【作者】 林鲁萍；

【导师】 王义权；

【作者基本信息】 厦门大学 ， 生物化学与分子生物学， 2006， 硕士

【摘要】 本研究利用前期研究中构建的pGEX-BgTX(P22-A31)质粒在大肠杆菌BL21(DE3)中表达并纯化后得到了较纯的重组α-银环蛇毒素同工毒素,得率约为1.225 mg/L。用重组蛋白与天然α-银环蛇毒素的抗原性一致。重组α-银环蛇毒素同工毒素的半致死剂量LD50为1.598 mg/kg,约为天然α-银环蛇毒素的1/5且有一定的镇痛药效,1/4 LD50剂量的镇痛百分率为55.2%,1/8 LD50剂量的镇痛百分率为20.5%,在外周镇痛作用中呈一定的量效关系。以上结果表明,本研究重组表达的α-银环蛇毒素同工毒素(P22-A31)具有与天然α-银环蛇毒素相似的生物学活性。α-银环蛇毒素cDNA序列的多态性及其引起这种多态性的原因一直存在争议。本文从同一银环蛇个体中克隆并测序了5个α-银环蛇毒素基因组序列,又在同一次反转录的cDNA中克隆神经生长因子的cDNA,并通过计算多个克隆间的突变情况,与同一体系中得到的12种α-银环蛇毒素cDNA序列突变率做比较,并统计了前人克隆到的多种α-银环蛇毒素cDNA序列突变的情况,得出结论:α-银环蛇毒素cDNA序列的多态性不是由于基因组不同而来的,也不是由RNA编辑造成,很可能是由于反转录过程,PCR,基因克隆及DNA测序过程人为引入的。运用RACE技术从银环蛇毒腺cDNA中克隆到2个C型凝集素基因的全长cDNA序列,BML-1和BML-2,其中BML-2是首次从银环蛇(Bungarus multicinctus)中克隆到。并将这2个蛋白135个氨基酸的成熟肽部分和137个氨基酸的成熟肽部分分别连入pET-His载体后在BL21(DE3)plysS中表达,重组蛋白以包涵体的形式存在,复性后的重组C型凝集素具有凝集红细胞活性。其中由135个氨基酸组成的重组C型凝集素以单体形式存在,由137个氨基酸组成的重组C型凝集素有50%以二聚体的形式存在,另50%以单体形式存在。Western blotting和亲和纯化结果表明,天然C型凝集素是以更小分子量的单体形成的二聚体。纯化到的4种分子量不同的蛋白中14.4KD左右的蛋白可以形成二聚体,认为是目的蛋白。银环蛇C型凝集素基因的克隆和表达研究对对阐明银更多还原

【Abstract】 α-bungarotoxin plays very important role in neuroscience research, clinical application and the pharmaceutical industry. In order to acquire quantites ofα-bungarotoxin, we expressed GST-α-bungarotoxin fusion protein using constructed plasmid pGEX-BgTX (P22-A31) in E. coli BL21 (DE3) cell and finally obtained recombinantα-bungarotoxin with yields of about 1.225 mg/L. The results of both ELISA and Western blot showed that recombinantα-bungarotoxin has the same antigenicity as naturalα-bungarotoxin. In vivo toxicity tests showed that the LD50 of recombinantα-bungarotoxin was 1.598 mg/kg, about 1/5 that of naturalα-bungarotoxin. Analgesis percentages with doses of 1/4 LD50 and 1/8 LD50 were 55.2% and 20.5% respectively, indicating that recombinantα-bungarotoxin possesses analgesic efficiency.It is disputed whether there is polymorphism in cDNA ofα-bungarotoxin, and what is the mechanism to result of this phenomenon. In order to further study this question, we cloned and sequencedα-bungarotoxin gene from the same individual and nerve growth factor cDNA from the same reverse transcription products by Wang et al, and analyzed their mutation rates. Those results indicate that polymorphism ofα-bungarotoxin cDNA is not transcripted from genomic DNA or RNA editing, but results from reverse transcription process, PCR, and gene cloning.C-type lectins are found in many animals and bind in a Ca2+-dependent fashion to mono- and oligosaccharides. In our research, we cloned two C-type lectin full-length cDNA from Bungarus multicinctus venom gland by RACE technology, named BML-1, BML-2. It is the first report to clone BML-2 cDNA from venom gland of Bungarus multicinctus. We also constructed expression vector using 135 and 137 amino acids of two C-type lectins ligated into pET-His plasmid and then expressed更多还原