人受精蛋白β整联蛋白配体区cDNA的克隆、表达及抗体制备  

Cloning, Expression and Antibody Production of the Disintegrin Domain of Human Fertilin β

作　　者：丁蓓蓓[1] 沈卫英[1] 蒋俶[1] 王健[1] 高尔生[1] 申庆祥[1] 

机构地区：[1]上海市计划生育科学研究所国家计划生育药具重点实验室,上海200032

出　　处：《生物化学与生物物理学报》2001年第3期303-308,共6页

基　　金：CONRAD/Mellon Foundation资助项目!(MFG 93 2 3)&&

摘　　要：从人睾丸中抽提mRNA ,合成双链cDNA ,利用合成的PCR引物扩增受精蛋白 β(fertilinβ)的整联蛋白配体区cDNA(hf2 79)。序列分析表明 ,该区编码 93个氨基酸 ,与文献报道完全相同。将hf2 79插入质粒 pGEX 4T 2 ,构建 pGEX hf2 79表达质粒 ,转化大肠杆菌BL2 1(DE3) ,表达菌株经IPTG诱导 ,可产生大量可溶性的表达蛋白GST HF93。SDS PAGE分析表明融合蛋白表观分子量为 38kD ,其含量占菌体可溶性蛋白的 5 0 %以上。表达产物经谷胱甘肽转硫酶 (GST)亲和层析柱纯化 ,得到 90 %以上纯度的融合蛋白。融合蛋白经凝血酶酶切 2h可得HF93肽。再经GST亲和层析柱去除GST ,得到纯度大于 80 %的HF93肽。将其和SDS PAGE凝胶上切下的HF93多肽条带一起用于免疫BALB/c小鼠 ,经ELISA检测 。A cDNA for the disintegrin domain ( hf 279) was isolated by PCR from human testis cDNAs. DNA sequencing indicated that hf 279 cDNA encoded 93 amino acid residues, and it was identical with the reported sequence of fertilin β. An expression plasmid, pGEX hf 279, was constructed by inserting hf 279 cDNA into plasmid pGEX 4T 2 containing gst gene. The expression plasmid was introduced into E.coli BL21(DE3) cells and a substantial amount of soluble fused protein GST HF93 was obtained by the expression strain HF93/BL21 induced with IPTG. SDS PAGE analysis revealed that the GST HF93 fusion protein had an apparent molecular weight of 38 kD and accumulated up to 50% of bacterial soluble proteins. The fusion protein was purified by glutathione S transferase (GST) Sepharose 4B column (purity>90%) and digested by thrombin to obtain the purified HF93 peptide (purity>80%). Polyclonal antibodies were obtained from the serum of mice immunized with purified HF93 which was isolated by GST Sepharose 4B column and SDS PAGE. ELISA and Western blot analysis showed its specificity to HF93. Therefore this antibody can be used in further studies on the function of HF93.

关 键 词：人受精蛋白β整联蛋白配体区 基因克隆 基因表达 大肠杆菌 抗体 

分 类 号：Q78[生物学—分子生物学]