on the published sequence of Genbank, we designed a pair of primers and amplified the ferredoxin gene with PCR method using genome as templates. After being purified, the gene was directionally cloned into plasmid pMD-18T simple vector, result in recombinant plasmid pMD-18T-Fd. The recombinant plasmid was then transferred into competent E coli JM109. The transformants were screened and identified by PCR and restriction analysis; additionally, the sequence of the coding region of theferredoxin gene in pMD-18T-Fd was confirmed by DNA sequencing. The difference of sequence between isolation strain from China and original standard from Genbank was analyzed. Results: Results had showed that the size of amplified ferredoxin gene from Sichuan was 306bp. Sequence analysis showed that its identity revealed 99% with T. vaginalis from the GenBank. Our work will provide experimental evidence for prevention and treatment on Trichomoniasis.The second part: Object: Expression of ferredoxin in E.coli and construction the eukaryotic plasmid. Methods: After a few times passage, the fresh clinical isolate of T. vaginal/swas sonicated and supercentri-fuged, the supernatant was soluble antigen. The concentration of protein in soluble antigen was detected by Bardford method. Polyclonal antibody against T. vaginalis isolated from Sichuan was generated in 3.0 Kg rabbit immunized with soluble antigen. Antibody titre was determined with ELISA method. The recombinant plasmid pMD-18T-Fd confirmed correct was digested by restriction enzymes HindR and Baift\ I and subcloned into the pUC19 vector that had been digested with same restriction enzymes, then transformed into E coli JM109. The protein was induced to express by IPTG and analyzed by SDS-PAGE and Western blot. At the same time, the prokaryotic cell expression recombinant plasmid pET3C-Fd presented by Dr. Johnson was transformed into E coli BL21 and the protein was induced to express with same method. Because T. vaginalis is eukaryotic cell, it is important to study expression of ferredoxin in eukaryotic cell. We constructed the eukaryotic cell expression recombinant plasmid containing ferredoxin gene of T. vaginalis pcDNA3. l(+)-Fd. Results :The concentration of protein in soluble antigen was 0.44g/L The polyclonal antibody obtained from rabbit had a titre of above 1:8000 and the antibody can be used as primary antibody of Western blot or ELISA assay. The result of SDS-PAGE showed that there was a new band at an apparent molecular of 14,000 Da. A specific hybridization signal was verified by Western blot. Besides, ferredoxin was also expressed in ï¿¡ coli transformed recombinant plasmid pET3C-Fd, which will facilitate to study function of ferredoxin. Eukaryotic cell expression recombinant plasmid pcDNA3.1 (+)-Fd constructed by us will be useful for the further study of the Ferredoxin.Conclusion: The ferredoxin gene was successfully amplified and cloned using DNA extracted from T. vaginalis of Sichuan as templates. Subsequently, ferredoxin was induced to expression. At last, a eukaryotic cell expression recombinant plasmid containing ferredoxin gene of T. vaginalis was constructed. All works are going to be used further study of function of the ferredoxin and provide study basis for prevention and treatment of Trichomoniasis.