Analysis of genes of C-type lectin domain-containing proteins with useful biological activities.

许多含有c型凝集素样结构域的蛋白质是从蛇毒中分离出来的。它们表达了多种多样的生物活动。IX/X-bp是从哈布蛇（Trimeresurus flavviridis）毒液中分离出来的异二聚体蛋白之一，由两个c型凝集素样结构域组成。两个c型凝集素样结构域被交换。在钙离子存在的情况下，该蛋白与凝血因子IX和X结合，抑制凝血级联反应。IX/X-bp的互补DNA克隆表明，IX/X-bp的每条链都由各自的基因编码。蛇毒c型凝集素样蛋白的基因结构尚未见报道。在本研究中，我们克隆了IX/X-bp A链编码基因。从黄病毒Trimeresurus的血液中制备总基因组DNA，用Sau3Al消化部分基因组DNA。使用BamHl预消化的Lambda EMBL3克隆载体构建基因组文库。培养含有IX/X-bp A链cDNA质粒的大肠杆菌克隆并制备质粒。用Sall和Notl酶切将插入的DNA从质粒上切下，用DIG标记作为探针筛选基因组文库。从基因组文库中分离到1个编码IX/X-bp A链的基因克隆，采用双脱氧核苷酸链终止法确定其核苷酸序列。IX/X-bp A链的基因结构不同于许多脊椎动物c型凝集素结构域，如asialalglyprotein receptor和lgE Fc ε receptor，它们的蛋白质结构与IX/X-bp非常相似，但Drickamer将IX/X-bp分类在VII类以外的组中。其中一个内含子插入到铰链区，提示内含子插入对结构域交换机制的贡献。

Many proteins that contain C-type lectin-like domains are isolated from snake venom. They express a great variety of biological activities. IX/X-bp is one of the heterodimeric proteins isolated from the venom of habu snake (Trimeresurus flavoviridis) consisting of two C-type lectin-like domains. Two C-type lectin-like domains are swapped. This protein binds to blood coagulation factors IX and X in the presence of Ca ions and inhibits the blood coagulation cascade. Complementary DNA cloning of IX/X-bp indicated that each chain of IX/X-bp is encoded by respective gene.. Gene structure of snake venom C-type lectin-like protein is not yet reported. In the present study, we cloned the gene encoding IX/X-bp A chain. Total genomic DNA was prepared from the blood of Trimeresurus flavoviridis, and partially digested with Sau3Al. A genomic library was constructed using a Lambda EMBL3 cloning vector predigested by BamHl according to the manufacture's protocol. E. coli clone containing IX/X-bp A chain cDNA plasmid was cultured and the plasmid was prepared. Inserted DNA was cleaved off from the plasmid by the digestion with Sall and Notl and labeled with DIG to use as probe to screen the genomic library. One gene clone encoding the IX/X-bp A chain was isolated from the genomic library and the nucleotide sequence was determined by the dideoxynucleotide chain-termination method. The gene structure of IX/X-bp A chain is different from that of many vertebrate C-type lectin domains such as asialoglycoprotein receptors and lgE Fc ε receptor whose protein structures are very similar to that of IX/X-bp but are classified in the groups other than group VII by Drickamer. One of introns is inserted in the hinge region suggesting the contribution of the intron insertion to the domain swapping mechanism.