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

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

Ciudad Autónoma de Buenos Aires

Jornada; Jornadas de Jóvenes Investigadores; 2018

Facultad de Veterinaria

Organisms of the genus ?Candidatus Liberibacter?, all vectored by psyllids, are generally recognized 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 the Asian citrus psyllid Diaphorina citri. During insect feeding, the bacteria are introduced into the phloem and colonize sieve tubes; though they eventually develop severe chlorosis and die, infected trees may remain asymptomatic for many years. After the onset of the symptoms (smaller, deformed fruit with uneven coloration, leaves and shoots develop yellow patches and branch dieback), citrus producers discard the plants and need to re-plant the affected area. Further complicating the issue, research on the genus is extremely difficult, for none of the HLB pathogens can be grown in culture. Even though the disease still hasn‟t reached the Argentine lemon producers, the development of both research and diagnostic tools are of the utmost importance to be able to stay ahead of the disease. In this context, our objective was to develop immunological tools that will allow the study and early diagnosis of HLB. To this end we selected, expressed and purified three proteins from CLas; once the proteins were obtained, we immunized a single lama every two weeks, for two and a half months. After that, an ELISA using the sera corresponding to the pre-immune condition, as well as the 4th and 5th immunizations was done. Three days after the last immunization, we purified lymphocytes from full blood, extracted and retrotranscribed RNA. 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 as template was done to further amplify the VHH segments and the PCR product was ligated into a phagemid vector. TG1 bacteria were transformed and colonies expressing the VHH were obtained. Following the production, purification and immunization of the selected antigens, elevated antibody titers were attained, this together with the results of retro-transcription, PCRs and transformation, allowed us to conclude that the lama was correctly immunized and the library was successfully built.