| Objective: The present study was conducted to construct the prokaryotic expression plasmid of cysticercus cellulosae antigen cYI, to observe its expression in E.coli DH5( and specific antigenicity. In order to provide the theoretical basis for further study of structure and function of antigen cYI and for developing diagnostic antigen and vaccine of cysticercosis. Methods: 1. The achievement of target gene cYI : Using the extracted positive plasmid DNA as templates, according to the sequence of cDNA designed primers, PCR was performed under the following condition: 94℃ for 5min, then 94℃ for 1min,56℃ for 1min and 72℃ for 1min per cycle ,The amplification was carried out for 30 cycles, followed by incubation at 72 °C for 10 min. The PCR products were digested with EcoR I and HindIII, and then identified with 1.2% agarose gel electrophoresis. 2. The recombination transformed into the E.coli DH5????The resulting PCR fragments and high –lever fusion expression vector PMAL-C2 were digested with EcoR I and HindIII. The fragments of interests were recovered from agarose gel, purified and ligated by T4 DNA ligase, which resulted in the recombination plasmid PMAL-C-cYI. Then the recombination was transformed into the E.coli DH5???According to the principle of ?-complement of β-galactosidase, selected the white colony on the LB culture medium with ampicillin, IPTG and x-gal. The positive clones were identified by enzyme digestion with 1.0% agarose gel electrophoresis. 3.The expression of recombination: To activate the E.coli DH5??harboring the recombination plasmid at culture medium at 37(C, then seeded them on LB/agar plates with 100 (g/ml ampicillin; Next day single strain was selected and inoculated into LB medium containing 100 (g/ml ampicillin overnight at 37 °C in an orbital shaker (220 rpm); Inoculated 100 ml rich broth with 1 ml of an overnight culture cells containing the fusion plasmid. Grown at 37(C with good aeration to 2(108 (OD600=0.5-0.6), added IPTG to the culture to give a final concentration of 0.3mM, inoculationed at 37(C for 4-6 h. Took 1ml sample at intervals and microcentrifuged for 2min. Discard supernatant and resuspend the cells in TE buffer. 4. SDS-PAGE: To freeze the negative control cells, induced and uninduced cells in buffer overnight at -20(C. The cells were placed in an ice-water bath and sonicated in shot pulses until the released protein reached a maximum, centrifuged at 9,000g(4(C for 20 min, then the supernatant and pellet were saved on ice respectively. Added 10(l 2(SDS-PAGE sample buffer to 10(l of the extract, and each sample was boiled for 5 minutes and then loaded into 10% polyacrylamide-SDS gel, electrophoresed for 2h. The gel was then stained with 0.25% coomassie brilliant blue R-250 witch determined the protein concentration. 5.Western blotting analysis: Protein samples of the negative control cells, induced and uninduced cells were separated by SDS-PAGE and transferred onto the nitrocellulose membrane. After blocked with 5% non-fat milk, the membranes were incubated with the antibody of diluted serum of normal human, cerebral cysticercosis patient and trichomonas vaginalis patient at 4°C overnight. After washed three times, the membranes were incubated with rabbit-anti-human polyclonal antibody at room temperature for 1 hour. Then discarded the second antibody and washed the membranes three times. The signals were developed with the 4-Chloro-1-NapHthol. Results: (1) PCR was performed with the designed primers and the templates of extracted positive plasmid DNA. The PCR products showed a single band about 550 bp on 1.2% agarose gel electrophoresis. (2) The positive clones that were selected with the principle of blue-white colony were identified by enzyme digestion with 1.0% agarose gel electrophoresis. The result of digestion showed that the recombinant plasmid PMAL-C-cYI was constructed successfully in this experiment, and the sequence of target gene of recombinant plasmid constructed was in accordance with the sequence of cYI cDNA reported. (3) Th...
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