| Cytotoxic T lymphocytes (CTLs) play an important role in T-cell-mediated immune response. CTLs recognize protein antigens in the form of the specific peptide fragment that are presented at the cell surface by major histocompatibility complex (MHC) class I molecules. When the antigen-specific T-cell receptor (TCR) on the T-cell surface interacts with the appropriate peptide-MHC complex, with the help of a second co-stimulatory signal, CTLs are induced to activate, devise and proliferate. So, CTLs carry out important effector functions, being one of the cell types that are responsible for destroying tumor cells, allogeneic transplant cells and all kinds of infected cells. For this reason, there has for many years been much interst in assay of detection and activation mechanism of the antigen-specific CTLs. However, there are many difficulties in studying the activity and functions of the antigen-specific CTLs ex vivo. It is hard to cultivate the antigen-specific CTLs. It will bring a lot of non-specific CTLs. Nevertheless, there is lack of the techniques that assay the frequency of the specific CTLs accurately and isolate them quickly. It is harder to assay the frequency and characterization of the T cell clone in vivo. The assays before, such as 51Cr release assay, ELISPOT assay and intracellular-cytotoxic staining and so on, require the CTLs to be re-stimulated and expanded in number in vitro. These assays are time-consuming, and their specification is very poor. Recently the soluble MHC-peptide tetramer-analysis becomes a new method to quantitative the antigen-specific T cells and sort them so as to confirm their functional ability. It is effective, sensitive and specific to study the T-cell-mediated immune response. At present, many specific tetramers were developed in America. They were used to monitor many viral infection and tumor such as HIV, EBV, CMV, melanoma and cervical carcinoma. Yet, thereare drawbacks in the course of producing the tetramers, for example, the renaturation rates is low and the operation is very complex. Moreover, they are too expensive. To overcome these drawbacks, we successfully produced the new antibody HLA-peptide polymers to study the activation and detection of the antigen-specific CTLs, then fixed the new antibody HLA-peptide polymers to screen the antigen-specific CTLs. These lay a foundation for new activation method and screening technique platform of the antigen-specific CTLs.We expressed and purified heavy chain (HC) and light chain ( 3 2m) of HLA-A* 0201. HC andP 2m engineering bacteria was induced with IPTG. The expression rate of HC and P 2m engineering bacteria was 43% and 47% respectively and their molecular weight is about 32KD and 13KD.The crude inclusion body was obtained after break of the bacteria by ultrasound, and with the patent method turned to refined inclusion body, which was then renaturated and ultrafiltrated, and purified with DEAE Sepherose Fast Flow and Superdex 75. The purity rate of HC reached toabout 95%.So,the HLA-A* 0201 HC after purity can be used as antigen to produce polyclonal antibody and monoclonal antibody.The antigen of HLA-A* 0201 heavy chain was used to immunize the hens assisted with Freud. The titer of IgY was 1:106 after three immunizations. IgY was purified from the egg yolk by water extraction, salt precipitation with ammonium sulfate and dialysis; SDS-PAGE showed that there are two bands on the gel. They were heavy chain and light chain of the IgY and their molecular weight is about 67KD and 30KD respectively. The purity of the IgY reached 88%. The protein concentration of purity IgY was 9.77mg/ml.The cell-ELISA showed that the IgY can response to Caski cell strain with HLA-A* 0201 positively. This indicated that IgY can combine the natural HLA-A* 0201 molecular and be used to prepare the antibody HL A polymer.The Balb/C mice were immunized with the purified recombinant HLA-A*0201 HC as antigen. The hybridoma cell line secreting anti-HC McAb steadily was obtained by using the hybridoma technique. The characterizatio... |
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