中文摘要：

目的：获得肠三叶因子（ITF）的原核表达产物及抗rITF抗体，为深入研究ITF的作用机制及其受体研究奠定基础。方法：常规提取人小肠组织总RNA，用RT—PCR获得ITF编码基因片段，克隆至质粒pET32a获得原核表达载体，双酶切和测序后转化至OrigamiB（DE3）用IPTG诱导表达，优化条件获得最大表达产量；用SDS—PAGE、Westernblot鉴定表达产物，亲和层析纯化获得的重组蛋白rITF皮下多点注射家兔，制备多克隆抗体，并用此抗体进行大鼠肠组织免疫组化研究。结果：测序证实PCR扩增获得ITF全长基因序列与基因文库中的完全一致，将该基因片段正确插入表达载体pET32a中、优化表达条件后，重组蛋白的表达量达到50mg／L；Westernblot证明重组蛋白具有良好的抗原性和特异性；通过Ni-NTA亲和层析、超滤离心后，得到90％纯度的蛋白；收集兔血清，纯化后获得特异性良好的ITF抗体，免疫组化染色肠组织显示ITF表达的部位定位于杯状细胞。结论：成功构建了表达载体pET32a-ITF，在大肠杆菌中表达并纯化获得纯度较高的rITF，并获得了生物活性较高的ITF抗体，ITF主要在肠道杯状细胞分泌表达。

英文摘要：

Objective： To express the gene fragment encoding for ITF in Escherichia coli and prepare the antibody, and also to lay the foundations for further study of its Mechanism and pharmaceutical function. Method： The gene fragment which encoding for mature peptide of ITF was obtained by RT-PCR, then inserted into the MCS of the expression vector pET32a to construct the recombinant vec- tor. After double enzyme digestion and gene sequencing, the recombinant vector was transformed into E. coli Origami B （DE3）, and rhlTF was expressed by IPTG induction, rhlTF was purified by Ni-NTA affinity chromatography, determined by SDS-PAGE and West- ern blot analysis. The antibody to ITF was prepared by injection the purified recombinant protein to rabbits, and then was used to Immunohistochemistry study of rat＇ s small intestine Result： Sequencing results showed that the gene fragment obtained by RT-PCR was consistent with the reference sequence （ GenBank Accession No. NM003226）, and then it was successfully inserted into pET32a to construct the recombinant vector. The rhlTF was expressed at a concentration of 50 mg/L. After purification, the purity of recombinant protein was above 70%. The result of western blot analysis showed that rhlTF had fine antigenicity and specificity. The ITF antibody collected and purified from the rabbits serum was confirmed to have good specificity, and expressed in the goblet cell by immunohistochemistry study. Conclusion： The expression vector pET32a-ITF was constructed and expressed in E.coli successfully, and the recombinant protein rlTF and its antibody was also purified in this study. ITF expressed in small intestine goblet cell.