Daintain-AIF-1的純化及其單克隆抗體的制備與鑒定.pdf

1、華中科技大學碩士學位論文Daintain/AIF-1的純化及其單克隆抗體的制備與鑒定姓名：趙燕英申請學位級別：碩士專業(yè)：生物化學與分子生物學指導教師：陳正望20060507IIAbstractDaintain/allograft inflammatory factor 1 (AIF-1) is a 146-aa residue hormone-likecytokine discovered recently that is encode

2、d within the HLA class III genomic regionwith a mass of 17kDa and an acetylated N terminus. Daintain/AIF-1 ， mainly expressedby activated macrophages and T lymphocytes，is involved in Transplant rejection,autoimmune disea

3、se and tumor progress etc.For further investigation of this peptide distribution and functions, Daintain/AIF-1was isolated from porcine intestine with acetic acid extraction ,alginic acid adsorption,and sodium chloride

4、 saltingout followed by sephadex G-25(fine)columnchromatography，DEAE anion exchange column chromatography and reverse-phaseHPLC purification, The molecular mass of the fraction determined by MALDI-TOF-MS was 16.692

5、kDa. After hydrolysised with trypsogen, the fragment separated by RP-HPLC was selected for sequence analysis as YSSDEDLSRK consensus with the 36-45aa residue of Daintain/AIF-1, So the fraction identifed was Daintain/AIF-

6、1.Daintain/AIF-1 purified was then used to immunize BALB/c mice as theimmunogen. Furthermore, Both hybridization technique and enzyme linkedimmunosorbent assay(ELISA) were performed to generate cell lines which producedm

7、onoclonal antibodies(Mabs) against Daintain/AIF-1. Isotypes, titer and affinity of theMabs belong to G1 subclass with κ light chain. Thereafter, the specificity ofmonoclonal antibodies was identified by Western blot and

8、 competitive inhibitionELISA.The Mabs against Daintain/AIF-1 prepared and identified in first time are usefultools for further investigation of transplant rejection, autoimmune diseases and cancerdiagnosis.Keywords ： cyt