| Urinary tract is one of the most common places to be infected by bacteria that accounts for more than 85% of urinary infection. Escherichia coli is its major pathogen. The sort of E.coli isolated from urine of patients with upper urinary tract infections, namely Uropathogenic Escherichia coli (UPEC), possesses a puissant ability to the attachment on the epithelium of mucous membrane of urethra. P fimbrae, an important virulent factor located on the surface of the UPEC, excerts its function of attachment upon the surface of mucosa cells by specifically binding to the receptors distributed on the epithelial surface of urethral mucous membrane, whereas adhesin PapG on the tip of fimbrea plays a key role in introducing the attachment. Immunological studies of adhesin protein encounter a great difficulty owing to several factors like various sera types of adhesins with different local distribution, susceptible population, infected sites and clinical disorders or symptoms although highly conservative, extremely too small amount of the total protein they occupy to intrigue immune reaction of the host and susceptibility of degradation by periplasmic protein. Present work is conducted to precisely identify the type of PapG adhesin of local UPEC (UPEC 132) in Tianjin followed by the construction and expression of recombinant fusion-protein to be used in the immunological and bacteriologicalstudies, to preliminarily observe the immunologically protective effects induced by this fusion-protein.Part One Cloning and Sequencing of Adhesin Gene papG of UPEC132It has been reported hitherto that there are three types of PapG adhesins based on the binding characteristics of PapG to their receptors. UPEC carrying type II papG alleles is of important significance in clinics. Co-existence of type II PapG with either type of F7-2, F10 or F11 of Pap A has been reported within a fimbria thus far, yet we have found no report of the coexistence of type II PapG with other type of PapA within a pilus.At present work, product encoded by papG of local UPEC 132 adhesin gene was identified to be a type II PapG adhesin by PCR using two different primers. Sequence analysis of PCR amplification of UPEC 132 revealed that homogeneities of the whole nucleotides and their putative amino acids were as high as 98.6% and 98.5% respectively between UPEC132 papG and UPECIA2 of Fll serum type, but as low as 57.1% and 45.9% between UPEC 132 papG and UPEC J96 of the same serotype, giving further evidence that papG of UPEC 132 belonged to II type/adhesin alleles. Thus, we can conclude that P pilus of UPEC 132 consisted of type II PapG and F13 PapA, with the latter proved by our previous work.Furthermore, there were 11 point mutations and one deletion mutation of tri-nucleotide codon in the whole sequence of PapG 132 gene compared to that of UPECIA2 type II adhesin. These mutations included: 1)7 synonymous mutations; 2) 4 missense mutations with the 2 sited in the specific receptor binding domain and the other 2 in PapD protein processing domain; and 3) numbers of amino acid residues of PapG protein were one less than those of PapGIA2 for the sake of tri-nucleotide codon deletion.In general, we reported here that 1) combination of local UPEC pilus was type II PapG and type F13 PapA, differentiated from all patterns of combination that had ever been found; 2) there were 4 point mutations, 7 synonymous mutations and a tri-nucleotide codon deletion in the whole sequence between UPEC 132 papG and papGIA2. These findings were fundamentally important for our further work on gene clone, fusion-protein production and immunologic stimulation.Part TwoConstruction and Analysis of Recombinant Expression Plasmid of papG from UPEC132One of the important virulent factors of bacterial pathogenicity is the colonization of bateria on the surface of mucous membrane epithelium by the adhesive pili. High conservation of minor subunits of pili associated with adhesion provides a possibility of adhesin vaccine preparation, although antigenicity...
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