抗内毒素单链抗体基因的构建、序列分析及表达

目的：构建抗内毒素（ＬＰＳ） 单链抗体基因， 并尝试其在Ｅ．ｃｏｌｉ 中的表达。方法： 采用ｌｉｎｋｅｒ Ｐｒｉｍ ｅｒ Ｍｉｘ ，按ＶＨｌｉｎｋｅｒＶＬ 的结构将鼠抗ＬＰＳ ｍ Ａｂ Ｃ３Ａ２ 的ＶＨ ，ＶＬ 基因拼接成单链抗体（ＳｃＦｖ） 基因；用ＰＥ３７３Ａ 型全自动ＤＮＡ序列分析仪测定其核苷酸序列。ＰＣＲ 扩增抗ＬＰＳ ＳｃＦｖ 基因并更换两端接头序列后，插入谷胱甘肽巯基转移酶（ＧＳＴ）融合表达载体ｐＧＥＸ４Ｔ１ ；转染Ｅ．ｃｏｌｉ ＪＭ１０９ ，以ＩＰＴＧ 诱导表达，ＳＤＳＰＡＧＥ 分析表达产物。结果：扩增出的ＳｃＦｖ基因长７３５ｂｐ ， 序列分析表明，该序列完整、正确；ＳＤＳＰＡＧＥ 显示，转染入重组质粒ｐ４ＴＣ３Ａ２Ｆｖ 的ＪＭ１０９ 菌经诱导后，有相对分子质量（ Ｍｒ） 约为５２ ０００ 的外源蛋白表达。结论：成功地构建了鼠抗ＬＰＳ ＳｃＦｖ 基因，并在Ｅ．ｃｏｌｉ ＪＭ１０９中表达了ＧＳＴＳｃＦｖ 融合蛋白。

Construction,sequencing ofthe anti-LPS ScFv gene and expression of GST-ScFv fusion protein

Aim: To construct anti LPS single chain Fv gene and attempt to express the GST ScFv fusion protein in high efficiency. Methods: The VH and VL gene of mouse anti LPS mAb were connected into ScFv gene by a flexible linker and the ScFv gene fragments were cloned into vector pGEX 4T 1. The recombinant plasmid were transfected into E.coli JM109 and the expression of targeted protein was induced with IPTG. The expressed protein was analysed by SDS PAGE. Results: The length of ScFv gene fragments was 735 bp. The sequencing for it showed that the cloned ScFv gene fragment was correct. SDS PAGE indicated that the recombinant plasmid p4T C3A2 expressed a new protein band with a Mr about 52 000. Conclusion: The ScFv gene was sucessfully constructed and GST ScFv fusion protein highly expressed in E.coli was obtained.