Screening Of Ligand Of CT813by Yeast Two-hybrid Technique

Chlamydia trachomatis（CT）is obligate intracellular parasites ofeukaryotic cell with a unique biphasic life cycle. The development cycleis EB→RB→EB, EB has a weak metabolic capability with srtonginfection, RB has a strong metabolic capability with no infection.Chlamydial infections in humans cause severe health problems, includingblinding trachoma and sexually transmitted diseases. It can also infringeinguinal lymph nodes and cause suppurative lymphadenitis or chroniclymphogranuloma venereum.Chlamydia trachomatis’ biosynthesis and assembly are conducted inthe inclusion body，Inclusion not only provides microenvironment forchlamydia trachomatis replication, but also protects duplicate CT fromthe host’s immune system to be recognized and eliminated. And CT mustabsorb the nutrition, metabolic intermediates and transform of signalsfrom the host cell via inclusion membrane. Inclusion membrane proteinscoded mainly by CT gene are the main component of inclusion membrane(inclusion membrane, Inc). Since1995, Rochey confirmedexperimentally for the first time that Chlamydia transports its own genesencoding proteins to inclusion membrane and then modifies the Inc. Withthe development of molecular biology technology, new inclusionsmembrane proteins have been constantly identified. The CT’s genome hasbeen sequenced. According to the properties of hydrophobic membraneproteins that have been identified, a computer aided program haspredicted36CT gene encoding proteins as candidates for inclusionmembrane proteins. CT813has been discovered and proven to be aninclusion membrane protein, but its biological function and role in the process of CT pathogenicity is still unclear. Therefore we use the yeasttwo-hybrid technique to search for the proteins that interact with CT813,for further in-depth study of the biological characteristics of CT813andexpounding important information of pathogenic mechanism ofChlamydia trachomatis.Frist, the sequencing primers were designed to clone the target gene(CT813) according to stdgen gene bank.Then the genes of CT813wereamplified from the genomic DNA of CT D by PCR, and PCR productswere digested by Restriction Endonucleases Sma I and Pst Ⅰ,theninserted into appropriate site of bait vector pGBKT7, then linked with T4ligase,the recombinants were transformed into competent cell (BL-21).These recombinant plasmids were transformed into yeast cells AH109and Y187respectively, after identification, neither had the ability ofself-activation or yeast cell toxicity.The yeast two-hybrid assays were performed with the HeLa cDNAlibrary and the bait vector pGBKT7-CT813. After positive recombinantyeast strain AH109compounding with Y187from cDNA library andclover or Mickey shaped zygote appearing, the compound will be coatedon SD/-Ade/-His/-Leu/-Trp with X-Gal for preliminary screening. Screenpositive colonies by detecting β-galactosidase activity and colony PCR,then the results were confirmed with backcross experiment. Plasmidssequencing and BLAST searching homologous proteins showed that:pGBKT7-CT813can interact with multiple gene-transcribe products ininteraction assays. The candidate proteins interacting with CT813screened by yeast two-hybrid technique include Galectin-1(LGALS1),cAMP response element binding protein-3(CREB3), ribosomal proteinL10a (RPL10a) and tubulin37E-16(RP1-37E16).