Study On Construction Of MHC-Peptide Tetramer And Its Technical Innovation

Background and objectiveCytotoxic T lymphocytes (CTLs) are one of the major components of cell-mediated immunity and kill target cells upon recognition of antigenic peptides presented in the context of MHC class I molecules. The recognition is also restricted by MHC class I molecules. It is necessary to analyze the antigen-specific CTL in order to understand the cellular immune function, exploring the disease mechanism and assessing the efficacy of the immunotherapy. At present the tetramer assay is the only one method to quantitate directly the antigen-specific CTLs. However, there are some disadvantages such as high prices and tedious procedures in the preparation of tetramers. To some degree, this assay is limited to a small scale application. Tetramers with different antigens are requested to meet the demanding of research and clinical application. Therefore, in order to improve its feasibility, the innovation of technology to construct tetramers should be developed. Also, it is necessary to develop tetramer products of independently intellectual property so that to meet the rapid development of our country's medical science.Chronic hepatitis B virus (HBV) infection is one of the most important health problems worldwide. HBV has many kinds of antigens, and each kind of antigen includes many epitopes. It is pivotal to construct tetramers with various epitopes of HBV.The tetramer with HBcAg107-115 was constructed here so as to investigate the frequency of HBV-specific CTL and for other applications. In order to set up a new method that was relatively easier to construct a tetramer, SCT was construced to form a tetramer with HBcAg107-115, using biotinylation.Methodes1. The construction of the tetramer with HBcAg107-115Upon addition of IPTG, A2-BSP and p2m were generated in E.coli BL21(DE3) transformed by the expression plasmid as inclusion bodies. After they were separated and solubilized in urea solution, respectively, the inclusion bodies showed correct size by SDS—PAGE analysis. The denaturant effect of the urea was removed by dilution in a refolding buffer. This allowed A2-BSP, p2m and HBcAg 107-115 to associate in a conformationally correct form. The refolded products were first concentrated and then were loaded on to the gel filtration column. The fraction eluting at 45KD were collected, based on the SDS-PAGE analysis. That was followed by biotinylation with the enzyme BirA at the unique site engineered to the C terminus of the A2-BSP. It should be done to perform a test to check the biotinylated proteins after the extra biotin was removed. Tetramers were generated by the biotinylated protein with streptavidin-PE at a molar ratio of4:l.2. Checking the activation of tetramers for HBcAg 107-115The splenocytes of two immunized transgenic mice were separated. One was immunized with by the recombination vector pcDNA3.0-HBcAgl07-115, the other with vector pcDNA3.0. Tetramers with HBcAg 107-115, the anti-CD8~+ antibodies that were labeled with the FITC and the anti-CD3~+ antibodies that were labeled with the PE-Cy5 were admixed to the separated splenocytes, and then the sample was analyzed using a flow cytometry.3. SCT was formed in the tetramer with HBcAg 107-115The DNA sequences of the PreScission protease site and the BSP were added to the 5' end and the 3' end of the plus strand of SCT, respectively, by overlapping PCR. The PCR product and the expression vector pET32c(+) were similarly digested with restriction endonucleases and ligated together with DNA ligase. The ligation reation was transformed into E.coli JM109 by the electroporation. After it was identified, the recombinant was transformed into the E.coli BL21(DE3). Upon addition of IPTG, the recombination protein was expressed to form the inclusion body, and to fuse with a His-tag. The inclusion body was extracted and dissolved in urea solution. After the denatured protein was refolded in vitro, the folding reaction was concentrated and added the PreScission protease. After cutting the fusion protein, the recombination was further purified using Ni-column and GST-column in order. The purified protein was further formed in the tetramer following the preceding method.4. Tetramers with HBcAg 107-115 were generated using the orthodox and altered methods, respectively. To find which one is superior, the differences between the two methods were compared. Results and conclusions1. Completion of the construction of the tetramer with HBcAg 107-115 using the orthodox and altered methods.2. The pSB1 vector was constructed.3. The altered method was more superior than the orthodox one.