α-银环蛇毒素基因的克隆与表达

Gene cloning and expression of α-bungarotoxin

与传统的捕蛇或养蛇提毒的方法相比,利用基因工程方法生产蛇神经毒素具有明显的潜在优势。本研究结合国内外的研究现状,以α-银环蛇毒素(α-bungarotoxin,α-BgTx)为例子来探讨蛇神经毒素基因在大肠杆菌表达体系中的表达情况及其规模生产的可行性。首先根据文献报道α-银环蛇毒素的氨基酸序列,推导出其DNA序列并设计成部分互补的4条寡核苷酸片段,利用DNA合成仪人工合成、纯化其寡核苷酸片段,通过片段退火、切口补平、连接、克隆、测序鉴定获得了α-银环蛇毒素基因;然后将α-银环蛇毒素基因克隆至pGEX-2T质粒中分别转化大肠杆菌DH5(和JM109进行表达分析,融合蛋白约占细菌总蛋白的30％～40％,其中部分为可溶性表达,部分以包含体的形式表达;对可溶性表达的条件进行了优化。最后以天然纯化的α-银环蛇毒素作为对照,分析融合方式表达的重组α-银环蛇毒素的活性,ELISA结果显示其与天然的α-银环蛇毒素比较具有相似的抗原性。

The genetical engineering methods to produce the snake neurotoxm obviously have potential advantages over the traditional methods of extracting neurotoxin from snakes. This paper was to study the expression and ine feasibility of scale production of snake neurotoxin in the routine expression systems such as E. coli with the α-bungarotox-in as an example. Firstly, on the basis of the reported ammo acid sequence of α-bungarotoxm, DNA sequence of α-bungarotoxin was deduced and four partial complementary oligonucleotide fragments were designed. The coding region of α-bungaroloxin was obtained by renaturing the DNA fragments, nick filling-in, ligation and PCR. The coding region of α-bungarotoxin was cloned into plasmids pGEX-2T, named as pDZ04, and transformed E.coil BL21 to study the expression of α-bungarotoxm gene. The results of SDS-PAGE analysis showed that the expression level of the re-combinant plasmid pDZ04 could express efficiently in BL21, the fusion protein took up about 30%-40% of bacterial total protein. Finally, The biological activity of the recombinant α-bungarotoxm and the fusion protein was studied with the natural α-bungarotoxin purified from the snake venom as control, ELISA results showed that they have the similar antigenicity.