Preparation of monoclonal antibodies against gamma-type phospholipase A2 inhibitors and immunodetection of these proteins in snake blood

Abstract

BackgroundThe gamma-type phospholipase A2 inhibitor (PLIγ) is a natural protein commonly found in snake serum, which can neutralize pathophysiological effects of snake venom phospholipases A2. Therefore, this protein is a potential candidate to the development of a novel antivenom. To the best of our knowledge, there is no antibody currently available for PLIγ identification and characterization.MethodsBioinformatics prediction of epitope using DNAStar software was performed based on the sequence of Sinonatrix annularis PLIγ (SaPLIγ). The best epitope 151CPVLRLSNRTHEANRNDLIKVA172 was chosen and synthesized, and then conjugated to keyhole limpet hemocyanin and bovine serum albumin for use as an immunogen and plate-coating antigen, respectively.ResultsEighteen IgG anti-PLIγ mAb hybridoma cell strains were obtained, and all the mAbs had positive interaction with recombinant His6-PLIγ and natural SaPLIγ. Moreover, the mAb from 10E9 strain was also successfully used for the immunodetection of other snake serum PLIγs. cDNA sequence alignment of those PLIγs from different snake species showed that their epitope segments were highly homologous.ConclusionsThe successful preparation of anti-PLIγmAb is significant for further investigation on the relationship between the structure and function of PLIγs, as well as the interaction between PLIγs and PLA2s.

Identification and characterization of the first endogenous phospholipase A2 inhibitor from a non-venomous tropical snake, Boa constrictor (Serpentes: Boidae)

“…However, another sbγPLInamed PIP for phospholipase A 2 inhibitor from Python -was soon described in the non-venomous and non-ophiophagus species Malayopython reticulatus (formerly Python reticulatus) [8]. Since then, a number of sbγPLIs were detected in colubrid from Asia: Dinodon rufozanatum [5], Elaphe carinata [5], E. climacophora [7], E. rufodorsata [5], E. teniura, Macropisthodon rudis [9], Synonatrix annularis [4], and Zaocys dhumnades [5], in addition to xenodermatid Achalinus rufescens [5]. A structurallyrelated PIP homolog was also described in the non-venomous rock python (P. sebae) from Africa, although with poor PLA 2 inhibition activity [23].…”

“…According to the presence of known domains from mammal proteins -C-type lectin-like, tandem leucine-rich repeats (LRRs), or three-finger motifs -sbPLIs were grouped into alpha (α), beta (β) or gamma (γ) structural classes, respectively [3]. Comparable inhibitors were later identified in a number of non-venomous species [4,5,6,7,8,9]. Whether venomous or not, some snake species express sbPLIs belonging to up to three different structural classes simultaneously [3,7,10,11].…”

“…SbγPLIs are the most widely distributed inhibitors among elapid and viperid species from the Old and New World [12,13,14]. Concerning non-venomous snakes, as far as we know, until now sbγPLIs were solely purified from Asian species [4,5,6,7,8,9]. With that in mind, we investigated the presence of this kind of inhibitor in Boa constrictor -a non-venomous tropical snake -popularly known as jiboia.…”