| X maltophilia belongs to theGram-negative bacteria. It can produce CG-like receptor that is like to the mammalian LH/CG receptor, which plays a vital role in reproductive physiology in mammalians. The bacteria can also secrete 48.5 KD xCG protein, which has certain homology with the 3 -subunit of hCG. X maltophilia is the first prokaryote in which both the CG-like receptor and the endogenous ligand xCG protein have been discovered.The CG-like receptor of X maltophilia has some similarity with rat LH/CG receptor in molecular weight and ligand-binding characteristics. But it hasn' t been known for the gene encoding CG-like receptor, its structure and function by now. In order to understandthe structure and biological function of CG-like receptor, it needs to study the gene encoding CG-like receptor, understand the gene structure and characteristic, and be compared with that of mammalian LH/CG receptor.This is important for learning whether there has homology between the CG-like receptor of X maltophilia with mammalian LH/CG receptor, and studying on the bacterial pathogenesis.Firstly, we identified the strain bought from American ATCC, and it was X maltophilia . We developed a rapid approach in extracting the genomic DNA from X maltophilia. The detergent SDS and CTAB were used to destroy the structure of cell membrane to liberate the cellular nucleic acids; and the CTAB/polysaccharide /protein complex was removed by using phenol/chloroform/isoamyl alcohol; then DNA was precipitated with isopropanol, and finally DNA was dissolved in TE buffer. This approach was simple, rapid and economical. The purity and concentration of DNA extracted with this approach was enough for digestion by restriction endonucleases, PCR amplification, and construction of genomic library.Secondly, we designed one pair of primers from thenucleotide sequence of LH/CG receptor transmerabrane domain in known species which has much homology. The genomic DNA of X maltophilia was used as template to PCR amplify based on above primers, and about 600bp aim product was cloned in a pUCm-T vector. After the recombinant plasmid was identified by restriction endonuclease digestion and PCR amplication, the recombinant plasmid pUCm-Rec was obtained. Using the DIG-labeled 600 bp fragment as probe, it was hybridized with pUCm-Rec///?a I and the genomic DNA of X maltophilia by DNA dot blotting, and they had positive hybridization signals. This showed that the PCR-amplified product had homology with the genomic DNA of X maltophilia, and about 600bp fragment was real PCR-amplified product, which using the genomic DNA of X maltophilia as template. The insert on the recombinant plasmid pUCm-Rec was sequenced. The PCR product was 593bp. This 593bp nucleotide and its translating 197 amino acid(aa) transmembrane domain of CG-like receptor hadn' t homology with that of mammalian LH/CG receptor transmembrane domain respectively. But the 5~196aa fragment of the 197 aa transmembrane domain of CG-like receptor, showed 51%identity with the 199~388aa fragment of 572aa putative protein from Caulobacter crescentus CB15. And its 16~ 76aa region had 33% identity with the 985~1046aa of 1137aa guanylate cyclase from Caenorhabditis elegans, this indicated that there maybe had a similar guanylate cyclase/cGMP signal system for X maltophilia, which provided a clue to study the signal transduction in / maltophilia.Finally, we constructed the genomic DNA library of X maltophilia, and screened the gene encoding CG-like receptor of X maltophilia from it. The genomic DNA of X maltophilia was partially digested by Sau 3A I and Pst I respectively, the 1 ~ 5 kb and 0.5~7 kb DNA fragment were purified from agarose gel electrophoresis. The vector pUC18 was digested by Bam H I and Pst I respectively, and was treated with CIAP. Then the treated pUC18/Bam H I and pUC18/pst I were ligated with the previous purified DNA fragment respectively. The two kinds of recombinant DNA molecules were transformed to the competent E coli JM109, and there were obtained about 4200 whi...
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