Expression Of Full-length Mouse/Human Chimeric Antibody Against Hantaan Virus In Arabidopsis Thaliana And Study On Its Immunological Properties

Hantaan virus (HTNV) is the main cause of hemorrhagic fever with renal syndrome (HFRS). Murine derived monoclonal antibody (mAb) may cause the production of anti-murine antibody in human body. And the usage of human derived mAbs is limited for the difficulties in manufacture. Researches on genetic engineering antibodies, expressed in eukaryotic system, the yield of antibodies are low. In prokaryotic system, the yield of antibodies is high, but the biological properties of its products are affected by their poor biological modification. The yield of antibodies expressed in plant systems is high, and their products can posses good biological properties, for plant systems have the ability to modify the products.Our research has constructed a full-length mouse/human chimeric antibody gene by technology of genetic engineering antibodies and expressed the antibody in Arabidopsis thaliana. The main methods and results are followed:1. By connecting variable region segment of murine derived anti-HTNV mAb 3G1 gene and constant region segment of human IgG1 gene, the plant expression vectors 3G1MH–pCAMBIA 2301 was constructed firstly. Then, 3G1MH–pCAMBIA 2301 was transformed into Agrobacterium tumefaciens GV3101 by TSS freeze-thaw method, and then the recombinant was transferred into wild type Arabidopsis thaliana by using the vacuum-transgenic method. In order to screen positive transformed plants, the genomic DNA of the generation T0 transformed plants was isolated and identified by PCR with primers for heavy and light chain genes of their parent's immunoglobulin. The full length heavy and light chain RNA were detected by Northern Blot with their specific probes, indicating that the transgenic plants were constructed successfully.2. Furthermore, the cDNA derived from the transgenic plants was tested by real-time PCR. The results showed that the quantities of RNA in different transgenic plants differ from each other. The plant's proteins extracted from 5 transgenic plants with relatively high transcripted RNA were tested by Western Blot with goat anti- human IgG (Kappa chain specific) antibody. All extracts showed the expression of the target protein in these plants, though they were proteolysed to some extent, for a ~45 KD band was detected in all the plants tested. IFA results showed the specific binding activity of the expressed protein.3. The research results proved that the transgenic Arabidopsis thaliana expressed the recombinant chimeric antibody against HTNV with specific binding activity.This work has provided important theoretical and experimental evidence for the research and development of genetic engineering antibody expressed in plant systems.