Cloning And Expression Of Gingipain K Catalytic Domain From Porphyromonas Gingivalis And Preliminary Study On Anti-periodontitis Immunization

Recent evidence strongly suggests that the primary pathogen responsible for chronic progressive periodontal disease is Porphyromonas gingivalis. The gingipains, a group of cysteine proteases produced by P. gingivalis, have received considerable attention. The gingipains are the important virulence factors of P. gingivalis. The gingipain K (Kgp) is one of them. The initial translation product is large precursor, which is composed of four functional regions (the signal peptide, the NH2-terminal prosequence, the mature proteinase domain, and the COOH-terminal hemagglutinin domain). The nucleotide sequence of the kgp DNA covers 6.4kbp. The nucleotide sequence (6,366 nucleotides) includes the complete coding region and parts of the 5'-and 3'-noncoding regions. The open reading frame consisting of 5,169 nucleotides was found to encode 1,723 amino acid residues with a calculated mass of 218 kDa. The amino acid sequence suggests that the first 22 amino acid residues containing a hydrophovic amino acid cluster may represent a signal peptide. The hydropathy of the the NH2-terminus of the precursor is high enough for it to function as a signal sequence translocating the proteinacross the membrane. The next 206-residue sequence is considered to be an NH2-terminal propetid. The amino acid sequence of the mature enzyme was found to start with the 229th Asp residue and to end with the putative site of the 717th Arg residue. The remaining COOH-terminal portion is thought to be the COOH-terminal prosequence, which contains the hemagglutinin-related sequence starting with the 738th Ala residue, identical to the NH2-terminal sequence of P. gingivalis.In the present study, we assessed whether the KGPcd could be vaccine candidate for prevention of oral bone loss in a rat model. Used the molecular biological techniques, gingipain K catalytic domain (kgpcd) was cloned, expressed in E.coli BL21 (DE3) and purified by Ni-NTA affinity chromatography. Thus mass recombinant proteins were obtained. Then the anti-KGPcd antibody was prepared to proceed the following study. At the same time, the eukaryotic expression recombined plasmid of pcDN A3.1+/kgpcd was constructed. Sprague-Dawley (SD) rats were vaccinated with eukaryotic expression plasmid by the route of submandibular gland-targeted injection. The specific immune responses and their protection against periodontitis were observed by ELASA and HE. So, the immunization effects were verified, paving the way for the further study.This paper consists of the following two parts:1 Cloning, expression of the gingipain K catalytic domain (kgpcd) gene from Porphyromonas gingivalis and purification of the recombined KGPcd protein and Preparation of anti-KGPcd antibody1.1 Cloning and sequencing of kgpcd gene from Porphyromonas gingivalisIn the study, P. gingivalis was routinely cultured and its genomic DNA was isolated as templates by bacteria genomic DNA isolation kit.The desired DNA products encoding gingipain K. catalytic domain were obtained from the total genomic DNA by PCR with the the designed specific primers. The PCR products, ie, these objective gene segments (1467bp), were inserted into cloning vector of pGEM-T easy Vector and the inserting plasmids were transformed into E.coli XL-10 and had the blue-white screening. The positive clones, ie, the white clones, were analyzed by restriction endonuclease mapping and DNA sequencing. The results showed that the sequence of kgpcd were consistent with that of the P. gingivalis 381 in GeneBank.1.2 Expression of recombinant gingipain K catalytic domain gene in E.coli BL21 (DE3)The gene fragment encoding kgpcd was inserted into prokaryotic expression vector pET-16b in which foreign gene is controlled by T7 promoters. The recombinant plasmid pET-16b/kgpcd was transformed into E.coli BL21 (DE3) and induced by IPTG in order to express the fusion protein His (Histidine)-KGPcd. The expressed fusion protein was purified by Ni-NTA affinity chromatography and then refolded by dilution and dialysis. After induction, a new anticipated 56kDa...