The Screening, Construction, Expression And Preliminary Identification Of Human Anti-BLyS ScFv Antibody

B lymphocyte stilmulator (BLyS) is a member of tumor necrosis factor (TNF) super family, and expressed by the monocytes and macrophages. BLyS is significant for the growth, differentiation, proliferation and apoptosis of B lymphocyte. Overexpression of BLyS is connected with various kinds of autoimmume diseases (AD), such as rheumatoid arthritis, myasthenia gravis, systemic lupus erythematous (SLE), and so on, especially in the in the development of SLE, BLyS plays an important role. It is an effective way to cure these diseases by blocking the signaling pathways of BLyS, and belimumab has been obtained successful effect in the treatment of SLE which is combining with BLyS. Single-chain fragment variable (ScFv) has many characteristic advantages compared to the full-length antibody: ScFv could bind antigen specifically only while not interact with the Fc receptor on the cells. The molecular weight of ScFv is only 1/6 of full-length antibodies’, so it can reach target locations easily. It seems that anti-BLyS ScFv have potential therapeutic value for AD and broad application prospect.Using mammalian cell antibody library to screen antibody is a novel technology in recent years, and it develop quickly. It makes the antibody screening transfer to the eukaryotic expression system which is supported by the mammalian cells. Just because of this, the problem of post-translational modification in prokaryotic expression system has been resolved, and the antibody expressed in mammalian cells is more similar to human natural antibody.In this article, we used the mammalian cell display human ScFv antibody library to screen bran-new human anti-BLyS ScFv which has high affinity, and then constructed the recombinant plasmids, expressed the proteins. At last we identified the active of human anti-BLyS ScFv antibody preliminarily.Part Ⅰ: Screening human anti-BLyS ScFv from the mammalian cell display human ScFv antibody library.In this part the human anti-BLyS ScFv were screened from the mammalian cell display human ScFv antibody library. First, the library was transfected into 293T cells to make all ScFv displaying on the cytomembrane; then these cells were collected and incubated together with the BLyS labeled by FITC (FITC-BLyS) for 1h. The 0.1% positive cells with the highest fluorescence intensities were sorted from the total number of cells by using flow cytometry. The plasmids were extracted and amplified from transfection to produce the library for the next screening. We got the high affinity human anti-BLyS ScFv through three screening which reduced the concentration of antigen in turn (1 μg/106cells-0.1 μg/106cells-0.01 μg/106cells). After got the sequences of the ScFv, we analyzed them in IgBlast and IMGT. Finally, we got two new sequences of human anti-BLyS ScFv, the VH and VL of the two ScFv are complete (contain framework regions and complementary determining regions) and procreative.Part Ⅱ: The construction, expression and preliminary identification of human anti-BLyS ScFv antibodyIn this part our main purpose is taking the two ScFv into the vector p3457-his and expressing the proteins, then identifying the proteins’ active. At first we constructed two recombinant plasmids which contained the two ScFv by gene recombination technology, and identified the recombinant plasmids by some methods, such as colony PCR, sequencing, gel electrophoresis and digestion. We named these two recombinant plasmids p3457 B6-ScFv and p3457 B10-ScFv. Second, we transfected plasmids into 293E cells and cultured for 6-7 days. Collected the supernatant and assessed the expression by SDS-PAGE. Purified the proteins by AKTA purifier and assessed the expression by western blot. Measure proteins’ concentration by BCA. Then we measured the two ScFv antibodies’KD values by ForteBio Octet QK. In this part, we constructed two recombinant plasmids p3457 B6-ScFv and p3457 B10-ScFv and expressed ScFv antibody successfully. The KD value of B-6 ScFv is 10nM and B-10 ScFv is 3.08 nM indicated that the two ScFv antibodies have high affinity and they all can bind BLyS specifically. Two human anti-BLyS antibody not only can bind BLyS successfully but also can inhibit BLyS combined with its receptors in vitro.In conclusion, in this research, we screened two bran-new human anti-BLyS ScFv sequences in mammalian cell display human ScFv antibody library and expressed two human anti-BLyS ScFv antibody with high affinity in vitro. This research would lay a foundation for later studies, and provided the ideas and methods for other human ScFv antibody screening.