Ⅱ型志贺毒素单克隆抗体可变区基因克隆及单链抗体的表达和功能鉴定

目的从分泌抗肠出血性大肠埃希菌Ⅱ型志贺毒素中和单克隆抗体杂交瘤细胞株S2C4中克隆抗体可变区基因，构建单链抗体（ScFv），进行原核表达，并对其功能进行鉴定。方法从杂交瘤细胞株S2C4中提取总RNA，逆转录成cDNA。在cDNA3’-OH末端添加poly．G。PCR扩增包括5’非翻译区和信号肽序列在内的抗体重、轻链可变区基因VH和VL,PCR产物装入T—A载体测序。根据测序结果，设计引物分别扩增VH和VL编码区，再通过重叠PCR，在VH和VL．编码区基因之间引入连接链，构建Scn基因，并克隆到表达载体pComb3xSS中。重组载体导入E．coliTop10F’进行表达，重组蛋白经纯化后，分别用ELISA和动物保护性实验鉴定其生物学活性。结果VH和VL编码区基因全长分别为396bp和378bp，ScFv基因能在大肠埃希菌中高效表达，表达产物的分子量为34000，用NiSO4亲和层析柱成功纯化。功能性实验表明纯化的重组蛋白可以与Stx2毒素有效结合，能保护动物抵御毒素分子的攻击。结论成功地克隆S2C4单抗可变区基因，并构建、表达其单链抗体ScFv，为下一步进行该抗体人源化奠定实验基础。

Cloning Variable Region Gene Against Shiga Toxin 2 （Stx2） and Preparation and Functional Characterization of Stx2 Single Chain Antibody

Objective To clone and contruct the variable region genes from the neutralizing antibody-secreted hybridoma S2C4 against Enterohemorrhagic Eseherichia coli Shiga toxin 2 （Stx2） , prokaryotically express and functionally characterize Stx2 single chain antibody （ScFv） . Methods Total RNA was extracted from hybridoma cell line S2C4, and used to reversely transcribe eDNA. 3＇ end of cDNA was extended by a poly-G-tail using Terminal Deoxynucleotidyl Transferase. Tailed cDNA was used as template to amplify variable region genes, which included 5＇ UT, signal peptide and mature variable region encoded sequences. PCR product was T-A cloned for sequencing. The mature variable region was amplified by PCR using a combination of 5＇ gene specific primers and 3＇ primers, which matched the constant K and CH1 domains, respectively. VK and VH were fused by peptide linker using an overlap PCR to construct the ScFv gene. The ScFv gene was inserted into pComb3xSS vector and expressed in E. coli Top10F＇ . After purified by nickel ion affinity chromatography, the functional activity of the recombinant protein was evaluated by ELISA and murine toxin neutralization assay. Results The mature VH and VK encoded sequences contained 396 bp and 378 bp, respectively. ScFv was efficiently expressed in E. coli with a molecular weight of 34 000. The ScFv protein was successfully purified to homogeneity by affinity chromatography scheme. It had specific antigen binding activity in ELISA and also protected mice against lethal doses of Stx2 challenge. Conclusion The variable region genes of S2C4 monoclonal antibody were cloned. Stx2 single chain antibody format was constructed and expressed, thus facilitating future humanization research.