Abstract
(Englisch)
|
The major objective of this project was the generation of an expression library system to screen with sera from naturally and experimentally infected dogs. The purpose of this is the identification of antigens, which will be recognized very early by sera from infected dogs. However, this antigen, as well as the complete library can be tested for potential protective vaccine candidates.WE HAVE NOW GENERATED CDNA LIBRARIES FROM CULTURED L. INFANTUM PROMASTIGOTES, WHICH HAVE BEEN TESTED PRIOR TO RNA ISOLATION FOR INFECTIVITY TO HAMSTERS. LIBRARIES WERE CONSTRUCTED IN PUC-VECTORS AND PHAGE LAMBDA GT11 BY CONVENTIONAL CDNA SYNTHESIS AS WELL AS BY USE OF SPLICED LEADER SEQUENCE PRIMERS. BOTH LIBRARIES WERE TESTED FOR INSERTS,FOR COMPLEXITY, AND FOR EXPRESSION BY USING RANDOMLY PICKED CLONES.
INITIAL SCREENS WITH POOLED DOG SERA YIELDED HIGH BACKGROUND AND UNSPECIFIC BINDING. A SECOND SCREEN WITH INTENSIVELY PRE-ABSORBED SERA ON BACTERIAL LYSATE YIELDED 20 CLONES. ALL TWENTY CLONES REMAINED POSITIVE AFTER THE SECOND AND THIRD SCREEN. SEQUENCING OF ALL CLONES REVEALED THE IDENTIDY OF ALL CLONES AND THE HISTONE 2A SEQUENCE. SINCE ALL CLONES WERE UNEQUIVOCALLY IDENTIFIED BY INFECTED DOG SERA AND WERE NOT RECOGNIZED BY UNINFECTED DOGS, WE PROCEEDED AND SUBCLONED THE INSERTS INTO THE PQE SYSTEM TO ADD A 6XHIS-TAG FOR IMPROVED PURIFICATION. WE ARE NOW CURRENTLY EXPRESSING THIS ANTIGEN FOR DETAILED ANALYSIS WITH SUBSEQUENT SERA FROM EXPERIMENTALLY INFECTED DOGS TO DETERMINE THE FIRST APPEARANCE OF ANTI-H2A-ANTIBODIES. SIMULTANOUSLY, WE ARE FURTHER SCREENING THE LIBRARIES FOR ADDITIONAL ANTIGENS.
IN ORDER TO IDENTIFY RELEVANT ANTIGENS FOR THE DEVELOPMENT OF CANINE VACCINES, WE APPLIED ANOTHER APPROACH. UNDER THE ASUMPTION THAT LONG TERM CULTURED LEISHMANIA STRAINS WHICH ARE NOT ABLE ANYMORE TO INFECT HAMSTERS LACK ESSENTIAL PROTEINS, WHICH MIGHT DETERMINE VIRULENCE, WE SUBSTRACTED MRNA FROM A LEISHMANIA INFANTUM STRAIN WHICH RAPIDLY INFECTS HAMSTERS, AND FROM A STRAIN, WHICH DOES NOT INFECT HAMSTERS. FOR THIS APPROACH, WE HAVE USED THE CLONTECH PCR-SELECT CDNA SUBSTRACTION KIT, WHICH YIELDED A FRAGMENTED LIBRARY, OF WHICH WE CURRENTLY SCREEN THE OBTAINED CLONES BY SEQUENCING, SEQUENCE HOMOLOGY SEARCHES, AND PLAN TO RECLONE THESE INSERTS INTO AN EXPRESSION SYSTEM FOR ANTIBODY SCREENING.
IN ORDER TO UNEQUIVOCALLY IDENTIFY LEISHMANIA PARASITES, WE ALSO DEVELOPED A RAPID GENOTYPING SYSTEM BASED ON PCR AMPLIFICATION AND SUBSEQUENT RFLP-ANALYSIS OF AN UNDEFINED REPETITIVE SEQUENCE, AND THE MINI-EXON SEQUENCE. WITH THE LATTER STRATEGY, WE ARE ABLE TO IDENTIFY BOTH NEW WORLD AND OLD WORLD LEISHMANIA. THIS TYPING SCHEME IS CURRENTLY WRITTEN UP FOR PUBLICATION.
|
