Expression Of Human Anti-HER2scFv-9r Fusion Protein And Reserch Of Its Activity

The skeleton is the most common organ to be affected by metastatic breastcancer，with the highest prevalence in breast cancer. The cases of metastaticbone disease probably account for65%to75%in the breast cancer. Once thebreast cancer has spread to bone, it is generally considered to be incurable.Breast cancer relapsing to bone are associated with substantial bone-relatedevents including chronic bone pain, pathologic fracture, spinal cord compressionand hypercalcemia.These serious complications may lead to a dramatic decreasein the quality of life for breast cancer patients. Even if there are so manymethods commonly used to treat the breast cancer relapsing to bone in clinical,such as surgical treatment, rediothearpy, chemotherapy, analgesic drug treatmentand hormonotherapy, it is difficult to obtain satisfactory results.Thus the cancer targeted therapy came into being. Recently, due to thespecific anti-tumor effects of cancer targeted therapy, targeted therapytechnology applied in the treatment of breast cancer bone metastasis, can significantly improve cancer treatment and provide new candidate molecules forgene therapy for HER2-positive tumors.The discovery of RNA interference (RNAi) can specifically reduce theexpression of the endogenous protein, so it is hoped that the use of RNAi cantreat refractory disease, such as many genetic diseases and tumors.As anessential factor in the progress of RNAi, small interfering RNA(siRNA)hasbroad therapeutic application prospects. However, due to the poorintracellular absorption, the weak stability in plasma and the non-specifictransfer, the siRNA delivery in vivo is still the biggest obstacle to widely useRNAi in clinical.Single chain variable fragment (scFv) retains antigen binding activity, itsmolecular weight is smaller and immunogenicity is lower, making scFv anexcellent targeting molecule suitable for delivery carriers. scFv fused with othereffector molecules,such as protamine and immunotoxin, is of multiple functions.Basic amino acid oligo-9Arg containing a large number of positive charges is akind of cell-penetrating peptides. It can rely on the interaction of positive andnegative charge, and bind the nucleic acid with negative charges.In recent years, the studies found that the CXCL12/CXCR4biological axisplayed an importment role in the breast cancer relapse to bone.HER2expressionwas positively correlated with CXCR4expression in most bone metastases frombreast cancer. CXCR4expressed at high levels often indicates poor prognosis incancer patients. Thus HER2and CXCR4become important targets in cancertargeted therapy. Based on the above, with the help of genetic engineering andprotein technology, we try to get human-derived anti-HER2single-chainantibody (scFv)/oligo-9-arginine (9R) fusion protein and obtain good carrierof siRNA delivery in vivo. We also try to make the specific siRNA reach and internalize into HER2-positive tumor cells under the guidance of antibodyfusion protein So as to targetedly inhibit the expression of CXCR4.This strategyis expected to provide a new method for the clinical treatment of breast cancerbone metastasis.Objective:To construct a human fusion protein gene, that recognizes HER2scFv/oligo-9-arginine, express and purify the protein in E. coli, and analyze itsbinding activity.Methods:Using the PCR method, we amplified fusion gene scFv-9R. The scFv-9Rwas then cloned into the expression vector pQE30and expressed in E. coli M15.Expressed product was detected by SDS-PAGE and Western blot, purified byNi-NTA chelating agarose, refolded by dialysis and concentrated byultrafiltration. The antigen-binding activity of the scFv-9R was analyzed byELISA. The ability of antigen binding and internalization function wereconfirmed by indirect immunofluorescence. The siRNA binding ability wasdetected by electrophoretic mobility shift assay (EMSA). The rate of scFvcombined with the siRNA, was estimated by siRNA binding assays.Results:We correctly constructed the anti-HER2scFv-9R fusion gene. The fusionprotein was successfully expressed in E.coli M15induced by IPTG inthe form of inclusion body. We identified the fusion protein by SDS-PAGE andWestern blot. We obtained a purity of about87%of the target protein by usingNi-NTA affinity chromatography. Specific antigen binding activity of scFv-9Rwas confirmed and indirect immunofluorescence staining. Indirect immunofluorescence staining also revealed that it was able to internalize intoHER2positive cells. EMSA assured that it had siRNA binding activity. siRNAbinding assay showed that one molecular fusion protein could combine withapproximately3molecules of siRNA.Conclusion:The scFv-9R fusion protein can specificly bind with both HER2antigen,internalize into HER2positive cells and bind siRNAs. These results show thatthis fusion protein may prove to be a potential agent for the targeted siRNAdelivery.