Analysis Of Biological Functions Of Mouse Anti-human CD44 Monoclonal Antibody-HI313 And Construction Of ScFv Based On HI313

Objective:To study the biological functions of mouse anti human monoclonal antibody-HI313 , desire to construct anti-CD44 single chain fragment variable based on HI313 monoclonal antibody (HI1313-ScFv). These results provide a potential target and basis for developing CD44-targeted therapy in AML.Methods:Through observations on HI313 and HI44a monoclonal antibody affinity and competitive inhibition by flow cytometry; to analyze the affection on some CD44+ leukemia cell lines caused by HI313, we use MTS and trypan-blue dye . Annexin V/PI and The Cycle TESE^TM PLUS DNA Reagent Kit detect the growing inhibition and cycle of the cells; Detect HI313 effect in reverse the differentiation blockage of AML cell. Amplified both heavy chain and light chain variable region gene of HI313 McAb by RT-PCR, than splice them into ScFv in form of VH-linker-VL.That recombinant gene fragment fragment was cloned into plasmid pET22b and induced to express the recombinant protein, HI313-ScFv, by IPTG in E.coli BL21.We purified the output by Ni Sepharose^TM, denatured/re-natured and detected it binding activity with natural cell through Flow cytometry.Result:The relative affinities of HI313 tested by THP-1 and KG1a are 1.2nM and 1.34nM, while the ones of HI44a are 3.5nM and 5.89nM. The affinities of HI313 and HI44a are both less than the normal 10nM.The FACS shows that there is no competitive inhibition between HI313 and HI44a although they are both monoclonal antibodies of CD44, which indicates that the two antibodies do not share the same epitope.The HI313 monoclonal antibody can significantly inhibit the proliferation of the leukemia cell line NB4 by blocking the cell cycle on the G₀/G₁ point, while has little effects on other cell lines.Inducing differentiation of AML hemocytes shows that HI313 can increase the expression of myelogenous mature antigens.The variable region gene of both HI313 heavy chain (339bp) and light chain (357bp) was cloned.To construct The spliced ScFv fusion gene with linker by overlap-PCR. All long and successfully cloned into PET22b plasmid to construct pET22b/HI313-ScFv expression vector. That can high express recombinant protein in E.coli induced by IPTG. The recombinant ScFv protein can bind nature CD44 molecular. Conclusion:Both HI313 and HI44a possess high affinity, HI313 monoclonal antibody can inhibit growth of NB4 cell and induced those cell cycle retained G0/G1 stage. After construction of HI313-ScFv. we should keep moving to study its potential usage and reform it for future clinical application.