GENERATION OF A LAMA GLAMA IMMUNE LIBRARY AGAINST ANTIGENS OF CANDIDATUS LIBERIBACTER ASIATICUS, THE HLB PATHOGEN

SPERAT W; GOMEZ JM; TORRES P; BIANCO I; GUDESBLAT GE; VOJNOV A; GONZALEZ C; IBAÑEZ LI

Ciudad de Buenos Aires

Jornada; Jornadas de Jóvenes investigadores; 2018

Facultad de veterinaria UBA

Organisms of the genus ?Candidatus Liberibacter?, all vectored by psyllids, are generallyrecognized as the cause of four serious plant diseases: HuangLongBing (HLB), Zebra Chip,Psyllid Yellows and Yellows Decline, which currently threaten and destroy the citrus,potato, tomato and carrot industries, respectively. Candidatus Liberibacter asiaticus (CLas)is one of the three etiological agents of the citrus HLB disease and is transmitted by theAsian citrus psyllid Diaphorina citri. During insect feeding, the bacteria are introduced intothe phloem and colonize sieve tubes; though they eventually develop severe chlorosis anddie, infected trees may remain asymptomatic for many years. After the onset of thesymptoms (smaller, deformed fruit with uneven coloration, leaves and shoots developyellow patches and branch dieback), citrus producers discard the plants and need to re-plantthe affected area. Further complicating the issue, research on the genus is extremelydifficult, for none of the HLB pathogens can be grown in culture. Even though the diseasestill hasn‟t reached the Argentine lemon producers, the development of both research anddiagnostic tools are of the utmost importance to be able to stay ahead of the disease. In thiscontext, our objective was to develop immunological tools that will allow the study andearly diagnosis of HLB. To this end we selected, expressed and purified three proteins fromCLas; once the proteins were obtained, we immunized a single lama every two weeks, fortwo and a half months. After that, an ELISA using the sera corresponding to the preimmune condition, as well as the 4th and 5th immunizations was done. Three days after thelast immunization, we purified lymphocytes from full blood, extracted and retrotranscribedRNA. We subsequently amplified all the VH genes and purified the smallest band (≈700bp),which corresponds to the VHH antibodies. A second PCR using the purified DNA astemplate was done to further amplify the VHH segments and the PCR product was ligatedinto a phagemid vector. TG1 bacteria were transformed and colonies expressing the VHHwere obtained. Following the production, purification and immunization of the selectedantigens, elevated antibody titers were attained, this together with the results of retrotranscription, PCRs and transformation, allowed us to conclude that the lama was correctly immunized and the library was successfully built.