blake

					Chlamydial inclusion membrane
   proteins: localization and
        characterization

          Nathanael Blake
       HHMI Summer Internship
       Mentor: Dr. Dan Rockey
              The Chlamydia
The Chlamydia are an group of ubiquitous intracellular
pathogens distinguished by a unique biphasic lifecycle.
    Chlamydia trachomatis
Two sites of infection: ocular and
genital.
  Ocular strains cause several million cases of
  blindness each year, mostly in poor nations.
  Genital strains common in Western nations.
    4 to 5 million cases per year in the US.
     Chlamydia pneumoniae
Infects the lungs.
  Ubiquitous, majority of humans are infected.
  All effects of disease not known.
     Asthma, chronic bronchitis?
Also, it has recently been linked to heart
disease and atherosclerosis.
        Chlamydophila caviae
Infects guinea pigs.
    Good animal model from which to extrapolate
    about C. trachomatis in humans.
                  Treatments
No vaccine.
Laboratory tests required to confirm infection.
  Rockefeller Foundation offered 1 million dollars for
  simple test for C. trachomatis.
     Closed after five years because no one succeeded.
Most infections asymptomatic.
Antibiotics easily cure almost all cases.
The Chlamydial Lifecycle
The Chlamydial inclusion
      membrane
               Proteins to be studied




I’m are seeking to confirm that these are localized to the inclusion membrane
and to examine their interactions with human proteins.
Hydrophilicity plots provide evidence that these proteins are
localized to the inclusion membrane.




            GPIC 425




                                                GPIC 426
Primers were designed for Ct 58, CWL 369, GPIC 425, and GPIC 426. They were ordered from Sigma-
Genosys and used to amplify portions of these genes from genomic DNA via PCR. Gel electrophoresis
was used to determine that they had worked and fragments corresponding to the size of the target
sequence were extracted from the gel with a QIAGEN gel extraction kit.

   Ct58    CWL369      Ladder                            GPIC425 Ladder GPIC426
The expression vector
              PCR screen of transformed E. coli colonies
                       426   425       369       58




I ligated the digested plasmid (P-Mal C2) with the targeted gene fragments
and then transformed E. coli with the result. I screened for successful
transformation with LB Ampicillin plates and then ran a PCR screen on the
colonies. True positives were found for all but GPIC 426.
I then induced 58, 425, and 369 and harvested the protein.
While 58 yielded very little product, 425 and 369 provided
useable quantities.
                 Lad      425      369




                       Current Status
 1. 425 and 369 are being sent off for antibody production.
 2. I’m working on inducing 58.
 3. 426 has not yet been transformed, despite repeated
    attempts.
                       Moving
We left Nash and
Microbiology for Dryden and
Vet Med.
          Continuing Research
1. Once 369 and 425 antibodies are received, I’ll use
fluorescent microscopy to determine their localization.
2. I will continue in my attempts to troubleshoot the
transformation of E. coli with 426 and the induction process for
58.
3. Finally, I will begin work on two-hybrid analysis of these
proteins to examine their interactions with human proteins.


This work will be carried on through the school year, via the
undergraduate research program.
                     Thanks to
HHMI.


Dan Rockey


Everyone in the Rockey lab.

				
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posted:12/2/2011
language:English
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