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我们用 Pharmacia公司的重组噬菌体抗体系统的组合方法制出一种对响尾蛇毒素特异的、具有高亲和力的单克隆单链抗体可变区 (sc Fv)。筛选出一个高亲和力的克隆 ,编号为 A1 0 G,对其 DNA进行了测序。A1 0 G蛋白显示出高的反应特异性 ,只与响尾蛇神经毒素、concolor毒素和 Mojave毒素有密切的关系。用第二组磷脂酶 A2 s (PLA2 s)筛选 ,与 1 1种显示出交叉反应。第一组磷脂酶 A2 s (PLA2 s)在 ELISA中有交叉反应。编码A1 0 G的基因被亚克隆到一个表达载体 ,结果表达非融合蛋白 ,标记为 A1 0 GPE,复性、纯化至表面均一性。A1 0 G与完整响尾蛇毒素和响尾蛇毒素基本亚单位的解离常数分别为 7× 1 0 - 1 0 M和 6.8× 1 0 - 9M。当 A1 0 GPE与响尾蛇毒素基本亚单位或响尾蛇全毒素以摩尔比 5 :1预先孵育 ,则没有抑制磷脂酶活性现象。然而 ,表达蛋白可以在小鼠中部分中和响尾蛇毒素同系物 -Mojave毒素的致死率
We used a combination of Pharmacia’s recombinant phage antibody system to create a monoclonal scFv that is specific for crotoxin and has high affinity. A high-affinity clone was screened, designated A1 0 G, and its DNA sequenced. The A1 0 G protein showed high reaction specificity and was only closely related to rattlesnake neurotoxin, concolor toxin and Mojave toxin. The second group of phospholipase A2 s (PLA2 s) screening, and 11 species showed cross-reaction. The first group of phospholipase A2s (PLA2s) cross-reacted in the ELISA. The gene encoding A1 0 G was subcloned into an expression vector and the result was expressed as a non-fusion protein, labeled A1 0 GPE, refolded and purified to homogeneity of the surface. The dissociation constants of A1 0 G and the intact crotoxin and crotoxin basic subunits were 7 × 10 ~ 10 M and 6.8 × 10 ~ 9 M, respectively. Phospholipase activity was not inhibited when A1 0 GPE was pre-incubated with either crotoxin basic subunit or rattlesnake whole toxin at a molar ratio of 5: 1. However, the expression of the protein partially neutralized the lethality of the mite toxin homolog, Mojave toxin, in mice