Expression Of Recombinant Active Segments Of Human Perforin And Its Effect On HIV Infected CD4～+T Cells

OBJECTIVE The cytotoxic cells(including CD8+ T cells and NK cells)play a major role in the elimination of viruses. But CD8+ T cells can not do a better job of controlling HIV infection. Many studies suggest that HIV-specific CTL deficiency could be partly mediated by a defect in perforin expression. However, the exact mechanisms of perforin in HIV infection are poorly understood. The aim of the present study was to investigate the role of perferin in the pathogenesis of AIDS and its potential function of the therapy by studying the effect of recombinant active segments of human perforin on HIV infected MT4 cells.METHODS (l)In the present study, five forms of recombinant perforin, full-length(leader peptide excluded), MACl(28-349aa), MAC2(166-369aa) C-100 and N-60 amino acid of human perforin were selected as candidate active segments of human perforin and designated as HP1 HP2 HP3 HP4 HP5. The target genes were amplified by PCR, using HP as template. The PCR products were individually subcloned into sequencing vector pGEM-T. The recombinants were analyzed by restriction endonuclease digestion and PCR. The positive recombinant plasmids were made sequence.(2)HP1 HP2 HP3 HP5 were subcloned into expression vector pET-DsbA, and PET-41a(+) was used as the expression vector of HP4. The positive expression recombinants were transfered into host bacteria(E coli BL21plyss-DE3), and the expression effusion proein was induced with IPTG. After a large-scale cultures, the fusion proteins were purified using Ni-NTA agarose affinity chromatography. (3)Activity assay of fusion protein was examined by the lysis of erythrocytes mediated by recombinant candidate active segments of human perforin using a hemolysis micoassay.(4)Rabbit was immunized with the fusion protein of HP 1,and the antibody efficiency was assessed by double immunodifrusion.The specific combination of HP1 HP2 HP3 HP4 HP5 and multiclonal antibody was tested by Westernblot. (5)The human MT4 cells (CD4+ cells) were infected by HIV-1 SPSS. The effect of the recombinant active segments of human perform on the MT4 cells and HIV-1 spaa infected MT4 cells were compared. The surface structure and inner structure of cells were observed by scanning electronmicroscope, the measurement of intracellular Ca2+ concentration was by laser confocal system, the proliferation change was analysed by MTT and flow cytometry, the expression of HIV p24 antigen was tested by sandwich enzyme immunoassay, and the immunohistochemistry was employed to examine the location of recombinant active segments of human perform on the MT4 cells. RESULTS (1) The recombinant cloning plasmid pGEM-T-HP1 HP2 HP3 HP4 HP5 were constructed successfully. And the size of HP1 HP2 HP3 HP4 HP5 was about 1605bp 963bp 609bp 300bp 180bp. The nucleotide sequence of the target gene was identical to that published in Genebank.(2) The expression plasmids were constructed successfully. The expression host (Escherichia coli BL21plyss) expressed fusion protein highly. Then the fusion protein was affinity purified from bacteria protein by Ni-NTA. The fusion protein of HP1 HP2 HP3 HP4 HP5 were about 81.7kD 58kD 45kD 44kD 29.6kD, and the final yield of recombinant protein from 1 liters of bacterial cultures was 15mg~20mg.(3) HP2 and HP5 had the function of hemolysis, and the hemolysis ratio of recombinant active segments of human perform was related to the concentration.(4) The efficiency of anti-serum was 1:32 by double immunodiffusion test.And Westernblot analysis showed that anti-serum can particularly reacted to recombinant candidate active segments of human perform.(5) Recombinant active segments of human perform (HP2 and HP5) were able to induce the formation of structural pores in target membrane. And the cells displayed swell, the dilation of mitochondria and other organelles could be seen.The increasion of intracellular Ca2+ was observed. The proliferation of cells was promoted by recombinant active segments of human perform in a certain concentration range. Afterthe recombinant activ...