AIM: To analyze the nucleotide and putative amino acid sequences of PIA genes isolated from N. gonorrhoeae and to construct the prokaryotic expression system of PIA gene.METHODS: The entire PIA genes from 9 strains of N. gonorrhoeae were amplified by using high fidelity PCR. The target amplification fragments were sequenced after T-A cloning. Homology comparison of the nucleotide and putative amino acid sequences of PIA genes from the isolates with the reported sequences in GenBank was then performed. A prokaryotic expression system of PIA gene was constructed. Different dosages of IPTG were applied to induce the expression of the target recombinant protein (rPIA) and 10% SDS-PAGE plus Bio-Rad Agarose Image Analysor was used to determine the expression level of rPIA. rPIA was extracted using Ni-NTA affinity chromatography and the purified effect was detected by SDS-PAGE.RESULTS: In comparison with the reported PIA gene sequences (GenBank No: L19962), the homologies of nucleotide and putative amino acid sequences of PIA genes from the isolates were 99.6%-100% and 99.1%-100%, respectively, which indicated that all the isolates were belonging to serovars IA6. Output of rPIA was as high as 50.1% of the total bacterial proteins. The purified rPIA only showed a single target protein fragment in gel.CONCLUSION: Serovar IA6 is dominant in the local N. gonorrhoeae isolates and sequences of the encoding gene are relatively conserved. The constructed prokaryotic expression system is able to express rPIA with high efficiency, which may lay a foundation for further development of serological detection kit and vaccine of N. gonorrhoeae.

Plasmids are the most commonly used gene carriers in the field of gene synthesis and sequencing. However, the main problems faced by traditional plasmid DNA extraction technology are low extraction throughput and high production cost, so they cannot meet the growing demand. In this study, a double-magnetic-bead method (DMBM) for plasmid extraction was developed based on the principle of plasmid extraction. The effects of the input of magnetic beads, the size of plasmid DNA fragments, and the volume of bacterial on plasmid DNA extraction were explored. In addition, the quality, throughput, and cost of plasmid DNA extraction were also compared between this technique and the commercial plasmid DNA extraction kits. The results showed that the DMBM can meet the needs of extracting plasmid DNA with different cell densities and fragment lengths. Moreover, the sensitivity and quality of plasmid extraction by the DMBM method were both superior to those of the centrifugal adsorption column method. In addition, this technique could be applied on a 96-channel automated nucleic acid extractor, resulting in higher purity of the extracted plasmid DNA, 80% reduction in extraction time, and 57.1% reduction in cost. It also reduces manual operations, achieving high-throughput and low-cost plasmid DNA extraction, thus may facilitate gene synthesis and sequencing.
Plasmids/genetics* ; DNA/genetics* ; Nucleic Acids ; Genetic Techniques ; Magnetic Phenomena