基于GFP分子的抗体设计与改造

发布时间：2019-05-24 02:21

【摘要】：基因工程单链抗体(scFv)是一种新型的小分子抗体,由于其独特的结果特点在人类疾病的诊断与治疗中发挥着极其重要的作用。但scFv在体外表达时常常以不溶性的包涵体形式存在,使得表达的抗体结构破坏,丧失了亲和力。为此,寻求一种能够真正解决抗体体外表达的新方法是很有必要的。 本研究以绿色荧光蛋白GFP为出发点,选择GFP分子中的loop9作为抗体片段基准插入点,构建了不同抗体片段(scFv, VH, VL, HCDR3和LCDR3)插入的融合表达载体,通过转化大肠杆菌BL21并经IPTG诱导表达纯化获得不同插入体的融合蛋白。荧光活性测定和ELISA检测结果表明,HCDR3和LCDR3插入并没有影响GFP的荧光活性,同时保留了与母本scFv相似的抗体活性和特异性,而且LCDR3插入体的的抗体亲和力要明显高于HCDR3。而其他三种插入不仅破坏GFP荧光活性,也丧失了抗体的活性。 为了进一步研究抗体与抗原的相互作用关系,我们将抗体分子的6个不同CDR区域插入GFP的loop9,表达纯化获得插入体抗体蛋白。ELISA结果表明,无论在重链和轻链中,CDR3是参与抗原识别的主要作用区域,而CDR1和CDR2在抗原识别中所起的作用较小。同时,重链和轻链CDR3三种不同形式的区域被插入到loop9中的相同位置。实验结果表明,仅CDR3loop区域插入时所产生的抗体亲和力是最高的,而把包含CDR3的β折叠区域插入GFP则降低了对应抗体分子的亲和力。 获得基于GFP的CDR3插入抗体后,我们对抗体分子进行了体外分子进化,以其提高抗体亲和力。首先利用丙氨酸扫描寻找LCDR3中与抗原相互作用的关键位点,它们分别是LCDR3的第二个氨基酸Q和第七个氨基酸R2,并利用赖氨酸和其他亲水性氨基酸突变获得亲和力提高的突变株R2/K,Q位点所有氨基酸突变都会导致抗体亲和力明显降低,而在R2位点,只有赖氨酸突变后抗体的亲和力是明显提高的。最后我们对该系统进行了通用性分析,将不同抗原的scFv分子的CDR3插入到GFP中的loop9区域,所有的CDR3插入体能够被很好的表达,纯化获得的抗体也具有一定的亲和力。实验结果证实,该系统具有很好的通用性,可以用于其他基于GFP的新型功能性抗体的制备。
[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，

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