Establishment And Preliminary Application Of A Double Sandwich ELISA Detection System For Soluble DC-SIGN

Dendritic cells (DCs) can absorb process and pass the antigen effectively, immature DC has strong migration ability, mature DC can activate initial type T cells, in the start, regulation and maintain a central part of the immune response. Although T cell-mediated resistance to infection, transplantation, tumor, but if DCs not correct instruction T cells can be severely damage. DCs foreign cells and infectious microbe antigen into short peptide, combining membrane protein major histocompatibility complex (MHC). The MHC-peptide compound formed in the cells, they act as ligands antigen specific T cell receptor (TCR) in the plasma membrane finally present.Mention natural immune molecules in the discussion of the gene encoding has several types of protein:macrophage receptors, such as mannose receptor (MR, CD206), the combination of specific intercellular adhesion factor3non integrin (DC-signs and CD209), Dectin-1, Toll sample receptor (TLRs), Complement receptor3(CD11b/CR3/CD18), nucleotide oligomerization domain1(NODI) and NOD2, CD14, P2X7, vitamin D nuclear receptor; Soluble C-type lectin, such as surfactant protein (sp-A) and (sp-D) and mannose combined with lectin (MBL); Phagocyte cell factor, such as tumor necrosis factor (TNF), interleukin-1β (IL-1beta), IL-6, IL-10, IL-12, and IL-18; Deprived of their freedom, monocyte chemoattractant protein1(MCP-1), RANTES, CXCL10; And other important innate immune molecules, such as induced nitric oxide synthase (iNOS) and solute carrier protein and al (SLC11A1).DC-SIGN molecule is a kind of II type transmembrane protein, its expression position widely. At first by scientists in the United States in2000in the study of human immunodeficiency virus (HIV) infection found in mechanism. The C-type calcium rely on lectin receptors (C-type lectin receptor, CLR), the carbohydrate recognition domain (CRD), a neck area participation tetramer oligomerization, membrane domain and cytoplasmic tail mediated endocytosis interaction for coordination body change. Signal mediated capture and internalization of the virus, bacteria and fungi pathogens and dendritic cells occur immune reaction, such as HIV-1, ebola virus, cytomegalovirus, dengue fever virus and hepatitis C virus. In DC-SIGN start-up area or-336C is CHB infection risk factors, and-139T rise to protect the body against the role of HBV.In2000, Geijtenbeek E. B.and the team has published several articles about DC-SIGN articles; An article on the DC-SIGN as dendritic cells (DC) of a C-type lectin protein, and T cell adhesion molecules ICAM-2interaction plays a regulation of the cytokine secretion, transshipment and regulating role； A is the first research hot research project HIV-1on the virus gp120protein can be combined with DC-SIGN and promote the HIV virus to T cell transfer; The same year also published in CELL research prove besides receptor LFA-1, DC-SIGN to Ca2+since the way combined with T CELL adhesion molecules on the surface of ICAM-3(intercellular adhesion molecule3, ICAM-3), and can be mannan block.In recent years, also have vitro DC-SIGN related signal study has confirmed that blocking DC-SIGN can rise to block HIV-1to CD4+T cells transfer.Due to the team expresses the sDC-SIGN-GST fusion protein already, using the protein immunization rabbit antiserum, purified antiserum get higher titer polyclonal antibody; Rebuilds the DC-SIGN extracellular segment (aa62-404, including the neck area and CRD) prokaryotic expression vector, and expressed in E. coli, and the main target protein is present in the inclusions. In urea, gradient dialysis to avoid protein denaturation during dialysis, to get the purpose protein, Western-bolt testing its specificity.Innovation building a extracellular vector of soluble DC-SIGN double sandwich detection system in the domestic successfully:capture antiboby is monoclonal antibody4H3, soluble DC-SIGN for the extracellular segment, detection antibody polyclonal antibody. The minimum detectable concentration in5ng/ml, detection limit:5ng-100ng; At the same time the system was applied to detected extracellular period of sDC-SIGN content in serum for testing among160healthy male in serum and HBVHBeAg, colon cancer, chronic hepatitis B(CHB). The research process and the main conclusions are as follows:Part I The preparation of polyclonal antibody by using the fusion protein of sDC-SIGN-GSTNatural antigen molecule often contains a variety of different antigen specific antigen epitope, this section successful experiment expressed fusion protein sDC-SIGN-GST and stimulate the immune system, then the body more B cell is activated, produce antibodies in actually contains a variety of different in epitope immune globulin, for its polyclonal antibody. Through the rabbit serum acquisition, its advantage lies in that role is comprehensive, and antigen, immune conditioning, ADCC, mediated complement dependent cell poison effect, and other important role, source widely, easy preparation. And this section experiment is consider using rabbit sources of polyclonal antibody, mainly consider the follow-up to establish ELIS A detection system, can have more antigen epitope recognition.The purification methods of polyclonal antibody selection process as follows: saturated ammonium sulfate purified polyclonal antibody, antibodies after purification were purified and gel analysis the result by SDS-PAGE, Using ELISA to detect serum titer. To get high purity of the polyclonal antibody. Experiment expressed the sDC-SIGN-GST fusion protein successfully, Obtain the high purity of the purified protein, And higher polyclonal antibody titer is obtained by immune rabbit.PartⅡ. PET17b-sDC-SIGN recombinant plasmid construction and its protein expression in this section.Recombinant protein were the two strains of mice against human monoclonal anti DC-SIGN antibody (4H3,1C6); the length of the sDC-SIGN gene is about1300bp, Using of the polymerase chain reaction (PCR) to obtain the sDC-SIGN coding region sequence with BamHI and EcoRl two enzyme for enzyme, when try to sDC-SIGN gene sequence directly transferred to PET17b, met a lot of difficulties. After many attempts to give up after the PET41a-sDC-SIGN on PCR products transferred to PET17b directly, and it was transferred into the carrier PMD18-T firstly, with connecting success after the double enzyme cut back to the BamHI and EcoRl double enzyme carrier after PET17b, The result is connected successful. Using the PCR, double enzyme parallel1%agarose gel appraisal, the purpose gene was connected to the carrier PET17b successfully.PET17b carrier contains strong promoters T7, therefore, express proteins induced by IPTG. PET17b-sDC-SGIN recombinant plasmid was induced after sDC-SIGN electrophoresis showed that after optimization the best protein expression conditions, sDC-SIGN for the expression of protein under this experimental condition mainly exist in inclusion body, we hope to receive it in soluble form and try to find the other ways, Not only to save complex deformation and renaturation of protein, it is more important as expressed in the form of soluble closer to natural protein, lay a foundation for the subsequent testing natural ingredients to better and more convincing, but despite the change and optimization of induction conditions, still failed to get a DC-SIGN the extracellular protein soluble form. So a lot of protein expression sDC-SIGN after renaturation, Using4M urea gradient dialysis for sDC-SIGN.Through the recombinant plasmid PET17b-sDC-SIGN into bacteria E.coli expression by Western-bolt to express specific identification of protein,, expression of protein sDC-SIGN with preparation of monoclonal antibody (4H3) as well as the commercialization of DC-SIGN monoclonal antibody. Specifies that the preparation of protein has the characteristics of the immunogenicity.PartⅢ.The third chapter sDC-SIGN double sandwich ELISA detection system and preliminary application.Based on the experimental work of the two strains obtained by sDC-SIGN sheet resistance and purification rabbit anti sDC-SIGN polyclonal antibody in combination, verify with single resistance to4H3as envelope antibody, polyclonal antibody as detected antibody against other combination can get better sensitivity, and the background is also lower. The second chapter of the protein may be due to the difference of protein folding, ELISA results show that the sensitivity of sDC- SIGN protein is weak, for the seak of the rusult is prokaryotic expression of protein and natural protein may be different, so do not use the original nucleus of protein expression as a standard product, while the use of commercialization from human kidney epithelial cells HEK293expression N end extracellul. Three diseases sample separately two independent sample rate of the chi-square test statistic data showed:chronic HBV infections sDC-SIGN content in serum and healthy people with no statistical difference (P=0.492>0.05); And with HBV HBeAg:P=0.001<0.05; With colorectal cancer patients (P=0.006<0.05).Maybe some disease detection and recovery can be used as potential clinical status indicators.