| CD8+ T cells are one of the major components of cell-mediated immunity and play a key role in the elimination of virus-infected, tumor and allograft cells. The analysis of tetramer-positive CD8+ T cells has provided a method in the field of cellular immunology. The traditional methods, such as 51Cr release assays and LDA, are limited not only as they requires proliferation and survival of the antigen-specific cells, but also as they underestimate the number tremendously and are of little sensitivity.Tetramer analysis is a novel technique in immunological research that has dramatically changed our knowledge of the immune response to pathogens, tumors and autoimmune diseases. Through the formation of MHC -peptide tetrameric complexes it can provide accurate counts of antigen-specific T-cells and it allows their phenotypic and functional analysis. The tetramer is composed of the human leukocyte antigen (HLA) heavy chain, fi-2 microglobulin (6-2 m), the nominal peptide, and streptavidine. The HLA heavy chain and 6-2 m are expressed in Escherichia coll But up to now, most of Labs express these two proteins by using IPTG (isopropyl 6-D-thiogalactopyranoside) l . IPTG is very expensive, and it is very tedious andlaborious to induce expression protein. So it is very difficult to scale up to express the objective protein.To address this problem, extracellular fraction of HLA-A0201 and B-2m (absent signal peptide) genes were cloned from PBMCs by RT-PCR. DNA coding for a Gly-Ser linker and a BSP were added to the COOH-terminus of the extracellular fraction of HLA-A0201 by PCR, using an HLA-A0201 as the template. Then the HLA - A 0201 -BSP and 6-2m genes were cloned into pBV220 vector and expressed induced by elevating the cultural temperature to 42 , respectively. The expressed proteins were purified, and detected by ELISA and Western-blot analyses. High-efficientexpressions of HLA-A0201-BSP and B-2m proteins lay a good foundation for further expression and purification in prokaryotic system and constructing MHC class I-peptide tetramer complexes to study the function of CTLs.Expression of the recombinant proteins in each of the two constructions was analyzed. It was shown that in pBV220-HLA-A0201-BSP constructions the transformed cells produced a large amount of new proteins (MW: about 33 kD) occupied 46% of the total bacterial proteins estimated from SDS - PAGE gel. Thesoluble analyses showed that HLA-A0201-BSP was mainly located in the insoluble fraction of the cell as inclusion bodies and the proportion was about 90%. Meanwhile, the analyses of B-2m showed that there was a new expression band at MW about 12 kD whose expressive level was about 26% of the total bacterial proteins and the percentage of the protein in inclusion bodies was above 85%. The expression level was highly related with the inducing temperature and the inducing time. The optimal growth temperature was determined to be 37?C and the cell culture was induced at 42 0 C for 4 h before harvesting. Not only the yield of the recombinant proteins was higher at 37 than that at 30 , but also the rates of expression inclusion body formation was higher at 37?C than that at 30?C . And our experiments indicated thatthe prolonged induction time did not result in a significant increase of the expression yield of the recombinant proteins from 4h to overnight. Meanwhile, to obtain overexpressed recombinant protein, we compared the expression level of recombinant protein using different prokaryotic expression vectors (pET-30a (+), pET-28b (+), pET-22b (+), pET-42b (+), and pKKH). The expression level in pET28 b (+) was the highest and the next was that in pET22b (+). We also tried to use other E. coli as transformed cell to get a more efficiency expression and found that the expressi9n level was similarly (data not shown).The inclusion bodies of HLA-A0201-BSP and 6-2m dissolved were charged in an S Sepharose FF column. The objective proteins appeared in uricombined peak and were collected. After passing through a Q Sepharose FF column the proteins were elu...
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