成熟志贺毒素A亚单位(ShT-A)全长与截短片段在E.coli中的表达及多抗制备

Expression and Antibody Preparation with Matured and Truncated of Shiga Toxin A in E.coli

根据G enB ank中Ⅰ型痢疾志贺菌ShT-A基因的核苷酸序列,设计合成了2对引物,分别进行PCR扩增,将PCR产物分别与pGEM-T连接构建了克隆质粒pGEM-TA1和pGEM-TA2。用B amHⅠ和K pnⅠ以及B amHⅠ和X hoⅠ双酶切pGEM-TA1和pGEM-TA2,均得到大小为895 bp的ShT-A基因片段,分别与pQE 30和pET 21a(+)原核重组表达载体连接,构建pQE 30-A2和pET-21A1原核重组表达质粒。工程菌M 15/pQE 30-A 2和BL-21/pET-21A 1经IPTG诱导,SDS-PAGE电泳观察,均没有目的蛋白表达。这表明全长ShT-A可能对E.coli细胞产生毒性作用。DNA s is软件分析ShT-A基因序列,发现在513 bp处有H indⅢ位点,以ShT-A上游引物的B amHⅠ和H indⅢ从pGEM-TA1切出1个513 bp的截短片段,重新插入表达载体pQE 30,经PCR和双酶切鉴定正确后,得到pQE 30-A513重组表达质粒,转化E.coliM 15经IPTG诱导,SDS-PAGE电泳观察,在20 000处出现1条特异性的表达蛋白带,与截短ShT-A513相对分子质量相符。经薄层扫描分析,该截短片段的表达量约占菌体总蛋白的37.4%,主要为包涵体形式。免疫印迹结果表明,表达的重组ShT-A513蛋白同抗ShT-A513鼠多抗有特异性结合。

To express the ShT-A gene fragment in E. coli, pGEM-TA1 and pGEM-TA2 cloning vector and prokaryotic pQE30 and pET21a （h-） expression vector were respectively digested with the endonucleases BamH Ⅰ and Hind Ⅱ （BamH Ⅰ and Xho Ⅰ ）,and then the fragment of ShT-A and the large fragment of linearized pQE30 and pET21a（h-） were ligated under T4 DNA ligase,resuhed in recombinant vector,named as pQE30-A2 and pET-21A1. The two recombinante expression vectors were selected and identified by the colony-PCR and endonucleases digestion,finally expressed in E. coli M15 and BL-21. The two recombinante expression vectors induced by IPTG. SDS-PAGE showed no recombinant proteins were expressed in E. coli M15 and BL-21 cell. A single Hind Ⅱ site was found in the ShT-A gene analyzing by professional software of DNAsis. A 513 bp fragment of pGEM-TA1 cloning plasmid was cut by BamH Ⅰ and Hind Ⅱ ,and cloned into the pQE30 vector,the resulted pQE30A513. The engineering strain of E. coli M15 with pQE30-A513 was expressed in the truncated ShT-A induced by IPTG. SDSPAGE showed the size of expressed ShT-A513 protein was similar to the designed molecular weight in 20000,and the target protein occupied percent 37.1 of total cell protein and in inclusion body form. Western blotting showed the truncated pQE30A513 recombinant protein was connected by a polyclonal antibody from the immunized Kunming mouse.