Cloning, Sequence Analysis And Expression Of The Outer Membrane Protein I Of Neissria Gonorrhoeae

Objective To construct T-A cloning recombinant plasmids of PI genes of the Neisseria gonorrhoeae reference strains, and analyze the outer membrane protein PI gene difference between the reference strains and clinical isolates; to construct recombinant expression plasmids of PI gene, induce the recombinant strains to produce PI protein, and study their expression characteristic and the function of the recombinant PI protein.Methods There are four parts in this dissertation.1. Construction of cloning recombinant strains of PI genes of Neisseria gonorrhoeae reference strains: First, the genome DNA of Neisseria gonorrhoeae reference strains 29400 and 29403 was prepared according to the method described by J. Lewington et al. PI genes were then amplified by PCR. The products of PCR were purified and added Adenine at the end of the sequences. These PI genes were ligated with pBS-T vector and transformed into competent cell E.Coli DH5α. Positive clones selected by ampicillin resistance and blue-white selection were incubated and extracted plasmids, which were digested by restrictive endonuclease EcoR I and Xho I to confirm whether the PI genes were inserted in.2. Sequence analysis on PI genes: Sequence the PI genes obtained in step 1, and compare them with sequences in GenBank database so as to find their similarsequences. Two sequences gained in this research and another four gained in our previous study were aligned by ClustalW software, in order to find the differences among them. The six sequences were compared with the previous studies on topology of their Amino Acid sequences and 3D structures were predicted in using an online structure protein prediction software.3. The construction of PI gene recombinant strains for expression: Recombinant pBS-T-PI plasmids gained in step 1 were digested by EcoR I and Xho I, and then purified by gel extraction. At the same time, the pET30b(+) vector was digested firstly by EcoR I and Xho I, then by CIAP, and was purified by gel extraction. The purified PI genes and vector were ligated and transformed into competent cell DH5a, and positive strains were selected by Kanamycin resistance and double restrictive digestion.4. Expression, purification and identification of the recombinant PI protein: BL21-pET-PI recombinant strains were induced by IPTG to study their PI protein expression ability. Expressed recombinant protein was purified by agarose resin carrying chelated nickel ions, with the reaction between 6>