Gene expression analysis of russell's viper venom and molecular cloning, expression and functional study of RV factor X activator

Abstract

Russell's viper, Daboia russelli, is a venomous snake widely distributed in several parts of Asia. RV bites from different geographical areas cause variety of signs and symptoms with severe injury or even death in human. This study was aim at (1) characterization the toxin composition of Thai RV venom by Expressed Sequence Tags (ESTs) analysis, (2) utilization of the medical useful toxin by DNA recombinant technology, and (3) production of toxin-specific antibody by DNA immunization. ESTs are an important and rapid tool to characterize the pattern of gene expression of organism. cDNA library was constructed from the snake venom glands to study the active genes in the venom. A total 135 ESTs have been generated from size-selected clones at random. Fifty-seven percents (77/135) of ESTs were significant matching to the entries in the database. Of these, several ESTs involved in the toxicity of the venom were obtained, e.g. phospholipase A2, factor X activator and factor V activator, BPP-CNP homolog, etc. Some of cDNA sequences of these genes showed a variation from the other reports from RV from other region. These ESTs data could provide a resource of the expressed pattern relating to the clinical pathology of RV bite. In the mean time, factor X activator (RVV-X), one of RV key toxin, was cloned, characterized, and expressed in Pichia pastoris. Northern analysis indicated that the mRNA of RVV-X heavy chain and light chain were about 2.5 kb and 1.0 kb, respectively. However, the expression of RVV-X was unappreciated in Pichia yeast. Only heterologous RVV-X light chain could be seen in SDS-PAGE while RVV-X heavy chain could not be detected. RVV-X light chain could also be detected by anti-His (C-term) antibody and RVV-immunized horse antibodies. Moreover, to generate toxin-specific antibody, RVV-X was cloned into pVaxSec mammalian expression vector for DNA immunization in mice. RVV-X disintegrin domain showed to be a good DNA immunogen in antibody production in mice while RVV-X with other domains gave unsatisfied results. Antibody from RVV-X disintegrin domain-immunized mouse serum could bind to the RVV-X in both reduced and non-reduced forms. In summary, this work has identified the Thai RV venom composition by characterization the RV transcripts and could produce heterologous RVV-X light chain in Pichia pastoris. In addition, RVV-X-specific antibody was successfully produced by DNA immunization in mice which may be developed for the large-scale antibody production in other mammals