1. Preparation Of Immunomagnetic Alginate Sodium Composite Microsphere And The Application In Early Diagnosis Of Tumor 2. Construction Of Expression Vector, Prokaryotic Expression Of BirA Enzyme And Identification Of Expressed Product Activity

Magnetic polymer microspheres (MMS) are those microspheres synthesized by polymer and inorganic material on molecule scale, which have the ability of magnetism and special structure. Since varied functional groups linking on the surface of microspheres, such as -OH,-COOH, -NH2 and so on, MMS are biocompatible and can conjugate with some ligands of bioactive compound,these give them the ability to recognize and bind to corresponding antigen, antibody, nucleic acid etc. Then this compound can be separated and enriched through the magnetic field. So MMS are promising for the new tool on varial areas such as cell separation, enzyme immobilization, drug targeting, immunodetection and so on. In this thesis, a kind of neotype nano- MMS were prepared, these nano-MMS had some characteristics when compared with ordinary MMS, such as strongly magnetic response, superparamagnetism, highly stability, largely specific surface area and so on. MMs can be taked as publicly carrier and separate tool. Then associated with immune analytical technique and chemiluminescence reaction, new system of magnetic enrichment and detedtion and magnetic chemiluminescence immune assay system were designed and exploited, and applied in the early diagnose of tumor. These establishments of such detection can bring out strongly social effects and economic returns.1. Preparation of magnetic Alginate Sodium composite Microsphere by means of reverse phase microemulsion and imbed process Take heptane as organic dispersed medium, AOT as surfactant, sodium alginate solution and Fe@C nanoparticle as aqueous phase, the system of reverse phase microemulsion was formed, and calcium chlorination and epichlorohydrin were been taked as crossing agents. Preparation conditions such as the concentration of sodium alginate, the concentration of AOT and stirring speed were described, and then the microsphere properties also were described in this paper. Results showed that the microspheres had a spherical appearance, with narrow size distribution, and the average diameter is 279nm, while the microspheres also showed strongly magnetic response.2. Immunomagnetic Alginate Sodium composite microspheres (IMM) were prepared in this part.The imbedding method of chemical group on the surface of MMS are discussed, and 6-Aminocaproic Acid with six carbon chain length were attached onto MMS as"spacer arm", then goat anti-mouse IgG antibodies were binded with"spacer arm"through cross-linking reaction by preparing immunomagnetic alginate microspheres. Preparation conditions and MMS properties were described. Results showed that 98.3% immunomagnetic microsphere showed successfully cross-linked actived antibody and there were 111.2μg antibodies immobilized onto 1mg immunomagnetic microspheres.3. Tumor cell magnetic enrichment and detection method were established and applied in the detection of tumor cells in peripheral blood of lung adenocarcinoma patients. IMM coated with goat anti-mouse IgG antibodies were prepared, system of magnetic enrichment and detedtion were established by combining immunomagnetic separation with immunocytochemistry technique. The specificity, sensitivity and stability of the system were identified. In the detection of tumor cells in peripheral blood of lung adenocarcinoma patients, three kinds of method (enrichment and detection method that established in this thesis, immunocytochemistry and RT-PCR method) were used to compare their variability.The result showed that Tumor cell magnetic enrichment and detection system had the abilities of high sensitivity, specificity and stability. In the detection of clinic patients, the new detection system had no significant difference compared with RT-PCR methods (p>0.05), and had significant difference compared with immunocytochemistry methods (p<0.05).4. Magnetic chemiluminescence immune assay system were established and applied in the detection of tumor marker AFP in peripheral blood.IMM coated with goat anti-rabbit IgG antibodies were prepared, magnetic chemiluminescence immune assay system was established by combined immunomagnetic separation with chemiluminescence reaction. The specificity, sensitivity and stability of the system were identified. In the detection of tumor marker AFP in peripheral blood, the result showed that system of Magnetic chemiluminescence immune assay had the abilities of high sensitivity, specificity and stability, and the result of AFP in the blood have matched with clinic diagnosis.In this thesis, neotype Immunomagnetic composite microspheres were prepared taking Fe@C nanoparticle as magnetic materials. This resolved the problem of poor magnetism when taking ferric oxide as magnetic materials. Considering stereo conformation between antibody and antigen reaction, antibodies were binding on microspheres through"arm"molecule. In this basis, nano- MMS are promising for the new tool in more areas besides of system of magnetic enrichment and detedtion and magnetic chemiluminescence immune assay system. Objective: To construct the expression vector of biotin-protein ligase (BirA enzyme) gene and express the BirA enzyme with bioactivity in E.coli BL-21 (DE3).Methods: The BirA gene was amplified from E.coli genome by PCR and it was cloned into pGEX-4T-2 to construct the recombinant plasmid pGEX-BirA. After being verified by DNA sequencing, the fusion protein was expressed under IPTG induction in the E.coli BL-21 (DE3). The expressed product was purified through Glutathione-agarose chromatography column. The enzyme activity of the expressed product was identified by ELISA and Western blot.Results: The recombinant prokaryotic expression vector pGEX-BirA was constructed and the fusion protein GST-BirA with Mr being 61300 and bioactivity was expressed successfully. After the expressed product was purified through Glutathione-agarose chromatography column, the results of ELISA and Western blot showed that the expressed product could make HLA-A2 peptide complex biotinylation.Conclusion: BirA enzyme with bioactivity is prepared successfully which provide an effective reagent for studying the interaction between protein and protein.