【摘要】：基因工程單鏈抗體(scFv)是一種新型的小分子抗體,由于其獨特的結(jié)果特點在人類疾病的診斷與治療中發(fā)揮著極其重要的作用。但scFv在體外表達時常常以不溶性的包涵體形式存在,使得表達的抗體結(jié)構(gòu)破壞,喪失了親和力。為此,尋求一種能夠真正解決抗體體外表達的新方法是很有必要的。 本研究以綠色熒光蛋白GFP為出發(fā)點,選擇GFP分子中的loop9作為抗體片段基準插入點,構(gòu)建了不同抗體片段(scFv, VH, VL, HCDR3和LCDR3)插入的融合表達載體,通過轉(zhuǎn)化大腸桿菌BL21并經(jīng)IPTG誘導表達純化獲得不同插入體的融合蛋白。熒光活性測定和ELISA檢測結(jié)果表明,HCDR3和LCDR3插入并沒有影響GFP的熒光活性,同時保留了與母本scFv相似的抗體活性和特異性,而且LCDR3插入體的的抗體親和力要明顯高于HCDR3。而其他三種插入不僅破壞GFP熒光活性,也喪失了抗體的活性。 為了進一步研究抗體與抗原的相互作用關(guān)系,我們將抗體分子的6個不同CDR區(qū)域插入GFP的loop9,表達純化獲得插入體抗體蛋白。ELISA結(jié)果表明,無論在重鏈和輕鏈中,CDR3是參與抗原識別的主要作用區(qū)域,而CDR1和CDR2在抗原識別中所起的作用較小。同時,重鏈和輕鏈CDR3三種不同形式的區(qū)域被插入到loop9中的相同位置。實驗結(jié)果表明,僅CDR3loop區(qū)域插入時所產(chǎn)生的抗體親和力是最高的,而把包含CDR3的β折疊區(qū)域插入GFP則降低了對應(yīng)抗體分子的親和力。 獲得基于GFP的CDR3插入抗體后,我們對抗體分子進行了體外分子進化,以其提高抗體親和力。首先利用丙氨酸掃描尋找LCDR3中與抗原相互作用的關(guān)鍵位點,它們分別是LCDR3的第二個氨基酸Q和第七個氨基酸R2,并利用賴氨酸和其他親水性氨基酸突變獲得親和力提高的突變株R2/K,Q位點所有氨基酸突變都會導致抗體親和力明顯降低,而在R2位點,只有賴氨酸突變后抗體的親和力是明顯提高的。最后我們對該系統(tǒng)進行了通用性分析,將不同抗原的scFv分子的CDR3插入到GFP中的loop9區(qū)域,所有的CDR3插入體能夠被很好的表達,純化獲得的抗體也具有一定的親和力。實驗結(jié)果證實,該系統(tǒng)具有很好的通用性,可以用于其他基于GFP的新型功能性抗體的制備。
[Abstract]:Genetic engineering single chain antibody (scFv) is a new type of small molecule antibody, which plays an important role in the diagnosis and treatment of human diseases because of its unique outcome characteristics. However, scFv often exists in the form of insoluble inclusion body when expressed in vitro, which destroys the structure of the expressed antibody and loses its affinity. Therefore, it is necessary to find a new method to solve the problem of antibody expression in vitro. In this study, using green fluorescent protein GFP as starting point, loop9 in GFP molecule was selected as the benchmark insertion point of antibody fragment, and the fusion expression vectors inserted with different antibody fragments (scFv, VH, VL, HCDR3 and LCDR3 were constructed. The fusion proteins of different inserts were obtained by transformation of E. coli BL21 and induced expression and purification by IPTG. The results of fluorescence activity assay and ELISA showed that HCDR3 and LCDR3 insertion did not affect the fluorescence activity of GFP, and retained the antibody activity and specificity similar to that of female parent scFv, and the antibody affinity of LCDR3 insert was significantly higher than that of HCDR3.. The other three inserts not only destroyed the fluorescence activity of GFP, but also lost the activity of antibody. In order to further study the interaction between antibody and antigen, six different CDR regions of antibody molecules were inserted into GFP loop9, to express and purify the inserted antibody protein. Elisa results showed that in both heavy chain and light chain, CDR3 is the main region involved in antigen recognition, while CDR1 and CDR2 play a small role in antigen recognition. At the same time, three different forms of heavy chain and light chain CDR3 are inserted into the same position in loop9. The experimental results show that the antibody affinity produced only when the CDR3loop region is inserted is the highest, while the insertion of the 尾-fold region containing CDR3 into GFP reduces the affinity of the corresponding antibody molecules. After GFP-based CDR3 was inserted into the antibody, we evolved the antibody molecule in vitro to improve the affinity of the antibody. Firstly, alanine scanning was used to find the key sites of interaction with antigens in LCDR3, which were the second amino acid Q and the seventh amino acid R2 of LCDR3, respectively. The mutant R2 鈮，

本文編號：2484462