hlaa2cd35scfv融合蛋白在昆虫杆状病毒载体系统中的表达与纯化.pdf

华中科技大学 硕士学位论文 HLA-A2/CD35scFv融合蛋白在昆虫杆状病毒载体系统中的表达与 纯化 姓名：闫洁 申请学位级别：硕士 专业：免疫学 指导教师：吴雄文 20090501 ? ? ? ? ? ? ? ? ? ? ? ? 2 HLA-A2/CD35scFv ????????????????? ????? ??ı[ ?? ??[ ł? ? ???? ?ˆ˜˘-˙…‰ HLA I ‚?{œ???9,??cfi ”;?? ˆ ???HC“ g?LC“?…‰?????‡???????j?;????˙ T ”M? ?+ HLA I ‚?{???…9??‡?O?j?…‰?/HLA w‡??? N??”;??????ˆ•+?? HLA ?{N??;? CD35 ?{?Z? 9?9 1CR1“ ??S?????‡˚„?ˆ???”gB ”g??”g ? ?+??”;?? CD35 ?{?????c? !> HLA ?{? CD35 ??…9??‡O HLA ?{??N?”;??O4???S;?? …‰??”7X???” 4? HLA-A2/CD35scFv ?‡˚„?4?t?w8˙? 9:?9.;Gw˙? ??•+ ?????+????;??!>@;?9 pFastBacTMDual ?ABCDEFG” sf9 &; HLA-A2 .?α1,α2,α3 ˝“D CD35 ??…9?‡4HLA-A2/CD35scFv“? β2m 4??+Hfi ”?IJ9 ??h HLA-A2/CD35scFv ;GKL?m!>M{vNOP?HU??Q??F>? $c;GRStTUQVWX? HLA-A2/CD35scFv ?‡˚„ ˆ0?,-D?? Y?[ SgpFastBacTMDual+[β2m+HLA-A2/CD35scFv]Z[????;?9?t3 ? ? ? ? ? ? ? ? ? ? ? ? 3 ?+PCR??\pET-28a+[HLA-A2/CD35scFv]?9t3?2 ?g?˜+qh??>??nj??˜˚„?;gQ??nj O?˜h?pFastBacTMDual+[β2m+HLA-A2/CD35scFv]s?E.coil DH10BacTM? PCRnj??67+q?9Bacmid1β2m?HLA-A2/CD35scFv??4 ??nj; G??˜h?Bacmid DNA??+th9suZ[sf9”?v>suW?˜??+ sf9”]^wu ??wuW?sf9”?x?˜˚„Nyz⁄—GRStT?HLA-A2/CD35scFv?‡˚„ ?pI/Mw?%??? [expasy]?$??˜˚„HLA-A2/CD35scFv????z?pI“?6.12?BSA??z? pI“?4.7?4????M{vNOP?HU?v>?1?‡˚„⁄—??Q??? +W6/32?…?β2m…9??@…??ELISA?oNQ?W?˚„?V14g/ml“ ?? ˙Western blotting67?{%?70kD???]??S? HLA-A2/CD35scFv?‡˚„ ???”;?CD35?{?‡?5?‡?W’???4.4% ???67?Z[????;?9??Fc??;A$cRStT? HLA-A2/CD35scFv ?˜˚„?!>?M{vNOP?HU?Fh1?˜˚„?yz ⁄ — c?Q? ?UQ ?W? ?‡ ˚„? ? ” $c Sj? ? ‡ - h HLA-A2/CD35scFv ?‡˚„!> CD35 ?{??????”c??‡??? ? ? FW????…‰??”7X???”?j‘? ?d???HLA-A2/CD35scFv???…9??????9;??M{vNOP ? ? ? ? ? ? ? ? ? ? ? ? 4 Expression and purification of HLA-A2/CD35scFv Fusion Protein by Baculovirus Expression System Candidate[Jie Yan Tutor: Prof. Xiongwen Wu Abstract HLA class amolecule contains a heavy chain associated noncovalently with a light chain and a peptide. It distributes in kinds of karyocytes in human, which presenting definite epitope to special T cell. The fusion of HLA classa molecule and single-chain antibody can attach the peptide/HLA complex to target cells like tumor cells. CD35 molecule (complement receptor 1) is a kind of single strand binding membrane protein, which distributing in the surface of erythrocytes, B cells, macrophages, neutrophilic granulocytes and so on. It is possible to attach HLA-A2 molecule to the surface of erythrocytes through binding of CD35scFv to CD35, then take it as artifical APCs or TCs. The CD35scFv is recombiation protein that composed of VL,VH and a linker. It’s advantages are small MW, low immunogenicity, short half-life, high in penetrability. We have constructed HLA-A2/CD35scFv fusion gene successfully in the previous study, the product of prokaryotic expression showed that it was difficult to refold in vitro because of its complicated structurd, which limited the production and application of HLA-A2/CD35 fusion protein. The aim of this study is to use baculovirus expression vector system to express HLA-A2 (α1,α2,α3 domain) and HLA-A2/CD35scFv in double express vector in sf9 cells. In order to produce the fusion protein high purity, we used ion exchange chromatography to purify the infect supernatant. The following is the brief outlines of this study: 1.Construction and idetification of baculovirus express vector of pFastBacTMDual+ [β2m+HLA-A2/CD35scFv] ? ? ? ? ? ? ? ? ? ? ? ? 5 The cDNA encoding the HLA-A2/CD35 fusion protein and β2m were amplified by PCR from pET-28a+[HLA-A2/CD35sc-Fv] and pET-22b+[β2m] respectively, and cloned into the pFastBacTMDual to form pFastBacTMDual+[β2m+HLA-A2/CD35scFv] plasmid. The positive clones were identified with enzyme cut and DNA sequencing. The results confirmed that the vector were sucessfully constructed. 2. Acquirement and identification of bacmid contains β2m and HLA-A2/CD35scFv gene, expression,purity and identification of fusion protein. The recombinant plasmid pFastBacTMDual+[β2m+HLA-A2/CD35scFv] was transferred into E.coli DH10BacTMand formed a recombined baculovirus bacmid containing the interest genes of β2m and HLA-A2/CD35scFv gene. The bacmid was extracted from positive clones and then transfected into the insect cell line sf9 with liposome Cellfection2000. The sf9 cells that infected by recombinant virus secrete recombinant protein to supernantant. Sandwich ELISA with W6/32 and anti-β2m antibody was performed to detect the expression and conformation of the fusion protein. The theoretic PI of HLA-A2/CD35scFv fusion protein is 6.12 and PI of BSA is 4.7 with the expasy.Therefore, ion exchange chromatography was used to purity target protein. Western blotting by specific antibody comfirmed that the product was 70kD, which was coincidence with expect the expected molecular weight of the fusion protein. The fusion protein is tested for its binding to red blood cells, result shows that it is able to bind to human red blood cells, but the binding efficiency is relatively low (4。