Expression Of Genetically Engineered Anti-Keratin Full-Length Human IgG In CHO(dhfr～-) Cells

As some investigations have shown that human anti-keratin auto antibody (AK auto Abs) generally exist in human and animal bodies, and compose of the basic part of immune system. AK auto Abs play an important role in immune homeostasis. In some pathological states such as psoriasis and contact dermatitis, the existence of AK auto Abs could inhibit the progression of the diseases and be beneficial for the recovery from the diseases. Animal experiments have proved AK auto Abs are involved in the pathophysiological process of some dermatogic diseases. The purified AK auto Abs inhibited the proliferation and differentiation of keratinocytes depending on the dosage. The infusion of human serum immuglobin containing AK auto Abs had obvious results in some psoriasis patients. The AK auto Abs which were extracted from animal serums showed the prospective therapy and prospective application. But The clinical application of AK auto Abs was limited by the reliability of blood agents.The phage-display technology makes us to be possible to produce human antikeratin antibodies not by means of immune methods. Many monoclonal antibodies were madesuccessfully from phage antibody libraries in home and abroad. Recently our laboratory has already screened out the Fab and ScFv of anti-keratin antibodies successfully from semisynthetic phage antibody library and obtained soluble Fab with high specificity and affinity which provides better means for the application and further study of AK auto Abs. In our research Some fragments of these antibodies have the function of inhibiting proliferation of cultured keratinocytes. The antibody fragments, which were screened out from phage antibody libraries, can meet the needs of application. But some biological functions, such as complement activation, antibody-dependent cellular cytotoxicity (ADCC) and immunoloregulation, are lost because of not having Fc fragments. In some conditions especially treatment in vivo, the full length of IgG antibody molecule is needed. So we select the Fab or ScFv with the highest affinity to clone the variable region genes and construct the eukaryotic expressive vector which is transfected to dihydrofolate reductase defective CHO cells with lipofectamine 2000. Under the pressure of MTX, the transfectants express the human anti-keratin full-length of IgG antibody and its biological activity is detected.Affinity is an important parameter of antibody. It is that the antibody with high affinity can play an effective biological role in vivo. In biological and technological field, the antibodies with high affinity have advantageous value in clinical application. So in order to select the antibody with good behavior, we should elevate the affinity of antibodies which is screened out from phage display library timely. Our laboratory have screened out seven anti-keratin single chain antibodies and four anti-keratin Fab antibodies. Their activity and specificity is detected with enzyme-linked immunoadsordent assay. The relative molecular weight of antigen binding is analysed with western blot. But it is the most important to estimate the affinity of antibody by sulforhodanide or biosensor and screen the antibody clone with the maximinal affinity. Phage single chain antibodies can not bond themselves. So we cannot express the antibodies fragment, predict the activity of antibodies and detect the affinity of antibodies with biosensor. So we utilized the four strains of anti-keratin phage Fab antibodies to induce the expression of soluble antibodies with IPTG. The antibodies were purified with the affinity with the nickel column of affinity chromatography and the affinityis measured with biosensor. We selected the antibody with the maximal affinity.The plasmid with the maximal affinity is sequenced in Shanghai biological engineering Corporation. We design the primers according to the sequencing results to amplify the variable region genes of light and heavy chain and connect the leader sequence. The variable region genes of light and heavy chain with leader sequences(...